Annual Report of Biomass Programs at the Center for

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					                                   ANNUAL REPORT

                                            FOR

                BIOMASS PROGRAMS OF THE CENTER FOR
                        NATURAL RESOURCES

                                         2001-2002


INTRODUCTION

The Center for Biomass Programs was established in 1980 to identify biomass program
opportunities and to plan, develop, and coordinate projects. Reading literature and conferring
with colleagues to generate plans facilitates this. In addition, scanning request for proposals,
maintaining contact with sponsors, and being present at appropriate meetings helps identify
support. As of July 1, 1999, the Center merged with the Center for Natural Resources.

The Center’s biomass research and education programs focus on biomass waste capture, biomass
energy crops, advanced energy conversion technology and fuel-use options. Besides this, we
explore non-conventional uses of domestic crops and innovative waste management
opportunities. This taps into the university’s many disciplines, bringing together the expertise
needed to conduct a comprehensive biomass program.

The Center coordinates a variety of programs to enrich academic curricula and to extend research
to users. A biomass seminar series, featuring faculty research and presentations by visiting
scientists, is conducted to keep teachers/researchers at the cutting edge of biomass energy
technology.


VISITING SCIENTISTS

The Center coordinates a variety of programs to enrich academic curricula and to extend research
to users. A biomass seminar series, featuring faculty research and presentations by visiting
scientists, is conducted to keep teachers/researchers at the leading edge of biomass energy
technology. This year, we were visited by:

1. Dr. Rufus Chaney, Senior Research Agronomist, from United States Department of
   Agriculture, Beltsville, MD. The visit during the July 2002 included meeting with faculty
   and the students for the scientific information and a presentation on "practical
   phytoextraction of soil cadmium.

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2. Professor Ramamurthi Rallapalli, Ex-Vice Chancellor Sri Venkateswara, Tirupati, India and
   Adjunct Professor, Department of Medicine, Robert Wood Johnson Medical School, New
   Brunswick, NJ. The visit included signature of the second five-year cooperative agreement
   between the Sri Venkateswara University and the University of Florida.

3. Professor Geetha Bali, Director of the Center for Clean Environment Technology, Bangalore
   University, Bangalore, India. The visit included the discussion of the exchange students and
   scientists from Bangalore University and the University of Florida.


CONFERENCES/WORKSHOPS

Center for Natural Resources, Biomass Programs organized an international workshop on "The
Remediation of Contaminated Soils and Water" held in Gainesville, FL during July 24-26, 2002.

We cooperated with FORCE (Florida Organics Recycle Center for Excellence) to organizing the
"Composting in the Southeast Conference and Exposition" held in Palm Harbor, Florida on
October 6-9, 2002. Dr. Aziz Shiralipour was the chairman of the program committee.


INTERNATIONAL ACTIVITIES

Background - The University of Florida (UF), Florida Atlantic University (FAU), Sri
Venkateswara University (SVU) and Bangalore University (BU) have a sustained interest in, and
active programs addressing biotechnology for clean environment and energy. Because of this
mutual interest, a memorandum of agreement (MOA) was signed between UF and SVU in 1995
and another MOA was signed between FAU and BU, with UF being a cooperator, in 1997. The
exchange of the MOA between the BU and FAU/UF took place between Dr. N. R. Shetty, Vice
Chancellor, BU and Dr. Aziz Shiralipour, Associate Director, Center for Natural Resources,
Biomass Programs, UF during a ceremony in Bangalore, India on August 5, 1998. The latest
MOA was signed between the BU and UF in May 2000 in Gainesville, Florida. Dr. Mike
Martin, Vice President for Agricultural and Natural Resources, UF and Dr. K. Siddappa, Vice
Chancellor, BU signed the MOA.

As the results of these agreements several workshops/conferences were held either in India or
Florida as follows:

1. Indo/U.S. Workshop on Eco-Friendly Technologies

As a part of the collaborative program between UF and SVU, the Center arranged a trip to SVU
in Tirupati, India, to attend a workshop during September 1996. The group included UF
researches, a representative from Florida Department of Environmental Protection, and Florida
private companies. While in India, the group visited with the Indian government dignitaries, US-
AID officials in New Delhi and attended a workshop at SVU during September 1996. At the
opening ceremony of the workshop, the Associate Director of the Center exchanged the
memorandum of understanding between SVU and UF with SVU Vice Chancellor.

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2. U.S.-India Collaborative Workshop on Environment and Energy

This was a 2-day workshop organized by the Center for Environmental Studies, Florida Atlantic
University and Center for Biomass Programs, University of Florida. It was held in Boca Raton,
Florida on April 3, 1997 and in Gainesville, Florida on April 4, 1997. Scientists from the
University of Florida, Florida Atlantic University, SVU, Bangalore University, India Embassy in
the U.S., local and state agencies, and private sector participated in the workshop.

3. U.S.-India-Trinidad Workshop on Energy and Environment

This was a 2-day workshop organized by the Center for Environmental Studies, AU and Center
for Biomass Programs, UF. It was held on May 5, 1998 in Boca Raton, Florida and continued on
May 6, 1998 in Gainesville, Florida. Scientists from UF, AU, SVU, BU, West Indies University,
Trinidad, India and Trinidad Embassies in the U.S., local and state agencies, and private sector
participated in the workshop.

4. Indo-US Workshop on Application of Biotechnology for Clean Environment and Energy

As a part of the collaborate program between these universities, a group of AU and UF
researches, a representative from Florida Department of Environmental Protection, and Florida
private companies traveled to India for one week to visit with the Indian government dignitaries
and private sector, and to attend a workshop during August 1998.

5. International Biotechnology for Energy and Clean Environment Workshop

This was a 2-day workshop organized by the Center for Natural Resources, Biomass Programs.
It was held on May 1-2, 2000 in Gainesville, Florida. Scientists from the UF, AU, SVU, BU,
local and state agencies, and private sector Participated in the workshop. During the opening
ceremony, a MOA was signed between the UF and BU for five years and the MOA between the
UF and SVU was extended for another five years.

6. International Workshop on Biotechnology and Environmental Security

As part of the collaborative program between the BU and UF, the Center co-sponsored the
International workshop on Biotechnology and Environmental Security, during January 12-13,
2001 in Bangalore, India. Dr. Joe Schaefer and Dr. Aziz Shiralipour participated in this
workshop. Dr. Mike Martin, Vice President for Agricultural and Natural Resources planned to
attend to be awarded with a Honorary Professorship. Since Dr. Martin could not attend, Dr. Joe
Schaefer, Director of the Center, accepted the award for him.

7. "The Remediation of Contaminated Soils and Water Workshop"

Center for Natural Resources, Biomass Programs organized an international workshop on "The
Remediation of Contaminated Soils and Water" held in Gainesville, FL during July 24-26, 2002.


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During the opening ceremony on the first day, Professors Ramamurthi Rallapalli from Sri
Venkateswara University in India, Geetha Bali from the Bangalore University in India, and
Satish Kastury from Florida DEP in Tallahassee, FL were named IFAS Scholars by Dr. Michael
Martin, Vice President for Agriculture and Natural Resources, University of Florida. Release of
a book by Dr. Michael Martin on "Environmental Technology" edited by Geetha Bali,
Ramamurthi Rallapalli, S. B. Sullia, Aziz Shiralipour, and Satish Kastury and another book by
Satish Kastury on "Biodiversity" edited Ramamurthi Rallapalli and Geetha Bali was included in
the ceremony. During the first day, scientists made presentations from Bangalore, Sri
Venkateswara, Florida Atlantic, University of Florida and USDA.

During the second and third days a training course was offered to Florida DEP staff, Students
and staff from Atlantic University and University of Florida. The training part was sponsored by
Center for Natural Resources and the Interstate Technology and Regulatory Council.

These workshops on related topics were very successful and have resulted in the initiation of
interesting programs. During the last workshop in Gainesville, FL representatives from the
participating universities discussed the possibilities and the mechanism of faculty and student
exchange between the participating universities.


OUTREACH AND COMMUNICATION

The Center for Natural Resources developed a new Biomass brochure to communicate the goals
of the UF Biomass programs to a wider audience. In addition, the Center designed a web page
for Biomass programs in order to foster the exchange of ideas and information. This site can be
accessed at: http://cnr.ifas.ufl.edu/programs/biomass.htm

Dr. Aziz Shiralipour was selected to serve on the editorial board of Compost Science and
Utilization, a quarterly scientific journal, which provides authoritative data on the most effective
implementation of composting principles and research (published by The JG Press Inc.).


ONGOING RESEARCH PROJECTS ONGOING RESEARCH PROJECTS

The Center developed or assisted in the development of four projects and continues to manage
and/or monitor their progress. Summary reports are below:

1. Effects of Compost on Arsenic Leachability in Soils and Arsenic Uptake by a Fern

Project Leaders: Aziz. Shiralipour, Center for Natural Resources, UF, Gainesville, FL
Lena Ma, Department of Soil and Water Science, UF, Gainesville, FL
Rocky Cao, Soil and Water Science Department, University of Florida (Post Doctoral Research
Associate)

Sponsor: Florida DEP

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Objective

The objective of this three-year project is to provide experimental data to evaluate the effect of
composted material in preventing or reducing arsenic leaching into ground water.

Special Tasks

1.      Evaluate the effectiveness of composted material in reducing the inorganic arsenic
        leaching into ground water.

2.      Screen the most effective compost in reducing arsenic leachability in soils.

2.      Examine the impacts of compost amendment on arsenic uptake by a fern plant.

4.      Examine the feasibility of fern biomass reduction via composting to reduce disposal cost
        and compare the cost with the chemical remediation.
        During the first year, task 1 was accomplished. Tasks 2 and 3 will be completed at the
        second year. Task four shall be completed during the third year.

Task 1. Evaluation of Effectiveness of Compost in Reducing Soil Arsenic Leachability

Two types of soils were used in this experiment: Contaminated soil collected from an arsenic-
contaminated site (CCA site) in Central Florida (soil 2), Non-contaminated soil collected from a
sandy soil nearby the CCA site. (Soil 1) Both soils were air-dried and passed through a 2    -mm
sieve.

Two different types of composts were utilized: Municipal solid waste compost (MSW) from
Sumter County Composting Facility (Compost 1), 2) Biosolids compost (Bio) from the Solid
Waste Authority of Palm Beach Facility (Compost 2). The compost samples were air dried and
passed through a 2-mm sieve. Three levels of inorganic arsenic, 0, 10 and 50 mg/kg were added
to columns containing soil 1.

The effect of compost on reducing arsenic leachability was tested using a column experiment.
Compost was added to the soil at an equivalent rate of 50 tons per acre. The plastic columns
used in this experiment are 12.8 cm by 2.75 cm, which hold 60 ml solutions. These columns
were packed to about 11-cm depth. Columns were packed with either soils alone or mixture of
soils and composts in the following manner:

Soils alone:
60 grams soil 1 or 60 g soil 2 alone

Soil/compost mixture:
Mixture of 6 grams compost 1 and 54 g soil 1
Mixture of 6 grams compost 1 and 54 g soil 2

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Mixture of 6 grams compost 2 and 54 g soil 1
Mixture of 6 grams compost 2 and 54 g soil 2

A solution of 1500 mg arsenic/liter was prepared and 0, 0.4 or 2 ml was added to soil 1 or soil
1/composts to provide 0, 10, or 50 mg/kg arsenic, respectively. To the columns containing soil 2,
only deionized water was added. The columns were set at room temperature for a week for
incubation at 80% field capacity before leaching. During each leaching event, 2 inches of
deionized water was added to the columns to leach the columns. Leachates were collected at
weeks 1, 2, 4, 8 and 16 and were sent to the laboratory for chemical analysis. Analyses included
the measurements of pH, electrical conductivity (EC), water-soluble organic carbon (WSOC or
OC) and As in each leachate. Samples were replicated so statistical analysis can be made for
comparisons. Totally 36 columns were set up (3 compost types × 4 arsenic levels × 3 replicates).

First year results indicated that the newly added 50 mg/kg As had greater leachability than native
As in soil at the end of the 16th week. More As was leached out at higher As addition at all
collection periods. The leachability of added As decreased with time, indicating As adsorption
by soil. However, native As did not change much with time, suggesting slow release of As from
soil during incubation. However, native As did not change with time, suggesting slow released
of As from soil during incubation. Composts at the rates used in this work do show some
inhibitory effects on As concentration in the leachates. However, the cumulative amounts of
leached As were dependent on the types of composts. The municipal solid waste compost
(MSW) reduced the leachability of As in soil, especially in the native soil As, whereas the
biosolids compost greatly increased the leachability of native As. Composted biosolids had
greater electrical conductivity (EC) and dissolved organic carbon (DOC) than the composted
municipal solid wastes (MSW) did, which facilitate As release from soil. The DOC in the
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leachates decreased with increased leaching times, but changed little after the 4 week. Leachate
pH and EC also showed similar trends to DOC. These findings indicate that composts with high
EC and/or high DOC are not suitable to be utilized for reducing the arsenic leachability. On the
other hand, composts with low EC and/or low DOC are effective in reducing arsenic leachability
in contaminated soils.


2. Human Health and Ecological risk assessment

Project Leaders: Joseph S. Schaefer and Aziz Shiralipour, Center for Natural Resources, UF,
Gainesville, FL.

Sponsor: Florida DEP

Solid and hazardous waste management and disposal methods generate many questions and
concerns about actual and potential impacts on the environment and human health. To alleviate
these concerns, University of Florida (UF) has the potential resources. Many UF faculty who
hold positions in a variety of disciplinary units have expertise in the different aspects of
hazardous waste from risks to humans and the ecology of natural systems, to remediation of
toxic waste sites. The Center for Natural Resources/UF serves to facilitate multi-disciplinary

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collaborations among UF faculty and external stakeholders to conduct a 3      -year project for human
and ecological risk assessments. The project started on July 1, 2001. The scope of work includes
two separate, but somewhat related tasks, training and proposal review that will occur each year.

Staff Training Sessions

CNR will plan, organize and conduct one 3-day training session for the staff of the Hazardous
Waste Regulation Section (HWRS) of the Florida DEP, each of the three years at locations that
will be convenient for DEP staff and instructors. The instructors will consist of experts from UF
and other appropriate organizations. Topics will cover both human and ecologically related
risks. CNR will also provide workbooks containing related materials for participants at each
training session.

Proposal Reviews

HWRS clients that need to remediate hazardous and toxic waste sites are required to submit
proposals recommending the procedures they would like to follow to eliminate existing
hazardous conditions. HWRS issues remediation permits based on the feasibility and scientific
validity of the proposed methods. HWRS will send up to 6 proposals involving human risks and
6 proposals involving ecological risks to CNR each year. CNR will provide expert review or out
source to the appropriate experts within or external to UF. CNR will be responsible for
administering funds, and coordinating and managing this project.


3. Organics: A Wasted Resource? An Extended Case Study for the Investigation and
   Evaluation of Composting and Organic Waste Management Issues. A Curriculum and
   Companion Video Project

Project Leaders : Jerry Culen, Department of Family Youth and Community Sciences, UF,
Gainesville, FL; Wayne H. Smith, School of Forest Resources and Conservation, UF,
Gainesville, FL; Aziz Shiralipour, Center for Natural Resources, Biomass Programs, UF,
Gainesville, FL.

Sponsor: EPA Region IV.

Objectives

Major objectives of this project include contributing to the public’s understanding of
the concept of composting, the key issues that are present relative to composting and the
important role they have in the remediation of these issues. An additional goal, and ultimately
one of the most important, is to help learners develop the competence and motivation required to
participate in the decision making process related to these issues.

Project Planning/Outline



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Project approval and funding started in October 1998. It was planned to be completed by the
April 2000. It was extended and completed in 2001.

The curriculum development portion of the project follows an instructional model known as the
Extended Case Study (ECS). This is a well-researched model that has demonstrated ability to
change learner behavior. A literature review, web search, and interviews with specialists in
waste management and composting also helped frame the curriculum content particularly in
Chapters 1-2. The script for the video portion of the project was developed to primarily support
Chapters 1- 3.

The student activity guide and teachers/leaders guide was also complete. The Table of Contents,
which serves as a framework for developing the curriculum, was modified slightly to enhance
and compliment the video. The content material for individual chapters was compiled and
edited. Graphics specific to the guide have been produced and linked to the individual lessons.
Digitized still photos from video segments have been included in the activity guide during final
design and layout. The student activity guide of the ECS has been designed and printed in two
colors with a full color cover of selected images from the video.

In January 1999, New Century Multimedia was contracted to shoot the video portion of the
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project. The goal of the video component of the project was t compile documentary footage
from several major compost producers, which show source, process, and utilization opportunities
based on field, and lab tests that show beneficial use. The video was designed to capture players
that are a critical part of understanding problems and issues, thus providing part of the
instructional basis for the curriculum guide. Video production was completed in November of
1999 with minor editing of credits to be completed in 2000. Copies of the video were reproduced
in 2000. A full color cover/jacket was produced for 500 of the tapes. Tapes were distributed
with the curriculum in early 2001.


4. Phytoremediation of Contaminated Sites Using Woody Biomass

Project Leaders : D. L. Rockwood, School of Forest Resources and Conservation, UF,
Gainesville, FL; L. Q. Ma, Department of Soil and Water Science, UF, Gainesville, FL; A. E. S.
Green, Department of Mechanical Engineering, UF, Gainesville, FL; and A. Shiralipour, Center
for Natural Resources, Biomass Programs, UF, Gainesville, FL

Research team for this period: D. L. Rockwood, G. R. Alker, R. W. Cardellino SFRC, UF, L.
Q. Ma and C. Tu, SWS, UF

Sponsor: Florida DEP


Objectives

The objectives of this project were:


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a) identify the most effective genotypes for chemical/metal uptake,
b) quantify their remediation potential,
c) develop guidelines for establishing and managing remediation systems using these
   genotypes and
d) develop environmentally benign methods of disposal and/or possible uses of biomass
   produced in phytoremediating systems.

Initially the project focused on identifying both woody and herbaceous species, which
accumulated contaminants at elevated levels within their tissues. Indigenous plant species
growing on CCA contaminated sites were sampled, and a greenhouse species screening trial was
conducted where tissue concentrations of contaminants were analyzed. The fern Pteris vittata
(Chinese brake fern) was identified as a hyperaccumulator of arsenic, and the tree Taxodium
distichum (baldcypress) was identified as a potentially suitable candidate for copper remediation,
but was not classified as a hyperaccumulator.

The SWS, following the discovery of the hyperaccumulating fern, focused its research efforts on
investigating the hyperaccumulation mechanism, treatment efficacy, and overall applicability of
using P. vittata for phytoremediation of CCA contaminated sites. A series of greenhouse
experiments were conducted to demonstrate the phytoremediation effectiveness of the fern. The
most significant findings from this research to date include: Elevated concentrations of As (50 to
100 mg As kg-1 ) increased biomass production of P. vittata. This is contrast to the response of
most plant species to As exposures of this level. Moreover, P. vittata reduced soil As
concentration by 20% from 97.7 to 73.1 mg kg-1 after 20 weeks of growth in CCA contaminated
soil. The majority of the As taken up by P. vittata was translocated to the above ground portions
of the plants, benefiting the phytoremediation potential of this plant, since the majority of the As
removed from the soil can be exported from the site by harvesting the above ground parts.
Results strongly suggest that the arsenic hyperaccumulating property of P. vittata could be
exploited to remediate soils contaminated with As on a large scale.

Recent research conducted by the SFRC has focused on the development of Short Rotation
Woody Crops (SRWC). Species such as Populus and Eucalyptus are currently being improved
by selection and breeding to increase biomass production.          Although the aim of tree
improvement is to develop feedstocks for energy production, mulchwood, and pulpwood, the
objective of increasing yields also complements phytoremediation research objectives, because
in the absence of hyperaccumulating trees, increasing yields potentially result in increased
contaminant removal.         Furthermore if the tree species, planting techniques, harvesting
techniques, silvicultural options, etc. are the same for bioenergy production and
phytoremediation, not only will this reduce phytoremediation development costs, but also
increase the ability to utilize crops grown on contaminated sites as feedstocks to the energy,
mulch, and paper industries.

Two field trials and two greenhouse experiments examined the use of SRWC to treat CCA
contaminated soil. At a CCA contaminated site near Quincy, Florida, 11 of the 97 cottonwood
(CW) clones had above-average growth, and clone ST201 exhibited good growth and high As


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tissue concentration. However, first-year growth and survival at Quincy was somewhat
disappointing, primarily as a result of poor soil conditions and inadequate storage of cuttings.
The reduction in soil As concentration of 31.3% in soil surrounding areas where trees survived
was impressive compared to the unplanted control area where As concentration had increased
slightly by 0.6%, but the reduction in soil arsenic could not be explained by plant uptake alone.

Growth and uptake of arsenic by CW at a 1 acre CCA contaminated site in Archer was superior
to that observed at Quincy, with upwards of 2 m height in five months during the second year.
The Archer site was much smaller and contained fewer clones than Quincy; also many of the
clones present at Archer were not present at Quincy and vice versa. Surface soil (0-20cm)
concentrations were considerably higher at Archer (156-184 mg kg-1 ) compared to Quincy (mean
of 20.3 mg kg-1 ); thus, CW growth differences between the two sites were unlikely to be caused
by metal toxicity and demonstrated the value of site specific pilot-scale plots to assess tree
growth and phytoremediation efficacy prior to the establishment of full scale treatment systems.

The projected maximum uptake rate of arsenic for CW was estimated at around 121 g ha -1 yr-1 ,
which is low compared to the uptake of certain hyperaccumulaters. For example, Thaspi
caerulescens can accumulate 78 kg Zn, 2.6 kg Cd, and 2.6 kg Ni ha -1 yr-1 . However, unlike
hyperaccumulator, the plant tissue concentrations found in CW were considerably lower than the
concentration required to classify the plant tissues as toxic waste according to the toxic
characteristics leaching potential (TCLP). Therefore, CW grown on CCA contaminated land
would not require specialist treatment or disposal and may provide an income for the landowner
in combination with a gradual cleanup of the site.

A greenhouse study investigating the effect of applying synthetic (EDTA) and biological
(histidine) chelating agents to CW plants grown in CCA contaminated soil found that both
chelates increased tissue As concentration, but EDTA also significantly increased mortality.
EDTA plus histidine increased tissue As concentration by 97% compared to the control without
significantly affecting growth or survival. EDTA is regularly used in phytoremediation systems
to increase the bioavailability of metals for hyperaccumulator uptake, but the combined effects of
histidine and EDTA on the uptake of fast growing tree species have not been reported. Soil
applied EDTA not only increases the availability of metals for plant uptake, it can also increase
the risk of metal leaching. Histidine is a naturally occurring amino acid, which is readily broken
down in the environment, and when applied as a foliar spray has the potential to increase the
uptake of metals without increasing metal leaching.

The second greenhouse study examined uptake-transpiration and uptake–growth relationships for
copper and nitrogen in CW plants using a hydroponics apparatus. The apparatus was designed to
control transpiration by adjusting the humidity in close proximity to the plant leaves. The
apparatus and technique were specifically developed for this project and underwent several
refinements before two experimental trials were conducted. The results suggest that the uptake-
transpiration relationship was more significant for Cu and N removal by CW at the elevated
levels supplied to the plants by the hydroponics nutrient solution. These findings may justify
focusing research on selecting plants or trees that have elevated transpiration rates or methods to
enhance transpiration. However, further work is required to confirm these findings.
11



A 2.8 ha SRWC plantation was established near Winter Garden in Spring 1998. Tree growth,
tree water-use, soil composition, and soil water composition were monitored since establishment.
Investigations into the effect of tree species and silvicultural treatment options on woody
biomass production and water treatment identified a high degree of system complexity. The
greatest height and woody biomass production was generated by Eucalyptus grandis (EG) with
effluent plus compost plus mulch (ECM), followed by EG with effluent plus compost (    EC), CW
with ECM, and CW with EC. After 3 years of growth, EG had produced on average 119% more
woody biomass compared to CW for EC and ECM treatments. Compost application increased
yields by 131%, mulch by 76% and mulch plus compost by 158% compared to reclaimed water
application alone (E). The significantly increased supply of macro- and micronutrients provided
by compost addition supplemented reclaimed water derived nutrients to augment yields in
compost treated plots.

CW trees treated with EC transpired a total of 1233mm between 17th May and 19th December,
accounting for 33% of the total water applied. Stem N concentrations were higher in CW
compared to EG, resulting in greater removal of N by CW after the second growing season.
However, by the third growing season, higher biomass production in EG overcompensated for
lower stem N concentrations, resulting in a 10% higher total N uptake in EG.

In the absence of trees, N leaching at 5’ below the soil surface increased by 53%. N leaching
was not significantly affected by species but was strongly controlled by soil amendments, such
that compost increased and mulch decreased N leaching from the root zone. Plots treated with E
and EM removed proportionally more N than water from the reclaimed water, such that the
concentration of the water leached from the root zone was lower than the N concentration of the
applied reclaimed water. However, the N concentration of water leaching from EC and ECM
treated plots was generally higher than the concentration of applied reclaimed water.

Selection of species and treatment options for SRWC plantations irrigated with reclaimed water
is highly dependent on the objectives of the system. To maximize woody biomass production
and/or water use, EG with ECM treatment is recommended, but if the principal objective of the
SRWC-reclaimed water system is to maximize N removal, EG with EM is recommended to limit
the input of additional compost derived N to the system.

This ‘Phytoremediation of Contaminated Sites using Woody Biomass’ research project has
investigated and developed two phytoremediation techniques for treatment of CCA contaminated
soil, using either a fern or fast growing trees. The former technique has potential for relatively
rapid, but somewhat less cost effective treatment, while the latter technique may be more
appropriate for sites where there is a lower risk to groundwater and human health and where
there is less financial incentive to treat the site. The project has also resulted in the selection of
species and silvicultural options for remediation of reclaimed water, offering a method to
discharge treated sewage effluent to the environment while utilizing useful nutrient and water
resources and protecting the groundwater against nitrate contamination.

Recommended future work includes:
1. Continued monitoring of the phytoremediation effectiveness of the Archer, Quincy, and
Winter Garden systems,
12


2. Monitoring of tree growth and hydrocarbon uptake at the Silvex site,
3. A field planting to evaluate the efficiency of interplanting P. vittata and cottonwood in an
agroforestry-style system on arsenic contaminated site,
4. Hydroponics experiments to evaluate the factors controlling contaminant uptake in trees and
hyperaccumulator,
5. Method refinement and protocol development of the leaf disk screening test,
6. Large-scale in-vitro phytoremediation screening of cottonwood and Eucalyptus clones and
progenies.

Greenhouse studies on the effects of As species and concentrations on As uptake by ferns
continued to show the promise of brake fern as an As hyperaccumulator. Arsenic species
including Methylarsonate, Dimethylarsonate, Arsenic acid, Arsenious acid, Ca3 (AsO 4 ) 2 ,
PbHAsO 4 , NaH2 AsO 4 , FeAsO 4 , and AlAsO 4 , and arsenic concentrations from 0 to 500mg kg-1
were tested. As concentrations were higher in the ferns fronds than in its stems and roots. Fern
propagation and culture methods were developed


SPECIAL PROJECTS

Special projects are supported by the center to position faculty to be competitive for extramural
funding and/or solve short-term problems. Summary of the annual report from the projects
supported in 20001-2002 follow:

1.      Biofuel Production and Carbon Sequestration in Slash Pine Plantations

Project Leaders: Janaki Alavalapati and G. Andrew Stainback
School of Forest Resources and Conservation, University of Florida

Objectives and Rational

It is widely recognized that forests play an important role in the global carbon cycle by
sequestering and storing carbon, enabling the switch from more energy-intensive materials such
as steel to forest products, and facilitating the substitution of biomass fuels for fossil fuels. It is
the role of forests that has prompted discussion of using forestry as a means to mitigate global
warming. There has been previous research investigating the economic potential of using slash
pine forest to sequester carbon from the atmosphere. This project extends that research to
include the production of biofuels. Wood waste from harvesting can be cofired with coal in a
coal electrical utility plant. By co-firing coal with wood the amount of coal needed to produce a
given unit of electricity is reduced thereby reducing net emissions of CO2 to the atmosphere.


Methodology

A dynamic optimization model to determine the optimal rotation age of a slash pine stand was
developed that internalized benefits from sequestering carbon in tree biomass and selling biofuel
in addition to timber. From this model the optimal rotation age of the stand was determined
13


assuming various prices for carbon ($0-$200 per metric ton) and biofuel ($0-$5 per metric ton).
From this information, the impact of a carbon offset market on the amount carbon offsets
supplied (through sequestration and biofuel production), timber supply and land values was
determined.

Results

The inclusion of biofuel production into the economic analysis of sequestering carbon in slash
pine plantations would not have a very large effect on forest management. Even though carbon
sequestration benefits would lengthen the optimal rotation age considerably, a biofuel market
would decrease the optimal rotation age only slightly. This decrease in rotation leads to a small
decrease in the sequestered carbon supply. However the decline in sequestered carbon would be
more than offset by the displacement of carbon emissions from fossil fuels in a short period of
time. Furthermore, the increase in land value associated with biofuel production would lead to
an increase in sequestered carbon on the extensive margin by encouraging landowners to plant
more forests. The impact of biofuel production on the supply of sawtimber and pulpwood is
very small. Thus producing biofuel in slash pine plantations would have very little impact on
forest management and timber markets but may play a beneficial role in mitigating
anthropogenic climate change.

Deliverables

1) Part of completed dissertation “Economics Of Carbon Sequestration In Slash Pine (Pinus
elliottii) Plantations” by G. Andrew Stainback
2) Extending and refining this work will prepare a research paper prepared for a referred journal.
The work is in progress.
3) Results of this research will be presented at the Southern Economics Association Annual
Conference in November at New Orleans.

Impacts/outcomes related to Florida FIRST

Provides insight into how Florida and the rest of the southeast can mitigate anthropogenic
climate change cost effectively by using forestry.

New Projects

Analytical techniques developed in this research are now being applied to study environmental
services of agroforestry in the southern U.S.


2.     Understanding Soluble Salts, Metals, and Metal Speciation in Effect on Foliage
       Plant Production and Interior Performance

Project Leaders: Jianjun Chen1 and Dennis B. McConnell2
1
  Mid-Florida Research and Education Center and 1,2Environmental Horticulture Department
14


Objectives

The objectives are to determine metal speciation; boron availability, pH, and soluble salt levels
in representative composts and to evaluate effects of potting media formulated using composts,
sphagnum peat, and pine bark on foliage plant production and interior performance.

Background

Composts as components of potting media have been shown to produce marketable foliage
plants. However, little information is available on the suitability of compost-formulated media
for plant growth under interior conditions. Quality plants produced in foliage nurseries using
compost-formulated media may perform poorly when transferred to building interiors. For
example, the quality of parlor palms grown in compost-formulated media deteriorated after being
placed in interior conditions. Consequently, foliage plant growers have been reluctant to use
composts for potting medium formulation because of the potential post-production problems.

The poor interior performance of foliage plants grown in compost-formulated media could be
due to elevated levels of boron, heavy metals, soluble salts, or a combination of these factors.
Soluble salt levels may increase as irrigation frequencies are reduced, the pH changes, and the
buffering capacity of the media decreases. As a result, physiological problems, such as necrotic
spots on leaves, may occur. Research on the forms or species of mineral elements in composts
could aid our understanding of the dynamic processes of elements in compost-formulated media
and their potential effects on containerized plant growth.

Procedures

Experiment 1: To determine initial soluble salt levels, pH, and metal species in composts or
compost-formulated media.

Three representative Florida composts (1) two parts of MSW mixed with one part of BS based
on weight (Sumter County Solid Waste Facility, FL), (2) YT (Consolidated Resource Recovery,
Sarasota, FL), and (3) three parts of YT with two parts of BS based on weight (AllGro, Inc.,
West Palm Beach, FL) were blended with sphagnum peat (SP) and pine bark (PB) in volumetric
combinations to synthesize three compost-formulated media (1) 25% peat + 25% bark + 50%
YT, (2) 10% peat + 10% bark + 80% MSW/BS, and (3) 10% peat + 10% bark + 80 % YT/BS.
Electrical conductivity (EC), pH, cation-exchange capacity, total carbon and nitrogen, and
extractable mineral elements of P, K, Ca, Mg, S, B, Fe, Cu, Zn, Mo, Al, Cd, Co, Na, Cr, Ni, and
Pb were determined. Based on the concentration of mineral elements in composts, a sequential
extraction procedure was used to determine the speciation of Cu, Fe, Mn, Zn, and B using (1)
water, (2) KCl, (3) Na4 P2 O7 , (4) NaOH, and (5) HNO3 . Species obtained following the
extraction orders were (1) soluble, (2) ion exchangeable, (3) coordinated, (4) organically bound,
(5) mineral particulate, and (6) residue, respectively.

Experiment 2: To determine the effects of compost-formulated media on foliage plant growth
and interior performance.
15


The aforementioned three compost-formulated media were used to fill 15-cm (6”) containers,
percolated with 1 liter of water daily for three days consecutively, and then used for growing
Chamaedorea elegans, Dieffenbachia ‘Camille’, and Peperomia obtusifolia ‘Variegata’ in a
shaded greenhouse. The greenhouse permitted a maximum photosynthetically active radiation
(PAR) of 284 ? mol? m-2 ? s-1 , relative humidity from 60 to 90%, and temperatures of 25 to 35o
C. Plants were watered twice a week through overhead irrigation. Two weeks after planting, 5 g
of a controlled-release fertilizer, 18N-2.6P-10K (Osmocote 18-6-12, The Scotts Co., Marysville,
OH), were applied to the surface of each container. The experiment was set as a completely
randomized design with 10 replications. After attaining marketable sizes, plant height and
widths were measured. A growth index (GI) was calculated based as GI = [(canopy widest width
+ width perpendicular) ? ? ? ? x plant height. Plants were also graded visually for overall
quality, where: 1 = poor, 2 = substandard/unsalable, 3 = good/salable, 4 = very good, and 5 =
excellent. Substrate shrinkage, the percentage of decreased depth of substrate relative to the
original depth, was determined. Leachates were collected using the pour through method, and
pH and EC of the leachates were determined. Five of the 10 replicates were harvested by cutting
shoots at the substrate surface, and shoot fresh weights were determined.

The remaining five replicates of each treatment were then placed in rooms designed and used for
evaluating interior plant performance. Plants grew under a PAR of 16 ? mol? m-2 ? s-1 provided
by cool-white fluorescent lamps with 12-hour lighting daily, relative humidity ranging from 50
to 60%, and temperatures between 21 to 24 o C. Plants were watered weekly with no fertilizer
application. Six months later, plant canopy height and widths were recorded. After overall
quality grading, substrate shrinkage was also measured.

Results
Experiment 1. The pH of the composts and compost-formulated media ranged from 5.8 to 8.1
(Table 1). Soluble salts in composted YT, YT/BS, and MSW/BS were 3.4, 6.9, and 11.1 dS/m,
whereas in compost-formulated medium 1, 2, and 3 were 1.7, 5.3, and 9.1 dS/m, respectively.
The C/N ratio of ranged from 14.3 to 28.6, suggesting that the composts or compost-formulated
media were either within or close to the maturity range. The composts and compost-formulated
media had increased concentrations of P, K, Ca, Mg, and S. Other tested elements were not in
excess except for Cu, Fe, Mn, Zn, and B, and sequential extraction for species determination was
focused on these five elements.

There was 34.2% of total Cu in the MSW/BS compost in the available form and the remaining in
the immobile form; whereas 54.4% of total Cu in the YT/BS was in the available form,
suggesting that more Cu may be leached out from YT/BS than MSW/BS if not absorbed by
plants (Table 2). Although Fe in the composts was high, more than 70% were in immobile form.
The majority of Mn in three composts was also in the immobile form. The high percentage of
immobile forms of Fe and Mn could not adversely affect plant growth if media pH could be
maintained above 5. Zinc in the available form, however, was 75.1%, 72.6%, and 70.8% of total
in MSW/BS, YT, and YT/BS, respectively. There was about 70% B in the composts in available
forms as well. Thus, a percolation of the potting media with water before transplanting should
reduce those available forms of elements and minimize potential toxic problems to plants.
16


Experiment 2. There were no significant differences in growth indices, fresh weights, and
overall quality ratings between the three plant species produced in the control and medium 1
(Table 3). However, these parameters were significantly reduced when the three species were
produced in the medium 2 or 3 that was formulated using MSW/BS or YT/BS respectively at
80%. Although growth and quality were reduced, plants produced from the medium 2 and 3
were still marketable because there were no any visible growth disorder problems. The reduced
growth in the two media is probably due to the high soluble salts that may be high enough to
effect growth but not high enough to cause toxicity problem on plants.

Shrinkage, the loss of bulk volume in container substrates has been another concern when
composts are used as components of potting media for foliage plant production. Substrate
shrinkage ranged from 12.1% to 41.5% (Table 4) at the end of production. Media with severe
shrinkage were those formulated with 80% MSW/BS and YT/BS. It is generally believed that
substrates with 15% or less shrinkage are acceptable in foliage plant production. The control
medium and along with the medium 1 had about 15% shrinkage (Table 4).

The growth indices and fresh weights of Chamaedorea elegans and Peperomia obtusifolia
‘Variegata’ increased during the interior evaluation (data not shown). However, Dieffenbachia
‘Camille’ requires higher light levels than the other two species, and only minimal growth
occurred although plant quality was maintained. No disease was observed, no detectable odors
were released from the substrates, and no further substrate shrinkage occurred during the six-
month interior evaluation period. Comparing the quality ratings graded before (Table 3) and
after six months (Table 4) in an interior environment showed overall quality decreased slightly,
but plant aesthetic appearances were still maintained. These results demonstrate that three
composts, after being appropriately mixed with sphagnum peat and pine bark, not only produced
salable foliage plants in production, but also sustained the quality of the plants under interior
conditions over at least six months. Understanding mineral element speciation in composts can
help minimize potential toxic problems to plants when compost-formulated media is used for
foliage plant production.

Summary

Composts generally have high concentration of mineral elements, consequently high soluble salt
readings. A simple concentration analysis cannot clarify the dynamic processes of elements in
compost formulated potting mixes. Information on speciation can help make decisions before
plant transplanting to reduce potential problems when compost formulated mixes are used for
foliage plant production. In this study, marketable foliage plants were produced using all
compost-formulated substrates, but plants exhibiting growth and quality comparable to the control
were those produced from medium 1, which had better physical and chemical properties for
foliage plant growth. Marketable plants after being placed in interior conditions had no growth
disorder problems when continuously maintained in compost-formulated media.

Deliverables

Results from our compost research were presented in the 2002 International Symposium on
Composting and Compost Utilization held in May 2002, Columbus, OH, and published in
17


Compost Science and Utilization (Vol. 10: 217-225). The results were also published in Edis (In
print), 2002, University of Florida.
18


Table 1. The pH, electrical conductivity (EC), carbon to nitrogen ratio (C/N ratio), cation-exchange capacity (CEC), and concentrations
of extractable nutrient elements of three composts and compost-formulated media.
______________________________________________________________________________________________________________

                       EC       C/N    CEC                     P             K             Ca            Mg             S         B
Medium      pH       (dS/m) (meq/100g)
_______     ___      ______    ___ _________               ______________________ mg•kg-1 _______________________________
Control      3.7        0.2        14.3         3.9            8.7           66.5          447.1         87.5          36.3       0.7
YT           7.2       3.4         23.7       16.2           234.6         1180.5        2005.8         394.6           77.6      1.8
MSW/BS       8.1       6.9         28.6       16.4           228.4         1267.7        2246.0         358.5          644.7      7.5
YT/BS        5.4      11.0         18.6       36.4           275.5         1706.8        5586.3         698.3          678.5     14.9
Medium 1    5.6        1.8         22.8       22.5           145.3          739.0        1844.6         297.7           68.3      1.3
Medium 2     7.4       5.5         26.7       27.0           129.9         1016.6        2105.3         315.9          526.3      5.9
Medium 3     6.5       9.1        18.0       21.5            256.8         1446.7        4731.8         620.8          581.3     10.5
______________________________________________________________________________________________________________

Medium 1 = 25% peat + 25% bark + 50% YT, Medium 2 = 10% peat + 10% bark + 80% MSW/BS, and Medium 3 = 10% peat + 10%
bark + 80 % YT/BS.
19
Table 2. Copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) species in three composts (% of total).
__________________________________________________________________________________________________________________

                        Water soluble       Exchangeable           Complexed            Organically      Solid particulate   Insoluble
Metal    Compost           (H2 O)              (KCl)               (Na4 P2 O7 )        bound (NaOH)          (HNO3 )         (residual)
____     ________      ___________          ____________          _________           ____________       _________           _________

Cu      MSW/BS             3.5                   6.2                 24.5                    12.4            53.2                0.2
         YT                5.0                   12.3                  1.7                    8.8            55.1               18.1
        YT/BS              0.7                    0.6                 53.1                   14.9            18.0               12.7
Fe      MSW/BS             0.0                  0.4                  25.6                    5.0             55.8               13.2
         YT                0.0                  0.1                   1.0                     0.0            26.4               72.5
        YT/BS              0.0                  0.1                  20.0                     7.2            43.5               29.2
Mn      MSW/BS             0.0                  0.0                  24.0                    7.5             60.7                7.8
         YT                0.0                  0.0                  14.9                      0.0           77.8                7.3
        YT/BS              0.0                  0.0                  13.5                     0.0            59.5               27.0
Zn      MSW/BS               0.7                0.4                  75.1                    2.9             10.6               10.3
         YT                0.0                  0.0                  72.6                    21.1             0.0                6.3
        YT/BS              0.0                  0.0                  70.8                    10.6             6.2               12.4
B       MSW/BS             0.0                  0.0                  68.0                    6.7              8.6               16.7
         YT                0.0                  0.0                  70.1                    5.1              2.5               22.3
        YT/BS              0.0                  0.0                  75.8                    9.6             10.8                3.8
__________________________________________________________________________________________________________________
20
Table 3. Growth index, fresh weight, and overall quality of Peperomia obtusifolia ‘Variegata’, Chamaedorea elegans, and Dieffenbachia
‘Camille’ produced in three container substrates formulated with composts from three Florida facilities.
___________________________________________________________________________________________________________________

                  Peperomia obtusifolia                         Chamaedorea elegans                   Dieffenbachia ‘Camille’‘
Medium No.z _________________________               __________________________      __________________________________
                GIy             FWx           QRw             GI             FW             QR              GI             FW           QR
___________________________________________________________________________________________________________________
  1             1134            257          5.0              634            650            4.8             1009           180          4.7
  2              995            224          3.7              512            485            3.7              893           145          3.5
  3              814            219          3.4              508            504            3.7              789           138          3.3
Control         1108            248          4.8              598            614            4.5             1131           182          4.4
LSD(0.05)        102              21          0.8               78           102            0.6               100            19         0.7
___________________________________________________________________________________________________________________
z
 Medium 1 = 25% peat + 25% bark + 50% YT, Medium 2 = 10% peat + 10% bark + 80% MSW/BS, and Medium 3 = 10% peat + 10% bark +
80 % YT/BS.
y
  Growth index (cm2 ) calculated according to GI = [(canopy widest width + width perpendicular) ÷ 2] x plant height.
x
  Fresh weight (g).
w
  Quality rating based on: 1 = poor, 2 = substandard/unsalable, 3 = good/salable, 4 = very good, and 5 = excellent.
v
  Mean separation in column by Fishers LSD, P ≤ 0.05.
21
Table 4. Electrical conductivity (dS/m), shrinkage (% of initial depth), and overall quality of
Peperomia obtusifolia ‘Variegata’, Chamaedorea elegans, and Dieffenbachia ‘Camille’ grown in
control and three container compost-formulated substrates after six months in interior
evaluation rooms.
_____________________________________________________________________________________________
______________________

                  Peperomia obtusifolia                           Chamaedorea elegans
Medium No.z _________________________                 __________________________
          __________________________________
EC                  SKy           QRx         EC               SK             QR          EC
_____________________________________________________________________________________________
______________________
  1                  0.8          13.0        4.5             904             12.1        4.6
  2                 1.0           35.4        3.8            1105             41.5        3.4
  3                 1.2           38.7        3.2            1137             36.8
Control             0.7            15.5       4.4              889            14.7        4.5
LSD(0.05)w          0.4            10         0.7              182            14.5        0.7
_____________________________________________________________________________________________
______________________
z
 Medium 1 = 25% peat + 25% bark + 50% YT, Medium 2 = 10% peat + 10% bark + 80%
MSW/BS, Medium 3 = 10% peat + 10% bark + 80 % YT/BS, and control = 50% sphagnum peat
+ 50% pine bark.
y
  SK = shrinkage of substrates.
x
  Quality rating based on: 1 = poor, 2 = substandard/unsalable, 3 = good/salable, 4 = very good,
and 5 = excellent.
w
  Mean separation in column by Fishers LSD, P ≤ 0.05.


3.        Identification of a pyruvate decarboxylase homologue from Clostridium
          acetobutylicum for use in the metabolic engineering of diverse bacteria for ethanol
          production.

Project Leader: Julie A. Maupin-Furlow, Associate Professor; Microbiology and Cell Science,
PO Box 110700, University of Florida; Gainesville, FL 32611-0700.
Tel: (352) 392-4095; Fax: (352) 392-5922; E-mail: jmaupin@ufl.edu

Objectives and rationale

     1. Express the Clostridium acetobutylicum pyruvate decarboxylase (PDC) homologue in
        gram-negative Escherichia coli and gram-positive Bacillus megaterium.

     2. Purify the C. aetobutylicum PDC homologue.

     3. Determine the biochemical properties of the purified PDC homologue.
22
       Commercially viable, large-scale production of ethanol from lignocellulosic materials
       requires a microorganism that is able to withstand the harsh conditions encountered in
       industry (e.g. high temperature, low pH, high concentrations of sugars and salts, toxins
       present in hydrolysates, etc.). The gram-positive bacteria have many of the above-
       mentioned features, which have lead to their widespread use in industry for a variety of
       processes including the production of cheeses, yogurt, proteases, and solvents.
       Unfortunately, these bacteria are largely lacking the ability to produce ethanol as a sole
       fermentation product.

Previous studies at the University of Florida have demonstrated that Escherichia coli and other
gram-negative bacteria can be genetically modified to produce ethanol from biomass using the
pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) genes from the gram-negative
bacterium Zymomonas mobilis (Ingram et al., 1999). Although feasible, similar approaches in
engineering gram-positive bacteria have been limited, probably due to differences in
transcription and translation of genes as well as protein degradation (Barbosa and Ingram, 1994;
Gold et al., 1996).

Dr. Ingram’s lab previously isolated a pdc gene from Zymomonas mobilis (Conway et al., 1987).
In collaboration with Dr. Ingram, we have now isolated three other bacterial pdc genes (i.e.
Sarcina ventriculi, Zymobacter palmae, Acetobacter pasteurianus) (Talarico et al., 2001; Raj et
al., 2001 and 2002). It would be advantageous in metabolic engineering to have a wide variety
of pdc genes available to channel pyruvate to ethanol; however, these are the only bacterial pdc
genes that have been characterized to date. The isolation and characterization of additional
ethanol production genes, such as the gram-positive C. acetobutylicum pdc homologue, are
predicted to broaden the diversity of bacteria which can be engineered for industrial production
of ethanol.

Procedures to accomplish objectives.

Expression plasmid pJAM425 was generated by PCR amplification of the C. acetobutylicum pdc
homologue       using       genomic      DNA       from      ATCC        strain     824,    5’-
GGTGACACATATGAAGAGTGAATAC-3’                             (oligo               1),           5’-
ATGGATCCCTAATTATTTTGATTTG-3’ (oligo 2) and Vent DNA polymerase. Primers were
designed for directional cloning of the pdc gene into pET24b using NdeI and BamHI. Plasmid
pJAM426 was constructed with the following modifications, oligo 2 was replaced with 5’-
GATACTCTCGAGATTATTTTGATTTG-3’ for directional cloning of the pdc gene into
pET24b using NdeI and XhoI. Plasmids pJAM425 and pJAM426 were used for expression of
the PDC homologue with and without a poly-His tag (-His6 ) in E. coli. For expression of PDC in
B. megaterium WH320 lac- xyl+, the BspEI-to-XbaI fragment of plasmid pJAM425 was ligated
with the SpeI and XmaI sites of plasmid pWH1520 to generate plasmid pJAM433. Plasmid
pJAM433 carries the pdc gene along with the Shine-Delgarno site and T7 transcriptional
terminator of the original pET24b vector. The Shine-Delgarno site of the inserted pdc gene was
positioned directly downstream of the xylA’ stop codon to allow for translational coupling in
which the ribosomes would presumably terminate at the stop codon for xylA’ and then reinitiate
23
at the pdc start codon. Fidelity of cloned DNA was confirmed by DNA sequencing using the
Sanger dideoxy method and a LICOR sequencer.

The C. aecetobutylicum PDC protein was expressed in recombinant E. coli and B. megaterium
using the plasmids described above. Cells were harvested by centrifugation (10,000 × g, 10 min,
4ºC), resuspended in 50 mM sodium phosphate buffer at pH 6.5 containing 1 mM TPP and 1
mM MgCl2 , and passed twice through a French pressure cell (20,000 lb per in2 , 4ºC). Cell lysate
was centrifuged (10,000 × g, 15 min, 4ºC). PDC was purified from soluble fractions using Q       -
Sepharose, Superdex 200, Phenyl Sepharose, and h   ydroxapatite chromatography. PDC inclusion
body aggregates were purified by washing the pellet twice with 20 mM Tris-HCl containing 1%
Triton X-100 and 10 mM EDTA at pH 7.5, resuspending the pellet in 0.3% N         -lauroylsarcosine
and 50 mM CAPS at pH 11.0 (15 mg protein per ml), and incubating for 15 min at RT.
Solubilized PDC was purified by centrifugation (10,000 × g, 10 min, RT) and refolded by
dialysis against 20 mM Tris-HCl buffer at pH 8.5 (2 × 3 h, 4ºC). Sample was filtered (45 ? m)
and applied to a Phenyl Sepharose column.           For purification of PDC-His6 , cells were
resuspended in 10 mM Tri-HCl at pH 8.0 containing 6 M guanidine hydrochloride and 100 mM
Na-phosphate (buffer A) and incubated with stirring (1 h, RT). Insoluble cell debris was
removed by centrifugation (10,000 × g, 15 min, 4ºC). Supernatant was applied to a Cu2+-NTA
column equilibrated in buffer A and washed with 10 mM Tri-HCl and 100 mM Na-phosphate
buffer containing 8 M urea (buffer B) at pH 5.9. The PDC-His6 protein was eluted with buffer B
at pH 4.5 and further purified by excision from SDS-PAGE. Gel slices were minced with a razor
blade in phosphate buffered saline and injected into rabbits for polyclonal antibody production.
Purification of PDC was monitored by SDS-PAGE, Western Blot, and decarboxylase activity
(i.e. PDC and IPD). PDC activity was measured by a coupled assay with baker’s yeast ADH
(Conway et al., 1987). IPD activity was measured as the production of indole acetaldehyde from
indole pyruvate (Koga et al., 1991).


Results

Identification of a C. acetobutylicum PDC homologue - C. acetobutylicum is a gram-positive
bacterium known to be especially hardy as demonstrated by its use in the industrial production of
solvents such as propanol, isopropanol, butanol, and acetone. The genome sequence of this
bacterium has been completed (Nolling et al., 2001). Comparison of the four bacterial PDCs to
the C. acetobutylicum genome revealed that this organism has the genetic capacity to produce a
PDC homologue. Structural analysis of the C. acetobutylicum PDC homologue revealed that all
amino acid residues known to be required for PDC activity were conserved. Thus, the C.
acetobultylicum PDC homologue was postulated to be a good candidate for engineering gram-
positive biocatalysts for bioethanol production.

Synthesis of the C. acetobutylicum PDC homologue in recombinant bacteria - The
identification of a C. acetobutylicum PDC homologue was somewhat unexpected since pyruvate
ferredoxin oxidoreductase, pyruvate formate lyase, and lactate dehydrogenase are predicted to be
the primary enzymes responsible for channeling pyruvate to the fermentation end-products
butanol, ethanol, acetone, acetate, lactate, and butyrate. To further examine the function of this
24
homologue, the putative pdc gene was cloned into expression vectors to generate plasmids
pJAM425, pJAM426 and pJAM433 (see methods). This enabled synthesis of PDC in
recombinant E. coli and B. megaterium with and without a C  -terminal histidine-tag (-His6 ). The
PDC was synthesized in both recombinant hosts as a 60-kDa protein on SDS-PAGE, consistent
with the molecular mass calculated from the gene sequence and similar to the masses of other
PDC proteins. Surprisingly, synthesis of the gram-positive C. acetobutylicum PDC in E. coli
was about 10- to 20-fold higher than B. megaterium.

Purification of the C. acetobutylicum PDC homologue - Based on SDS-PAGE, the majority
(98%) of PDC and PDC-His6 formed inclusion bodies when recombinant strains were grown at
temperatures ranging from 25º to 37ºC. Neither PDC nor IPD activity was detected in the
soluble or insoluble fractions. The soluble PDC fraction was further purified using procedures
which our lab has established for four known bacterial PDC enzymes. This initial approach
resulted in a low yield of soluble PDC which had no apparent decarboxylase activity (i.e. PDC or
IPD activity). The improper folding of PDC and/or the high LDH activity of the host strain may
have contributed to the apparent inactivity of the purified protein.               An E. coli
BL21(DE3)/pRARE ldh mutant strain was constructed and will be used for future production of
the PDC homologue. This approach is expected to minimize host LDH activity, which would
otherwise generate a high background by oxidation of NADH in the PDC/ADH coupled assay.

Our second approach was to purify, solubilize, and refold the PDC protein that had formed
inclusion bodies in recombinant E. coli. The advantage of this approach was that there was an
extremely large amount of PDC protein synthesized in this form, which could serve as starting
material for purification (approximately 50 mg protein per 500 ml culture). The PDC protein
was denatured and refolded into a conformation that remained in solution in the presence of
cofactors; however, neither PDC or IPD activity was detected.

An additional approach was to purify the PDC-His6 tagged protein using denaturing conditions
in combination with Cu2+-NTA chromatography. SDS-PAGE was used to further purify the
protein for polyclonal antibody production in rabbits. Anti-PDC was generated and was used for
immunodetection of PDC by Western blot. These results suggest that Western blot can be used
as an additional tool to monitor purification of the PDC homologue from the native organism C.
acetobutylicum ATCC 824 where it is expected to fold correctly. Column chromatography, as
described for the other PDC proteins, will be used for purification.

Biochemical properties of the C. acetobutylicum PDC/IPD homologue - This portion of the
project is important for assessing the usefulness of the PDC enzyme for the industrial production
of ethanol from biomass. Once we have purified a larger quantity of soluble PDC using the
methods described above (in progress), we will perform biochemical and kinetic analysis of the
purified PDC enzyme. This will include the: identification of the type of reaction(s) catalyzed
(e.g. decarboxylation of pyruvate and/or indole-3-pyruvate), determination of the optimal assay
conditions for these reaction(s), estimation of the Km and Vmax values under optimal conditions
as well as conditions predicted for a gram-positive bacterial cytosol during fermentation of
lignocellulosic hydrolysate, and characterization of the stability of the enzyme at various
temperatures, pH values, solvent conditions, etc.
25

Deliverables supported entirely or in part by this grant

     •   A pyruvate decarboxylase homologue has been cloned by PCR from the gram-positive
         bacterium C. acetobutylicum to generate expression plasmids pJAM425, pJAM426 and
         pJAM433.
     •   Using these plasmids, PDC was expressed at high-levels in recombinant E. coli and at
         low levels in Bacillus megaterium.
     •   The PDC has been purified from recombinant E. coli and used as antigen for generation
         of polyclonal antibodies in rabbits.

Talarico, L. T. and J. A. Maupin-Furlow. 2002. Bacterial pyruvate decarboxylases and their use
in ethanol production in Gram-positive bacteria. Southeastern Branch American Society for
Microbiology Abstracts.

Impacts/outcomes related to Florida FIRST

This research project utilized a systems approach (i.e. genomics) for the investigation of
fundamental aspects of microbial physiology and metabolism relevant to biotechnology and
microorganisms of environmental importance. It also enhanced our knowledge toward the
development of novel processes and biocatalysts for the conversion of agricultural residues into
alternative fuels and value-added chemicals.

New projects resulting from this grant

NSF and DOE proposals will be submitted within the next year that will incorporate the results
of this project.


4.       Pesticide Screening in Commercial Compost in Florida

Project Leader(s): P. Chris Wilson, Sandra B. Wilson, and Peter J. Stoffella

Objective(s) and rationale: The purpose of this study was to evaluate the presence of selected
pesticides in raw yard waste, ground yard waste, and finished compost at a commercial
composting facility in Florida. Pesticides may present production and health risks to non-target
plant and animal species.

Procedures to accomplish objectives

Samples were collected from raw yard waste, ground yard waste, and finished compost in
December 2001, at a commercial composting facility located in South Florida. Composite grab
samples were collected at a depth of 0.3 – 0.6 m. At each sub-sampling location, 7,079 cm3 of
materials were measured and placed in a cleaned 18.9 L bucket. Six sub-samples were collected
and composited into two separate duplicate samples for each type of material sampled. The sub-
26
samples were collected at several locations throughout each type of material. Samples were
stored on ice and delivered to the lab within 5 h of collection. The raw yard waste heaps
contained a variety of materials, including tree branches, grass clippings, palm fronds, and tree
leaves. Grass clippings were specifically targeted for sampling in these materials since they have
the highest likelihood of being associated with pesticide use in lawns.

Pesticides were extracted from the samples and analyzed using standard EPA methods. A
listing of analytes, analysis methods, and detection limits is presented in Table 1. Due to
expensive analytical costs, analytical methods were chosen that would provide information on as
many pesticides as possible. Sample extracts were analyzed using a HP 6890 gas chromatograph
(Agilent Technologies, Palo Alto, CA, USA), equipped with DB35MF and DBXLB columns and
dual electron capture detectors. Injector and detector temperatures were 250 and 350 °C,
respectively. Following a 0.5 min. hold time, oven temperatures were ramped from 110 to 320
°C @ 15 °C per min., with a final hold time at 320 ºC for 2 min. Any samples with positive
detections using this system were then confirmed using a HP 5973 GC-Mass Spectrometry
system (Agilent Technologies, Palo Alto, CA, USA) equipped with a DB5MF column and
operated under similar conditions as previously discussed.        Within the MS detector, the
quadropole and source temperatures were maintained at 106 °C and 230 °C, respectively.


Results

Atrazine was the only herbicide that was detected in any of the samples (Table 1), and it was
only present in raw yard waste. The mean concentration of atrazine in the duplicate samples was
1.6 mg/kg (PPM). This is not surprising since grass samples were specifically targeted and
atrazine is commonly available to homeowners and commercial businesses as an active
ingredient in “Weed and Feed” fertilizer/herbicide combinations.

Alpha chlordane(5.7 µg/kg), gamma chlordane (6.8 µg/kg), 4-DDE (7.0 µg/kg), and endosulfan I
(2.3 µg/kg ) were the only insecticides detected in samples. These insecticides were also only
present in raw yard waste samples. Alpha chlordane, gamma chlordane, and 4-DDE are
organochlorine insecticides that are no longer labeled for use in North America.           These
compounds are extremely persistent in the environment and have been known to move globally
in the atmosphere and deposit by atmospheric deposition. Endosulfan I is one of two molecular
forms (isomers) present in technical endosulfan. It is labeled for commercial use in the
production of several vegetable, fruit, and ornamental crops. The detection of endosulfan I in
raw yard waste was unexpected since it is not labeled for use on lawns and turfgrass areas.
However, it is possible that endsulfan from neighboring agricultural areas drifted from sites of
application and were deposited on the grass clippings. It is also possible that the endosulfan
contamination was from plant materials adjacent to where the grass clippings were located, since
the piles were not neat or segregated.

None of the pesticides were detected in ground yard waste or finished compost, possibly due to a
variety of reasons, including: a) timing, b) dilution, and c) degradation. In a study such as this
one, the seasonal timing of sample collection is very important since many of the pesticides have
27
fairly defined periods during the season when they are more likely to be applied. In the case of
the raw yard waste grass samples, those samples were fresh and still green and moist at
collection. However, the ground yard waste materials had possibly aged for several months,
during which degradation may have occurred. In addition, the ground yard waste was a
composite of a higher proportion of leaves, trees, and materials other than grass clippings.
Therefore, it is highly possible that these other materials diluted pesticide concentrations (if they
were present) to below the detection limits for the analytical methods. This may especially be
true in the case of the finished compost since it was only 50% yard waste, 50% biosolids (wt:wt).
Unfortunately, funding restraints prohibited seasonal sampling in this study.


Deliverables supported entirely or in part by this grant:

a)     Extension publications

       Note: Funding restraints limited the ability to gather enough data for development of
       sound extension publications. More information is needed to provide a more complete
       picture.

b)     Extension presentations

c)     Publications – refereed and non-refereed

      Refereed article submitted to: Compost Science and Utilization

d)     Abstracts and presentations

Impacts/outcomes related to Florida FIRST (e.g., Research – enhanced knowledge in (give
description), Extension – increased knowledge of target audience, affected change relative to
(give description of issue)

This project increased our knowledge regarding the presence of selected pesticides in finished
compost and composting feed-stocks. It also re-enforced the justification for more detailed
studies assessing potential environmental risks associated with pesticide use and compost safety.

New projects resulting from this grant: A more detailed project is in the developmental stages,
but no potential sources of funds have been identified.
.
28
Table 1. Pesticides analyzed, analytical methods, and detections in raw yard waste, ground yard
waste pre-composted, and finished compost from a South Florida composting facility.
Common Name                EPA          Raw Yard Waste        Ground Yard       Finished Compost
                          Method      [Conc.] (STDEV)*          Waste
                                           Herbicides
Atrazine                  525.2        1.6 mg/kg (0.3)           BDL                 BDL
Alachlor                  525.2              BDL                 BDL                 BDL
Bromacil                  525.2              BDL                 BDL                 BDL
Cyanazine                 525.2              BDL                 BDL                 BDL
Fluridone                 525.2              BDL                 BDL                 BDL
EPTC                      525.2              BDL                 BDL                 BDL
Simetryn                  525.2              BDL                 BDL                 BDL
Ametryn                   525.2              BDL                 BDL                 BDL
Metribuzin                525.2              BDL                 BDL                 BDL
Metolachlor               525.2              BDL                 BDL                 BDL
Napropamide               525.2              BDL                 BDL                 BDL
Norflurazon               525.2              BDL                 BDL                 BDL
2,4-D                     8151B              BDL                 BDL                 BDL
2,4,5-T                   8151B              BDL                 BDL                 BDL
2,4,5-TP (Silvex)         8151B              BDL                 BDL                 BDL
Dalapon                   8151B              BDL                 BDL                 BDL
2,4-DB                    8151B              BDL                 BDL                 BDL
Dicamba                   8151B              BDL                 BDL                 BDL
Dichloroprop              8151B              BDL                 BDL                 BDL
Dinoseb                   8151B              BDL                 BDL                 BDL
MCPA                      8151B              BDL                 BDL                 BDL
MCPP                      8151B              BDL                 BDL                 BDL
Hexazinone                525.2              BDL                 BDL                 BDL
Propazine                 525.2              BDL                 BDL                 BDL
Trifluralin               525.2              BDL                 BDL                 BDL
Terbacil                  525.2              BDL                 BDL                 BDL
Tebuthiuron               525.2              BDL                 BDL                 BDL
Molinate                  525.2              BDL                 BDL                 BDL
Prometon                  525.2              BDL                 BDL                 BDL
Pronamide                 525.2              BDL                 BDL                 BDL
Propachlor                525.2              BDL                 BDL                 BDL
Cycloate (Beets)          525.2              BDL                 BDL                 BDL
Butylate (corn)           525.2              BDL                 BDL                 BDL
Diphenamid                525.2              BDL                 BDL                 BDL
Pebulate (tomatoe         525.2              BDL                 BDL                 BDL
herbicide)
Terbutryn (grain           525.2            BDL                  BDL                 BDL
herbicide)
Vernolate (agonomic)       525.2            BDL                  BDL                 BDL
Chlorpropham –             525.2            BDL                  BDL                 BDL
herbicide, aquatic
weeds, fruit, veg.

                                           Insecticides
Chlorpyrifos               525.2             BDL                 BDL                 BDL
Ethoprop                   525.2             BDL                 BDL                 BDL
29
4,4-DDE                   3550/8081       7.0 µg/kg (1.7)            BDL                   BDL
Alpha chlordane           3550/8081       5.7 µg/kg (1.2)            BDL                   BDL
Gamma chlordane           3550/8081       6.8 µg/kg (0.9)            BDL                   BDL
Endosulfan I              3550/8081       2.3 µg/kg (0.3)            BDL                   BDL
Methyl paraoxon             525.2              BDL                   BDL                   BDL
Mevinphos (citrus,          525.2              BDL                   BDL                   BDL
fruits, vegetables)
                                              Fungicides
Fenarimol                   525.2              BDL                   BDL                   BDL
Triademefon                 525.2              BDL                   BDL                   BDL

                                                  Miscellaneous
Dichlorvos                      525.2                BDL               BDL                    BDL
Atraton                         525.2                BDL               BDL                    BDL
Butachlor                       525.2                BDL               BDL                    BDL
Stirofos                        525.2                BDL               BDL                    BDL
Tricyclazole                    525.2                BDL               BDL                    BDL
NOTE. BDL= below the minimum detection level. The minimum detection level (MDL) for each method were:
525.2 (0.3 ug/kg), 3550/8081 (0.1 ug/kg), and 8151B (0.3 ug/kg).
*STDEV=standard deviation of three replicate sub-samples analyzed from the composited sample.




5.0.    Variation in and Sampling Protocol for Arsenic Uptake by Cottonwood Clones


Project Leaders: DL Rockwood and LQ Ma, School of Forest Resources and Conservation, UF,
and Soil and Water Science Department, UF, respectively.


Background
 Recent studies have identified the potential of fast growing eastern cottonwood (Populus
deltoides) clones for metal and hydrocarbon phytoremediation in Florida ( Rockwood et al. 2001,
Cardellino 2001). To extend these studies, this project had four specific objectives: a) Assess
within tree variation in leaf, branch wood, branch bark, stem bark, and stem wood arsenic (As)
concentration, b) Quantify variation in these concentrations between clones within and across
studies, c) Determine variability in leaf As concentration over time, d) Evaluate field application
of chelating agents for increasing As uptake by cottonwood clones.


Procedures
To assess variation in these As concentrations, 17 clones were selected for sampling in existing
studies in Florida at Archer and Quincy (Table 1). Various clones had previously been estimated
to be relatively superior or inferior for growth and/or As uptake in the Archer, Quincy, and other
studies. Clones 112016, ST-229, and ST-240 were common to both studies. In early October
2001, samples of 3-5 leaves and a typical branch were collected from upper-, mid-, and lower-
30
crown positions from 30 ramets representing 16 clones at Archer and\or Quincy. Stems of 17
ramets of 11 clones at Archer were also felled to collect stem bark and wood samples from the
lower- and middle-third stem positions.
Table 1. Number of ramets per clone in the Archer and Quincy studies (no./no.) contributing
leaf, branch wood, branch bark, stem bark, and stem wood samples from lower (L), middle (M),
and upper (U) crown or stem positions in October 2001 and May 2002.


                                       October 2001                                    May 2002
              Leaf           Branch Wood     Branch Bark       Stem Bark Stem Wood       Leaf
 Clone L       M    U       L    M     U    L     M    U        L    M    L    M       M     U
110412 0/1 0/1 0/1         0/1 0/1    0/1 0/1 0/1 0/1
111829              0/1                               0/1
112016 2/0 2/0 2/1         2/0 2/0    2/1 2/0 2/0 2/1          2/0   2/0   2/0   2/0   2/0   2/0
KEN8 0/1 0/2 0/4           0/2 0/2    0/4 0/2 0/2 0/4
 S4C2 0/1 0/1 0/1          0/1 0/1    0/1 0/1 0/1 0/1
 S7C1          1/0 1/0           1/0  1/0         1/0 1/0      1/0   1/0   1/0   1/0   1/0   1/0
  ST1 2/0 2/0 2/0          2/0 2/0    2/0 2/0 2/0 2/0          3/0   3/0   3/0   3/0   2/0   3/0
 ST12 0/1 0/1 0/1          0/1 0/1    0/1 0/1 0/1 0/1
ST121                                                          1/0   1/0   1/0   1/0
ST153 2/0 2/0 3/0          2/0   2/0  3/0    2/0   2/0  3/0    3/0   3/0   3/0   3/0   2/0   2/0
ST183 0/1 0/1 0/1          0/1   0/1  0/1    0/1   0/1  0/1
ST197 1/0 1/0 1/0          1/0   1/0  1/0    1/0   1/0  1/0    1/0   1/0   1/0   1/0   1/0   1/0
ST201 0/1 0/1 0/1          0/1   0/1  0/1    0/1   0/1  0/1
ST229 2/0 2/0 2/2          2/0   2/0  2/2    2/0   2/0  2/2    3/0   3/0   3/0   3/0   3/0   3/0
ST240          1/0 0/1           1/0  0/1          1/0  0/1    1/0   1/0   1/0   1/0   1/0   1/0
ST273               0/1               0/1               0/1
 ST71 1/0 1/0 2/0           1/0 1/0   2/0     1/0 1/0 2/0       2/0 2/0 2/0 2/0 1/0 2/0
   Total 10/6 12/7 13/15   10/7 12/7 13/14   10/7 12/7 13/15   17/0 17/0 17/0 17/0 13/0 15/0
To determine variability in leaf As concentration over time, leaf samples from the three crown
positions of 15 ramets of eight clones at Archer were also taken on May 31, 2002 . All harvested
material (286 component-position samples) were rinsed with deionized water, dried at 60? C for
10 days, weighed, ground to 2mm, and then stored in sample jars for analysis of As content by
PPB Environmental Laboratories.


Results
Trees in the Archer and Quincy studies differed considerably in vigor (Table 2). At a first
coppice age of 10 months in October 2001, the Archer trees averaged 5.7m in height and 4.4cm
in DBH and as 7-month-old second coppice in May 2002 were 1.5m tall. The more vigorous
ramets at Archer were up to 7m tall as first coppice and reached 2m in height seven months after
felling the first coppice. In contrast, very few of the 8-month-old first coppice trees at Quincy
were 1m tall in October 2001, due primarily to the compacted, poorly drained clay soils.
31
Table 2. Average tree height (m) and DBH (cm) and As concentrations (:g/L) of leaf, branch
wood, branch bark, stem bark, and stem wood from lower (L), middle (M), and upper (U) crown
or stem positions for all ramets sampled in the Archer and Quincy studies in October 2001 and
May 2002.
                          Leaf       Branch Wood Branch Bark Stem Bark Stem Wood
Study Date Ht DBH L        M    U    L    M    U L M      U   L    M     L   M
Archer 10/01 5.7 4.4 36.7 24.5 17.0 5.8 6.1 6.2 8.0 9.1 11.0 7.3 7.2 2.5 2.5
        5/02 1.5          22.1 12.5
Quincy 10/01 <1      26.0 26.3 19.3 3.9 4.4 4.9 8.7 8.8 6.7
Across all sample trees, within tree variation was substantial (Table 2). As concentration was
highest in the leaves, next highest in branch bark, followed by stem bark, then branch wood, and
lowest in stem wood. Position was significant for leaf concentration as, at Archer, leaves from
the lowest part of the crown had more than twice the concentration of leaves from the upper
                                                                     ?
crown. Bark from upper branches tended to concentrations some 6 g/L less than upper leaves.
Crown or stem position had little influence on As concentrations in branch wood, stem bark, or
stem wood in the Archer trees in October, but in the Quincy trees, branch wood As concentration
increased gradually from lower to upper crown positions.


For six Archer clones with complete sampling in October 2001, these within tree trends were
very similar (Figure 1). Lower leaf concentrations of nearly 40? g/L decreased to less than half
as much in the upper leaves. Branch bark concentrations increased from just more than 8? g/L in
the lower crown to almost 11? g/L in the upper crown. Branch wood, stem bark, and stem wood
concentrations changed little with crown or stem position.


Variability over time for As concentration in leaves appears relatively small but seasonal (Table
2). For the six Archer clones resampled in May 2002, As concentrations in middle- and upper-
crown                                                                          leaves were both
about                                                                          3? g/L less than
                       Arsenic Concentration (ug/L)




in October                 50                                                  2001,      perhaps
reflecting a                                                                   tendency for As
increasing                 40                                                  in older leaves,
as was also                                                                    suggested       by
previous                   30                                                            sampling
(Cardellino                                                                    2001).
                                                      20

                                                      10

                                                       0
                                                            L        M        U
                                                      LF   38.4     26.4    15.4
                                                      BW    5.3     6.6      5.5
                                                      BB    8.1      10     10.7
                                                      SW    2.8     2.9
                                                      SB    6.6     6.7

                                                           Crown or Stem Position
32



Figure 1. As concentrations (? g/L) of leaf (LF), branch wood (BW), branch bark (BB), stem
bark (SB), and stem wood (SW) from lower (L), middle (M), and upper (U) crown or stem
positions for nine ramets of six clones sampled in the Archer study in October 2001.


Variation among clones appears large for the tree components that have the highest As
concentrations (Table 3). A threefold difference was common between the clones with the
lowest and highest concentrations in leaves at Archer and Quincy at the two sample times.
Twofold differences in branch bark concentrations common at Archer were that magnitude and
larger in the Quincy clones. The ranges for branch wood, stem bark, and stem wood
concentrations were of similar extent.
Table 3. Minimum and maximum clonal means for As concentrations (? g/L) of leaf, branch
wood, branch bark, stem bark, and stem wood from lower (L), middle (M), and upper (U) crown
or stem positions in the Archer and Quincy studies in October 2001 and May 2002.


                          Leaf       Branch Wood          Branch Bark Stem Bark Stem Wood
Study Date            L    M    U    L    M    U          L M      U    L   M    L    M
Archer 10/01 Min     19.5 13.2 11.3 3.8 2.4 2.4          5.5 4.4 8.9 4.2 3.7 2.5      2.5
             Max     55.8 39.9 25.8 10.1 20.6 10.6      10.6 14.7 12.3 9.2 10.4 4.1   3.8
       5/02 Min           12.8 6.7
             Max          35.5 16.3
Quincy 10/01 Min     10.7 10.6 10.7 3.1 2.4 2.7          3.0 6.9 2.8
             Max     58.4 45.7 39.1 5.1 6.1 6.2         23.7 11.5 9.2

Based on cumulative results, several clones are notable. Of the Archer clones, ST-153, ST-197,
and ST-229 had higher As concentrations in all tree components, and 112016 was consistently
low.      At Quincy, Clones 110412, 111829, ST-201, and ST-229 tended toward high
concentrations while 112016 was uniformly below average. Thus, across these two studies and
in conjunction with previous sampling (Cardellino 2001), Clones ST-153 and ST-229 may be
classified as high As concentrators, and Clone 112016 appears to be a low accumulator of As.
Negative correlations of tree size with concentration suggest a tendency for larger trees to have
lower As concentrations in virtually all tree components (Table 4). In the October Archer
sample, tree DBH was significantly inversely correlated with lower-leaf, upper-branch wood,
middle-branch bark, and middle-stem wood, and tree height was negatively associated with
lower-branch bark. In the May sample, tree height had a significant negative association with
upper-leaf concentration.


A conclusion derived from these tendencies - that fast-growing clones have lower As
concentrations – would suggest that total As uptake by cottonwood will be a balance between
33
biomass production and As concentration. However, certain clones, such as ST-229, that
combine fast growth with high concentration would maximize uptake.


Correlations among As concentrations suggest various sampling necessities (Table 4). No single
component-position appears to adequately characterize the whole tree, but the mid-crown leaf As
concentration was virtually always positively and frequently significantly correlated with all
concentrations except stem bark. Because all correlations among leaf concentrations were
positive and many were significant, the mid-crown position could characterize the leaf
component. The mixed correlations among the branch bark and branch wood concentrations do
not permit designation of a branch component-position to represent branches. Similarly, the
wide range and non-significance of correlations among stem components and positions preclude
identification of a stem sampling protocol.


Based on the results of this project, a quick sampling protocol for clonal screening could consist
of a single crown sample and multiple stem samples. For the tree crown, a mid-crown leaf
sample of 3-5 fully developed, vigorous leaves may suffice. A lower stem wood sample may
adequately depict As concentration in all stem wood because of relatively low levels with little
vertical variability. Several stem bark samples may be more suitable due stem bark As
concentration being at least twice as high as stem wood and varying unpredictably up the stem.


Biomass accumulation patterns over time and the As concentrations determined in this project
suggest that annual harvesting of cottonwood clones will result in the highest As removal from a
site. Because leaf and branch components tend to reach a constant biomass at young ages and
are the components with highest As concentrations, one-year-old cottonwood should be
harvested before leaf fall. To maximize As removal from a site, high density planting, e.g., 1 x
1m, of high concentrating clone(s) should be practiced.


A planned evaluation of the effectiveness of field application of chelating agents to increase As
uptake by cottonwood was not done due to resource limitations. Based on the above results,
Clones ST-153 and ST-229, as high As concentrators, and Clone 112016 as a low accumulator
warrant inclusion in such a study.        Should histidine plus EDTA uniformly increase As
concentrations in the various biomass components of different clones by twofold, as the
combination of the two agents did in a greenhouse study (Cardellino 2001), As uptake by
cottonwood leaves could exceed 100? g/L.
34
Table 4. Correlations among tree DBH, tree height, and As concentrations in lower-, middle-, and upper-leaf, lower-, middle-, and upper-
branch wood, lower-, middle-, and upper-branch bark, lower- and middle-stem bark, lower- and middle-stem wood in October 2001 (OD,
OH, OLFL, OLFM, OLFU, OBWL, OBWU, OBBL, OBBM, OBBU, OSBL, OSBM, OSWL, OSWM, respectively) and tree height and As
concentrations in middle- and upper-leaf in May 2002 (MH, MLFM, and MLFU, respectively) in the Archer (above the diagonal) and
Quincy (below the diagonal) studies (bold = significant correlation at the 5% level).
            OH OLFL OLFM           OLFU OBWL OBWM            OBWU OBBL OBBM             OBBU OSBL OSBM       OSWL     OSWM      MH MLFM MLFU
OD          0.79 -0.67 -0.56       -0.31 -0.05 -0.36          -0.80 -0.59 -0.83          -0.50 -0.18 -0.48     0.08    -0.86   0.28 -0.17   0.20
OH               -0.47 -0.56       -0.47 -0.15 -0.03          -0.57 -0.66 -0.43          -0.31 0.21 -0.11     -0.45    -0.51   -0.01 -0.23  0.43
OLFL                    0.76        0.30  0.31  0.88           0.82  0.51 0.73            0.62  0.00  0.60     0.09     0.91   -0.10 0.59   0.19
OLFM              0.93              0.73 0.78   0.51           0.66  0.60 0.68           0.86   0.16  0.28     0.34     0.80   0.24 0.84 -0.30
OLFU              0.67 0.89               0.62  0.06           0.31  0.40  0.34           0.47  0.13 -0.17     0.62     0.25   0.36 0.76 -0.48
OBWL             -0.28 -0.04        0.27        0.17           0.30  0.41  0.38          0.77   0.50  0.10     0.27     0.58   0.38   0.64 -0.26
OBWM              0.79 0.94         0.97 0.18                  0.56  0.13  0.54           0.43 -0.03 0.63     -0.11     0.74   -0.09 0.46   0.32
OBWU              0.23 0.17         0.08 -0.82 0.10                 0.73 0.86            0.68   0.45  0.41     0.00     0.85   -0.27 0.45   0.08
OBBL              0.21 0.53         0.85 0.57   0.75          -0.07        0.54           0.47  0.36  0.31     0.23     0.49   -0.42 0.61   0.18
OBBM              0.47 0.18        -0.19 -0.68 -0.11           0.25 -0.54                0.79   0.58  0.52    -0.31     0.86   -0.26 0.31 -0.05
OBBU              0.56 0.64         0.58 -0.11 0.68            0.35 0.32 -0.30                  0.47  0.22    -0.02     0.79   0.20   0.54 -0.28
OSBL                                                                                                  0.18    -0.51     0.17   -0.44 0.06   0.24
OSBM                                                                                                          -0.55     0.69   -0.63 0.15   0.50
OSWL                                                                                                                   -0.19   0.57   0.58 -0.37
OSWM                                                                                                                           -0.07 0.40 -0.06
MH                                                                                                                                   -0.07 -0.77
MLFM                                                                                                                                        0.39
35
References:
Cardellino, R. W. 2001. Phytoremediation of arsenic contaminated soils by fast growing eastern cottonwood Populus deltoides
(Bartr.) clones. M. S. thesis, University of Florida. 81p.

Rockwood, D. L., L. Q. Ma, G. R. Alker, C. Tu, and R. W. Cardellino. 2001. Phytoremediation of contaminated sites using woody
biomass. Final Report to the Florida Center for Solid and Hazardous Waste Management, June 2001. 95p.
36




6.     Compost Amended Soils for Enhanced Quality and Timing of Specialty Flower
       Production

Project leaders : Everett R. Emino, Professor of Environmental Horticulture; Rick Schoellhorn,
Assistant Professor of Environmental Horticulture; and Gladys Zinati, Postdoctoral Research
Associate, Department of Environmental Horticulture.

Objectives and background

Specialty cut flowers are a small but important industry in Florida. Sunflower, Helianthus
annuus ‘Sunbright’ and Zinnia, Zinnia elegans ‘Benary Giant Mix’ are model specialty cut
flowers to test the hypothesis that adding mature compost to intensely managed specialty cut
flower cultural systems will enhance flower productivity, quality and timing.

       A. To determine the influence of compost amended intensively managed flower beds on
          the growth, quality and timing of yield of model crops.
       B. To study the soil characteristics.
       C. Report to the scientific community and the specialty cut flower industry.

Procedures to accomplish objectives

Three flower beds four feet wide by about100 feet long were established at the Department of
Environmental Horticulture greenhouse complex. Each bed was divided into four equal sections
of approximately 100 square feet each. In a randomized complete block design compost was
added at the following rates: 1. Zero cubic feet or no addition (control). 2. Five cubic feet of
compost incorporated into the top six inches of soil. 3. Ten cubic feet of compost incorporated
as previously described. 4. Twenty cubic feet of compost added an incorporated as described.
Both zinnia and sunflower seeds were sown in a commercial potting mix in plug trays which
created a small transplantable plant. These plants were placed into the experimental plots at a 15
cm spacing and grown until flower. Similarly soil samples were taken and sent to an analytical
                                      research laboratory for analysis.

                                      Although the work is still in progress some results have
                                      been obtained.
37




Figure 1                                            Figure 2
Figure 1 illustrates Ms. Viviana Baiz holding a flat of sunflower transplants ready to be put into
the experimental area. Figure 2 illustrates Everett Emino, senior PI on the project, measuring
sunflower quality.

Figure 3
Figure 4

Figure 3 shows the early establishment of the
growing area and Figure 4 shows the cut flowers
prior to the initial harvest.
38




Table 1. Illustrates the growth of sunflowers from the 4 level in the growing beds illustrated
above. As compost rate increased growth increased. These values represent the means of two
planting dates.




           0
           4
           6
           5
39
  2
  3
  0
  5
  .
  0




Figure 2. Shows the cation exchange capacity of the 4 treatments. This chart represents the soil
just after it was amended.

Recomendations: None

5. Deliverables: The following papers were presented or will be presented as indicated.
Additional deliverables are planned.

1. Baiz, Viviana and E. Emino, 2002. Growth Response of Sunflower to Municipal Solid Waste
Compost Amended Media. Presented at the SR-ASHS Meeting in February.
40


2. Zinati, G. and E. Emino. 2002. Effects of Co-compost on Sandy Soil Properties for Specialty
Cut Flower Production. Presented at the SR-ASHS Meeting in February.

3. Emino, E. R. 2002. Compost Amended Media for Bed Culture of Specialty Cut Flowers
Positively influences Time of Harvest, Growth and Quality of Sunflower (Helianthus annuus)
‘Sunbright.’ To be presented at the joint ASHS, ISHS, and CSHS meeting in Toronto, August.


Leverage

This funding leveraged Florida Agricultural Experiment Resources with the support of Viviana
Baiz as an Experiment Station Undergraduate Intern, also it leveraged FORCE, the Florida
Organics Recycling Center for Excellence, and appreciation is extended to Sumter County for
contributing the compost for amending the beds and to Dr. Danny Colvin of the Plant Science
Unit for arranging for transporting the Compost from Sumter County to Gainesville.


7.     Increasing biomass production through intensive silviculture

       Project Leader(s): Shibu Jose

Objective(s) and rationale

The higher demand for hardwood biomass in recent years has resulted in attempts to increase
productivity by several folds from unit land. The acreage under fast growing hardwood species
such as sycamore (Platanus occidentalis L.), sweetgum (Liquidambar styraciflua L.) and eastern
cottonwood (Populus deltoides Bartr.) has been increasing steadily in the U.S. for the past
several years. It has become apparent in recent years that management techniques such as
fertilization and irrigation can increase biomass yield as much as four times compared to
traditional forest management practices. Forest industries in the southeastern U.S. are keen on
growing trees under such intensive fertilization and irrigation (fertigation) regimes.           The
economic and biological sustainability of such a fertigation system depends on several factors
such as species planted, fertilizer response, above- and below-ground carbon allocation and
insect or disease attacks. The knowledge base to manage such a system for maximum growth
efficiency (amount of biomass produced per unit leaf area) with the optimum level of resources
(light, water, and nutrients) is just developing. For example, it is well known that productivity is
closely correlated to leaf area, and leaf area has been used for decades as a measure of forest
productivity. However, growth models incorporating leaf area are yet to be developed for many
of the commercially important species, especially hardwoods.

Tree leaf area regulates productivity through its influence on canopy light interception and
resulting photosynthesis. Variation in leaf area, light interception, and resulting productivity can
be explained by site specific resource (light, water, and nutrients) availability. However, the
relationship between resource availability and resource use efficiency is seldom taken into
account in making fertilizer or irrigation recommendations in intensively managed biomass
plantations. The timing and quantity of fertilization and irrigation will play a crucial role in
41


determining the leaf area index and canopy nutrient content of a stand, which, in turn, will
determine the canopy photosynthetic efficiency and the biomass production potential. There

is a need to understand the temporal patterns of fertilizer and water requirements and resource
use efficiencies of different species in order to estimate the proper rate and timing of application.

Another important aspect often ignored is the below-ground carbon allocation. Fertilizing and
irrigating a forest stand often result in greater above-ground biomass production. The below-
ground carbon allocation may also change in response to variations in resource availability.
Since as much as 50% or greater of net primary productivity in a forest stand can be allocated to
below-ground biomass pool it is important to understand the below-ground fine root dynamics
especially in intensively managed plantations.

Hence, the objectives of the proposed study will be:

     (1) to examine the above- and below-ground biomass allocation patterns in three fast
         growing hardwood tree species in response to varying levels of fertilization and irrigation
     (2) to determine the optimum resource (light, water and nutrient) need and use efficiency to
         attain the best economically sustainable productivity

Specifically, the following questions will be asked for each species:

1. What is the relationship between woody biomass production and leaf area index?
2. What is the temporal pattern of below-ground biomass allocation and its relationship with
   stem wood biomass production and resource (light, nutrient and water) availability?
3. How does resource (light, nutrient and water) availability regulate nutrient and water use
   efficiency?


Procedures to accomplish objectives

The study is being conducted at a six-year-old fertigation trial established by International Paper
Corp. in Santa Rosa county, FL. Comparisons of the study parameters is being made among the
following species of interest:

        1. Cottonwood (Populus deltoides)
        2. Sycamore (Platanus occidentalis)
        3. Cherrybark oak (Quercus pagoda)

The treatments included control, irrigation only, irrigation with 56, 112, and 224 kg N ha-1 yr-1
fertilization.

Specific methods for each of the questions to be answered are described in detail below.

Question 1. What is the relationship between stem wood production and leaf area index?
42


Historic growth data (for past six years) is utilized for assessing biomass accumulation. Stand
leaf area index and canopy light interception were measured at monthly intervals from March
through November, 2001 using a Decagon AccuPAR Ceptometer. Leaf area index from
Ceptometer will be calibrated against litter trap data. Appropriate regression equations will be fit
to define relationship between stem wood biomass production and leaf area index.

Question 2. What is the temporal pattern of below-ground carbon allocation and its relationship
with stem wood production and resource availability?

Sequential root coring is used to quantify fine root production at bimonthly intervals. Five root
cores (2 inch diameter and 1 ft deep) are collected and washed to separate fine roots in each plot.
Roots are dried at 65o C to estimate biomass. This method will provide an accurate estimate of
fine root production rates. Further, nutrient investment in fine root biomass production can also
be calculated using this data.     Structural root biomass was quantified for all species in all the
treatments through destructive sampling during summer 2001.

Question 3. How does resource availability regulate nutrient and water use efficiency?

Canopy nutrient content at bimonthly intervals will be determined using foliage biomass and
foliar nutrient concentrations from March through November in 2002. Foliage biomass at
monthly intervals will be quantified non-destructively using foliage biomass-light interception
regression equations developed. Canopy nutrient use efficiency will be calculated as the amount
of biomass (wood, foliage, and fine root) produced per unit nutrient in the canopy. Water uptake
will be measured using Dynamax sap flow gauges on selected trees and will be scaled up to the
stand level. Soil water and temperature will also be monitored periodically to complement the
water uptake data. Water use efficiency will be calculated as the amount of biomass produced
per unit water taken up. Soil nitrogen mineralization will also be quantified on a monthly basis
using the in situ buried bag technique.


Results as of June, 30, 2002

The results are preliminary at this point. We have observed increased aboveground biomass
production in all three species in response to fertigation. Among the three species, cottonwood
has the highest biomass production, followed by sycamore and cherrybark oak. Leaf area index
(LAI) showed an increasing trend with increasing resource availability. Both biomass and LAI
seem to peak between 56 and 112 kg ha -1 yr-1 nitrogen treatment. The highest level of nitrogen
fertilization is unnecessary to attain maximum production potential and growth efficiency for the
three species in our study.

Fertilization increased rates of gross nitrogen mineralization in soil. The rate of nitrogen
mineralization was positively related to the initial content of nitrogen. There was a marked
seasonal pattern in monthly rates of mineralization related to the soil temperature and moisture.
The influence of fertilization on nitrogen mineralization was more pronounced in cottonwood
and cherrybark oak stands than in sycamore and loblolly pine stands.
43


Belowground carbon allocation does not follow any specific pattern. Water uptake and canopy
nutrient content are being quantified during the summer of 2002. Data collection will be
continued for one more year.

Deliverables supported entirely or in part by this grant:

A manuscript is being written for a referred journal article. An oral presentation of the results is
planned for the Ecological Society of America Annul Conference in August 2002. Another oral
presentation will be made at the Soil Science Society of America Annual meeting in Indianapolis
in November 2002. A Ph.D. dissertation will be completed in December 2004 based on this
project.

Oral presentations planned in 2002:

                                                                                     h
Henderson, D., Jose, S. and Ramsey, C. 2002. Leaf area-productivity relationships in t ree fast
      growing hardwood plantations along a fertigation gradient.      Ecological Society of
      America Annual Meeting, August 4-9, Tuczon, AZ.

Lee, K.H., and Jose, S. 2202. Nitrogen mineralization along a fertilization gradient in hardwood
       plantations. Soil Science Society of America Annual Meeting. November 10-15,
       Indianapolis.

Leveraging or cost sharing of CNR funds - In-kind FTEs or additional support from
another source received to enhance or expand project

Further funding ($12,000) was secured from the William Paul Shelley Sr. Memorail Fund at the
University of Florida. International Paper Corporation also provided funds ($10,000) and in-
kind contribution (worth $5,000).




8.     Issues of Cofiring Biomass with Coal at Electric Utilities

Project Leaders: M. Rahmani, A.W. Hodges, C. F. Kiker, and J. A. Stricker
Food and Resource Economics Department, Polk County Cooperative Extension Service
IFAS, University of Florida


Executive Summary

Direct combustion of biomass materials has long been considered as an option for producing
renewable energy. However, economic as well as technical feasibility of converting biomass to
electricity have faced serious challenges. Capital cost of power plants that convert biomass to
electricity, cost of biomass feedstocks, procurement of necessary biomass feedstocks within an
44


economical distance from power plant, and cost of energy produced are among issues weighing
on this option.

Cofiring biomass with other feedstocks, mainly coal, is seen as a feasible solution. This way, the
existing power plant facility can be used to blend biomass (up to 5 percent) with coal or inject
biomass separately (up to 20 percent) into the boiler. The percentage of biomass that is used
depends mainly on type of boiler and type and availability of biomass. While the practice of
cofiring wood wastes with coal has been considered as the most economical way to use biomass
for energy, it has also shown certain environmental benefits. Various studies have shown that
cofiring biomass with coal reduces SO2 , NOx, and CO2 emissions. Within the past ten years
successful cofiring of wood with coal has been tested or implemented in several electric power
plants in various parts of the United States.       In Florida, there are some electric utilities that have
tried or are trying cofiring biomass with coal as a pilot project.

Cofiring has its own challenges as well. Cofiring faces certain problems such as ash deposition,
corrosion, and carbon burnout. The issues of feedstock selection, procurement, handling and
processing, and high moisture content of biomass feedstocks are challenges to electric utilities
trying to implement cofiring. At the present time, there are two techniques for cofiring biomass
with coal: blending biomass with coal on the coal pile and feeding the blend to the pulverizer and
then the boiler; or preparing the bio-fuel separately from the coal and injecting it directly into the
boiler. Selecting the right technology considering all other technical properties of a power p   lant’s
boiler is of crucial importance to a successful implementation of the cofiring process. In this
project we studied the results of cofiring biomass with coal experience in several power plants in
Florida and other states by reviewing relevant publications on this issue combined with the
results of our own survey of power plants cofiring biomass with coal. Based on information
from previous work and conversations with some of the people involved in cofiring biomass with
coal, a survey questionnaire that addressed issues of cofiring was prepared. The questionnaire
addressed capacity of power plant, type of boiler, type of coal used, type of biomass used for
cofiring, cofiring techniques used, percent biomass used as cofiring, source and distance of
biomass feedstock, the results of emission reduction due to cofiring, and data on costs of
cofiring. The questionnaire also inquired about technical problems that power plants may have
experienced while cofiring biomass with coal.

Overall, 36 power plants tried or are trying cofiring some kind of renewable feedstock with coal
in the United States. Most of these power plants used cofiring as a test or demonstration effort to
investigate the technical and environmental effects of cofiring. Only a few power plants
continued cofiring beyond the test phase and for a whole year. Some power plants use cofiring
only during the off-peak season. However, cofiring efforts stopped as soon as cost and
procurement of biomass feedstocks became a burden.

While various types of feedstock were used for cofiring with coal, there were only two occasions
where biomass crops were tried. Wood chips, waste papers, pallets, railroad ties, pet coke,
willow bush, and switchgrass were mostly the feedstocks used in cofiring with coal. Heat
indexes of these feedstocks ranged from 5000 to 8000 Btu per pound. Collected information on
cost of cofiring was not sufficient for statistical analysis. However, it could be seen that the high
cost of growing, harvesting and transporting biomass crop to power plants could be the major
45


limiting factor in using biomass crops for cofiring with coal. High moisture content of biomass
crops can be another limiting factor. Certain types of boilers allowed separate injection of up to
20 percent of other feedstocks with coal.

Results of cofiring experience have shown certain achievements with regard to emission
reduction and waste reduction. The results have also shown some problems and limitation in
cofiring biomass with coal. While a few respondents stated no effect or no significant effect of
cofiring on CO2 , NOx, and SO2 reduction, most claimed that cofiring had a favorable effect on
emission reduction. High moisture content of waste or biomass used for cofiring, high cost of
procurement and transportation of biomass feedstocks, slagging, corrosion, carbon burnout, and
lower efficiency are problems power plants face when they consider cofiring.

In conclusion, cofiring has not become a common practice even with power plants that had
successful experience. Cofiring was tested mostly when certain advantages such as availability
of suitable feedstock with no cost or negative cost were available. Once the particular advantage
faded, cofiring was discontinued. For any type of biomass crop to become part of an actual
continuous cofiring process, several combined and coordinated efforts by the public and private
sectors need to be undertaken. It is the environmental advantage of cofiring that constitute the
major force for such an effort. Certainly green energy costs more at least for the time being and
with present resources.      The cost must be shared by direct users and the public institutions
concerned and interested in environmental advantages of green energy.
46


                                             Introduction

Cofiring biomass with coal is used to produce electricity at electric utilities. Today, cofiring has
become a more practical way of converting biomass to electricity. While cofiring of wood
wastes with coal appears to be the most economical way to use biomass for energy (Hughes,
2000), there also appears to be environmental benefits of cofiring in the form reduced pollutant
gases. Various studies have shown that cofiring biomass with coal reduces SO2 , NOx, and CO2
emissions (Tillman, 2000). Within the past ten years successful cofiring of wood with coal has
been tested or implemented in several electric power plants in various parts of the United
States(Cobb et al., 2000; Tillman et al., 1999; Tillman, 2000). Except for a very few cases,
almost all of these cofiring projects have been with coal, which can be of interest to more than
1500 coal-fired stoker boiler plants in the United States (Cobb et al., 1998). In Florida, there are
some electric utilities that have tried or are trying cofiring biomass with coal as a pilot project
(Segrest et al.,1998).       The benefits of generating green power, reduced greenhouse gas
emissions, reduced fuel cost, reduced wood wastes, extending the life of existing boilers, and
providing economic activity in rural regions are among advantages of cofiring (Giaier and
Eleniewski, 1999). Additionally, biomass waste materials could provide 3-5 percent of US
electrical power needs (Hughes, 2000). The advantage of urban wood wastes as a feedstock for
cofiring is that utility plants can receive tipping fees that would otherwise be paid for land filling.
These tipping fees range from$20 to $200 in various states (Cobb et. al., 1998). Tipping fees can
offset some of the handling costs of biomass feedstocks, resulting in a lower cost fuel than coal.

While there are advantages of cofiring biomass with coal, there are also some problems
associated with this practice.      Cofiring faces problems such as ash deposition, corrosion, and
carbon burnout (Baxter and Robinson, 1999). The issues of feedstock selection, procurement,
handling and processing, and high moisture content of biomass feedstocks are challenges to
electric utilities trying to implement cofiring. At the present time, there are two techniques for
cofiring biomass with coal: blending biomass with coal on the coal pile and feeding the blend to
the pulverizer and then the boiler; or preparing the bio-fuel separately from the coal and injecting
it directly into the boiler (Tillman and Battista, 1999). Selecting the right technology considering
all other technical properties of a power plant’s boiler is of crucial importance to a successful
implementation of the cofiring process.

Objectives

The objectives of this research project were the followings:

1.      To collect information from electric utilities regarding cofiring biomass with coal.
2.      To document the problems of using biomass for cofiring in electric utilities.
3.      To analyze and evaluate ways and means by which present problems of cofiring may be
        reduced in order to develop cofiring of biomass with other feedstocks at electric utilities.
4.      To lay out a systems framework for cofiring biomass.
5.      To estimate how much biomass is being cofired currently in Florida.
47


                                                                           o
While most of the preset objectives of the project were achieved, due t the lack of available
information, the quantity of biomass that was cofired at various power plants in Florida could not
be estimated.

Methodology

Based on information from reviewing previous work and conversations with some of the people
involved in cofiring biomass with coal a survey questionnaire that addressed issues of cofiring
was prepared. The questionnaire sought information such as capacity of power plant, type of
boiler, type of coal used, type of biomass used for cofiring, cofiring techniques used, percent
biomass used as cofiring, source and distance of biomass feedstock, emission reduction due to
cofiring and costs of cofiring. The questionnaire also inquired about technical problems that
power plants may have experienced while cofiring biomass with coal. A copy of the
questionnaire attached as appendix to this report. In addition, previous studies on cofiring were
obtained and reviewed in order to get acquainted with the issues of concern. In Florida, contacts
and personal interviews were made with power plants involve in cofiring other feedstocks with
coal. Telephone or e-mail contacts were made with the power plants trying cofiring in other
states. Information from various sources(Battista and Hughes, 2000) as well as responses to our
questionnaire were combined, analyzed and evaluated.

Results and Analysis

Collected information showed that there are 36 power plants in the United States that have tried
or are experimenting with some kind of cofiring.           Information was collected on general
characteristics of these power plants as well as the results from cofiring various feedstocks with
coal. Power plants with experience in cofiring use various types of coal. The type of coal they
use does not seem to have contributed to their decision to try cofiring. Bituminous was the
dominant type of coal used by these power plants, however, sub-bituminous, North Dakota
lignite, Power River Basin, and Shoshone coals were also used by some power plants cofiring
with biomass.

Cofiring objectives. The main objectives of cofiring can be summarized as waste reduction,
emission reduction, and feasibility study. Most of these power plants used cofiring as a test or
effort to see the technical and environmental effects of cofiring. Only a few power plants ( 15
percent) continued cofiring beyond the test phase and for a whole year. Some power plants use
cofiring only during the off-peak season. Mixing wood wastes with coal started as early as 1990
in some power plants. However, cofiring efforts stopped as soon as cost and procurement of
biomass feedstocks became a burden. Cofiring was performed in various boiler types such as
wall or tangentially fired pulverized coal, cyclone, stoker, and fluidized bed.

Type of feedstocks. While various types of feedstock were used for cofiring with coal, there
were only two occasions where produced biomass crops were tried. Wood chips, waste papers,
pallets, railroad ties, willow bush, and switchgrass were the types of feedstocks used in cofiring
with coal. Heat indexes of these feedstocks ranged from 5000 to 8000 Btu per pound. Some
power plants blended other feedstocks with coal and some separately injected other feedstocks
into the boiler.       Usually those injecting biomass separately for cofiring could use higher
48


percentages than those that blended biomass with coal. Feedstocks blended for cofiring ranged
from one to 10 percent based on heat value (mostly less than 5 percent). Blending other
feedstocks with coal could not be technically feasible to mix more than 5 percent. Separately
injected feedstocks could reach as high as 20 percent based on heat value. Certain types of
boilers allowed separate injection of up to 20 percent of other feedstocks with coal. Overall,
two-thirds of the plants blended feedstocks with coal and one third used separately injected
cofiring. About 70 percent of the power plants used wood wastes as the feedstock for cofiring.

Environmental benefits. Reductions of various gases were considered as a main objective of
cofiring. Reducing the amount of CO2 , NOx, and SO2 can help toward cleaner air and is an
environmental benefit. Most of the power plants that tried cofiring reported some reduction in
CO2 , NOx, and SO2 , however, some power plants reported reduction in gas emission as non-
significant or zero. There was also a case of an increase in the amount of some gases as the
result of cofiring. The types of feedstocks used for cofiring could contribute to lowering certain
gas emissions. Those power plants that used railroad ties mostly did not show favorable results
regarding emission reduction.

Problems associated with cofiring.       While there were some benefits from cofiring bio-based
feedstocks with coal, certain problems also emerged as the result of cofiring. Lower efficiency is
one major problem that was reported by most cofiring power plants. Higher moisture content of
bio-fuel used for cofiring, plugging, and impact on the coal handling system caused lower
efficiency. Consistency of bio-fuel at a cost comparable to coal was reported as another major
problem. Handling and chipping wood feedstocks at the right size for blending was yet another
problem. Dry wood generates dust that is hard to work with. Higher percentage blending( more
than 5 percent mix) has caused various technical problems such as feeder jam, lower efficiency,
and wood build up in various parts, for most power plants cofiring bio-fuel with coal. Although
the issue of ash from cofiring bio-fuel with coal is still under study by some power plants, there
are reports that ash from cofiring is less favorable for using in cement plants due to its
composition. In addition, there are regulatory considerations.

Monetary Cost and benefit of cofiring. Collected information on cost of cofiring did not provide
the necessary data for a statistical analysis and conclusion. However, it could be seen that the
high cost of growing, harvesting and transporting biomass crops to power plants could be the
major limiting factor in using biomass crops for cofiring with coal. Without giving a concrete
number, high cost of handling and processing, particularly chipping wood wastes were reported
as a major drawback to cofiring efforts. Complaints were made by several power plants
regarding high capital cost for modifying handling equipments for blending as well as separate
injection. “Not cost effective”, “not worth the extra labor and handling”, and “procuring and
handling costs of wood wastes are higher than cost of coal” were statements made by some of
the power plants managers that tried cofiring. However, those power plants that could get bio-
fuel delivered at their sites with no cost and even with some tipping fees, found it beneficial to
do cofiring as long as this kind of feedstock could be delivered.


Conclusion
49


Results of cofiring experience have shown certain achievement with regard to emission reduction
and waste reduction. The results have also shown some problems and limitation in cofiring
biomass with coal. While few stated no effect or no significant effect of cofiring on CO2 , NOx,
and SO2 reduction, most claimed cofiring had a favorable effect on emission reduction. High
moisture content of waste or biomass used for cofiring, high cost of procurement and
transportation of biomass feedstocks, slagging, corrosion, carbon burnout, and lower efficiency
are problems power plant faced when they tried cofiring. In conclusion, cofiring has not
become a common practice even with power plants that had successful experience. Except for a
few cases, whenever advantages emerged out of certain circumstances (availability of suitable
feedstock with no cost or even some extra financial reward), cofiring was performed just as a
test. Once the particular advantage faded, cofiring was discontinued. For any type of biomass
crop to become part of an actual continuous cofiring process, several combined and coordinated
efforts by the public and private sectors need to be undertaken. It is the environmental advantage
of cofiring that constitute the major force for such an effort. Certainly green energy will cost
more at least for the time being and with present resources. The extra cost must be shared by
direct users and the public institutions concerned and interested in environmental advantages of
green energy.

Acknowledgments

The investigators would like to express their appreciation for the financial support by the Center
for Natural Resources, University of Florida. The advice and help of Dr. Evan Hughes from
Electric Power Research Institute in sharing information on cofiring and facilitating our contact
with selected power plants cofiring biomass with coal is greatly appreciated.             We are also
thankful to the managers of several power plants in responding to our survey questionnaire.

References

Battista, Joseph J., and Evan E. Hughes, 2000. “A Survey of Biomass Cofiring Experience in the
United States”, Cofiring Alternative and EPRI, December 29, 2000.

Baxter, Larry, and Allen Robinson, 1999. “Cofiring Biomass with Coal”, Proceedings of the
Fourth Biomass Conference of the Americas, Oakland, California, August 29-September 2, pp.
1277-1284.

Cobb, James T. Jr., and William E. Elder, 2000. “Cofiring Urban Waste with Coal in Stoker
Boilers”, Proceedings of the Bioenergy 2000, Moving Technology Into the Marketplace, The
Ninth Biennial Bioenergy Conference, Buffalo, New York, October 15-19.

Cobb, James T. Jr., William W. Elder, Mark C. Freeman, Robert A. James, Lew R. McGreery,
William Biedenbach, and William E. Burnett, 1998. “ Demonstration Program for Wood/Coal
Cofiring in Western Pennsylvania”, Proceedings of the Bioenergy ‘98, Expanding Bioenergy
Partnership”, Madison, Wisconsin, October 4-8, pp.251-261
50


Giaier, T. A., and M. A. Eleniewski, 1999. “Cofiring Biomass with Coal Utilizing Water-Cooled
Vibrating Grate Technology”, Proceedings of the Fourth Biomass Conference of the Americas,
Oakland, California, August 29-September 2, pp. 13431348.

Hughes, Enan, 2000. “Biomass cofiring: economics, policy and opportunities”, Biomass and
Bioenergy, Vol.19, 6, December 2000, pp.457-465.

Segrest, S. A., D. L. Rockwood, J.A. Stricker, A.E.S. Green, W. H. Smith, and D. R. Carter,
1998. “Biomass Cofiring with Coal at Lakeland, Florida, Utilities”, Proceedings of the
Bioenergy ‘98, Expanding Bioenergy Partnership”, Madison, Wisconsin, October 4-8, pp. 315-
325.

Tillman, David A., “Cofiring benefits for coal and biomass”, Biomass and Bioenergy, Vol.19, 6,
December 2000, pp. 363-364.

Tillman, David A., “Biomass cofiring: the technology, the experience, the combustion
consequences”, Biomass and Bioenergy, Vol.19, 6, December 2000, pp.365-384.

Tillman, David A., and Joseph R. Battista, 1999. “Cofiring Coal in a Boiler Using Direct
Injection of Wood Waste”, Proceedings of the Fourth Biomass Conference of the Americas,
Oakland, California, August 29-September 2, pp.1309-1313.

Tillman, David A., Sean Plasynki, and Evan Hughes, 1999. “Biomass Cofiring in Coal-Fired
Boilers: Test Programs and Results”, Proceedings of the Fourth Biomass Conference of the
Americas, Oakland, California, August 29-September 2, pp. 1287-1297.
51


Appendix

               Issues of Cofiring Biomass with Coal at Electric Utilities
Project Leaders : Mohammad Rahmani, Alan W. Hodges, Clyde F. Kiker, and James A. Stricker
       Food and Resource Economics Department, Polk County Cooperative Extension Service
                                  IFAS, University of Florida

                                            Survey Questionnaire
                         “For electric power plants cofiring various biomass with coal”

        “This survey is being conducted by the University of Florida’s Institute of Food & Agricultural
        Sciences, as part of a research project to evaluate the issues of cofiring biomass with coal at
        electric utilities. The survey is being sent to those electric utilities that have experience cofiring
        biomass with coal. It is important that you provide information for your electric utility, so that
        your cofiring experience is represented in this study. All information obtained in this survey
        about your particular business will be kept strictly confidential; only averages or totals for all
        survey respondents will be disclosed. Your identity will be kept confidential to the extent
        provided by law. You do not have to answer any questions that you do not wish to. Your
        participation is voluntary and you can withdraw at anytime without penalty. There is no
        compensation nor anticipated risks for participating in this survey, however, you will receive a
        copy of the final project report if you wish. The project is sponsored by the Center for Natural
        Resources, University of Florida. If you have any questions about this survey, you may contact
        the investigator (Mohammad Rahmani, PO Box 110240, Gainesville, Fl 326511,Ph. 352-392-
        1881 x315, or MRahmani@mail.ifas.ufl.edu). Please sign below that you have read the procedure
        described above and you agree to participate in the survey.”

        Signature.................................


        Name of electric power plant
        Location of electric power plant

Question 1: What is the capacity of your power plant? .......... MW

Question 2: When was plant built?.........

Question 3: How many years in operation? ........years

Question 4: What type of boiler you have?
?                      a. Pulverized coal boiler
?                      b. Pulverize coal boiler: wall-fired
?                      c. Other type pulverized coal
?                      d. Cyclone boiler
?                      e. Other(please specify)



Question 5: What type of coal do you use?
52


?                        a. Pennsylvania bituminous ......%
?                        b. Illinois bituminous        .......%
?                        c. Wyoming subbituminous .......%
?                        d. North Dakota lignite        .......%
?                        e. Other(please specify) ..........................................   .....%

Question 6: When did you start cofiring? m....y......, and for how long......m or.......y

Question 7: What type of biomass have you used (are using)for cofiring?
?                      a. Wood wastes
?                      b. Agricultural wastes/residues
?                      c. Biomass crops (please specify)
?                      d. Other (please specify)

Question 8: What percent biomass used as cofiring?
?                     a. Based on weight? ......%
?                     b. Based on heat?      .......%

Question 9: What cofiring techniques using(used)?
       ?                a. Blending with coal
?                       b. Injected separately

Question 10: How much biomass used for cofiring? .........dry ton

Question 11: What is (was)the source of biomass feedstock?
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I.                      a. Waste management companies
II.                     b. Sawmills
III.                    c. Other wood industry mills or manufacturers
IV.                     d. Pulp and/or paper mills
V.                      e. Biomass crop producers
VI.                     f. Others (please specify)

Question 12: What is (was) the distance of the source of biomass feedstock to your power
              plant?         9 Up to 20 miles       9 20 to 50 miles      9 over 50
              miles

Question 13: What is (was) biomass heat index? ....Btu/dry lb.

Question 13a: What was the moisture content of the biomass fuel?
I.                   a. As received ........% (by weight, wet basis)
II.                  b. As fired .......% (by weight, wet basis)

Question 14: What is (was) the cost of biomass or wood wastes used for cofiring?
              $........./ton (as received), $....../dry ton, $....../MBtu

Question 15: How much did you spend as capital cost of conversion, modification or
       extra equipment for cofiring? $.........

Question 16:What kind of emission reduction occurred as the result of cofiring?
I.                    a. CO2 : before cofiring ..........; after cofiring.........
II.                   b. NOx: before cofiring ..........; after cofiring.........
III.                  c. SO2 : before cofiring ..........; after cofiring.........
IV.                   d. CO: before cofiring ..........; after cofiring.........
V.                    e. Opacity: before cofiring ..........; after cofiring.........
VI.                   f. Particular matter (PM10 or specify): before cofiring ..........; after
              cofiring.........

Question 17: Did you experience any change in cost of energy as the result of cofiring?
I.                     NO
II.                    YES: before cofiring $......./kWh; after cofiring $......./kWh

Question 18: What kind of technical difficulties did you experience?
I.                    a. Blending biomass with coal
II.                   b. Biomass moisture content
III.                  c. Biomass procurement
IV.                   d. Transportation of biomass feedstock
V.                    e. Slagging/fouling/deposition/etc.
VI.                   f. Corrosion,
VII.                  g. Carbon burnout
VIII.                 h. Others (please specify)
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Question 19: Could you sell cofiring ash to cement plants?
I.                    YES
II.                   NO

Question 20:Did you experience any economic drawbacks to cofiring?
I.                    a. High capital cost of modification or equipment change
II.                   b. High cost of blending biomass with coal
III.                  c. Cofiring resulted in higher cost of energy
IV.                   d. Other (please specify)




9.     Improving seed production and preserving seed of tall castor bean ecotypes

Project Leader: Gordon M. Prine, Professor of Agronomy

Background

In the last several years we have found that endemic tall castor bean ecotypes have
potential to produce high biomass yields of woody stems. In January 2000, we harvested
plots from a castor stand near Lakeland, FL that was less than one year old that had an
average biomass yield of 66 Mg ha-1 (29 tons/A) of oven dry stems. The plant height was
over 7 meters (25 feet) tall. It is nearly impossible to harvest seed from such tall plants
and usually plants this high have few seed per plant. If we are going to make the tall
castor bean an energy crop, we must have an adequate supply of seed.

We planted plots for studying the seed production at Green Acres farm in April, 2000.
Our treatments reduced height of tall castor plants, but a killing frost occurred before
seed matured. We got new seed and made planting at Plant Science Research and
Education Unit (PSREU) near Citra in 2001. The plants at Citra grew normally, but seed
at planting were bad and poor stands were obtained. We had to give cutting treatments to
individual plants instead of plots as originally planned. This study in still underway and
seed will be harvested in January, 2002. We now want to collect seed of tall castor
genotypes through Peninsular Florida and, select the best ones and preserve these seed for
the future. Sites for some of the best wild ecotype stands are endangered by urban
development. We will use techniques for shortening the collected tall plants to make
seed for storage in 2002 season.

Objectives

To continue to find methods to improve and increase the seed production of tall castor
bean ecotypes. To collect, increase and preserve the best tall castor bean genotypes.

Procedure
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 Seed of tall red and green castor bean ecotypes were collected at PSREU in the fall of
2001 and winter of 2002 for planting in May, 2002. We contacted county agents and
USDA NRCS workers in peninsular Florida to collect castor seed and/or direct us to
where we could collect seed of local castor ecotypes. This survey and collection trips in
late January and February resulted in genotypes collected at 20 sites scattered over the
Florida peninsular south of I-4. We planted these 20 ecotypes at PSREU at Citra in May
2002. Seed of most genotypes came up and plots of each ecotype are doing well at the
present time. We plan to allow to intercross and collect seed from all ecotypes this fall
and winter. This will conserve the genetic material in these ecotypes..

Expected Results

 We should continue to manage the tall castor plants to harvest enough seed to properly
evaluate the crop for energy use. We will conserve seed from the best tall castor
genotypes and integrate them for future use.

Deliverables

 Castor bean could become an important energy crop in Florida, particularly in the
peninsular area and in other humid subtropics and tropics. We prepared a paper on castor
bean for bioenergy use at 5th Biomass Conference of Americas at Orlando (meeting
cancelled because of September 11 event but abstract was published) in December, 2001
and ASA-CSSA-SSSA meeting at Charlotte, NC in October, 2001. We expect to present
a poster paper on castor bean at Bioenergy 2002 conference in Boise, Idaho this
September, 2002.

Budge

 We have spent all of our budget and we have an expensive period ahead to grow and
collect seed from our planted castor ecotypes at PSREU. We need about $1800 to
complete this project in next fiscal year.




10.     Feasibility Analysis of Biomass Conversion and Nutrient Recover Concept to
        Improve Water quality and Promote Ecosystem Sustainability

Investigators: Mark Clark (project leader), Joseph Prenger, Ann Wilkie.

Background

Increased development, fertilization, and alteration of upland landscapes in many areas
have led to increased nutrient and sediment loads to downstream aquatic ecosystems.
These watersheds are open, allowing excess nutrients to be transported downstream to
accumulate in freshwater ecosystems and eventually discharge to coastal environments.
Maintenance of high intensity lawns and crops is only possible with continuous nutrient
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inputs from external sources and often results in nutrient runoff in excess of pre-
intensification conditions. Increased primary productivity in response to runoff of these
nutrients into aquatic systems can result in increased autochthonous sedimentation rates,
shifts in species composition, change in habitat type, and loss of habitat and desired use
of the system for fauna including humans.                 Nuisance exotic species add to the
degradation of habitat by exploiting these nutrients and amplifying the response of a
system to these additions. When present, nuisance species can significantly compound
ecosystem trophic degradation by speeding up impacts and reducing resiliency of the
system. These conditions appear to be most pronounced in tropical and subtropical
ecosystems where light and temperature are rarely limiting to production and the spread
of exotic species is not restricted by freeze boundaries.

In many areas, limited funds and limited technical resources curtail efforts to manage and
improve degraded aquatic ecosystems, resulting in a successional shift in species
composition and function in response to the increase in nutrients and presence of exotics.
In some instances, although the immediate downstream ecosystem may be impacted by
excessive vegetative growth, increased biomass production and burial in sediments can
act as a nutrient sink (at least temporarily) thereby reducing the availability of nutrients to
ecosystems further downstream.         Thus, downstream water quality may benefit by
increased vegetative growth, but conditions in directly adjacent water bodies may be
undesirable or incompatible with designated uses. In areas where resources are available
for active management, most efforts within the aquatic system are focused on extirpation
                                                         f
or reduction of exotic species, often through the use o herbicides. The use of herbicides
is very effective for control of the extent of nuisance species, however, as a result of plant
mortality, much of the nutrients assimilated are released back into the water column to
potentially degrade downstream systems or promote native or exotic species growth in
subsequent seasons. In those cases where harvesting of nuisance species has been
conducted, costs of harvesting, transport, and disposal of biomass are often considered
prohibitive, principally due to the fact that costs are only amortized for exotics removal
and not amortized for the reduction of nitrogen (N) and phosphorus (P) from the system,
or future need to dredge accumulated sediments.

Linkages between watershed runoff, internal cycling of nutrients, and downstream
impacts are well defined in the literature and major pathways are expressed in Figure 1a.
In this diagram, input of nutrient from the watershed increase available water column and
soil nutrients within an aquatic ecosystem. These nutrient pools are then utilized by
vegetation for growth, and upon senescence some of the assimilated nutrients are released
back into the water column or accumulate in sediments. In open water bodies, those
nutrients not stored in the sediment or within biomass may flow downstream to stimulate
growth there if nutrients are limiting.       When management resources are available,
harvesting or herbicide treatment of nuisance species can increase the rate of nutrient
cycling between the biomass and water column or soil nutrient pools.

In this project, we will evaluate the feasibility of creating a watershed-scale resource
recovery loop that will cycle nutrients stored in biomass back up into the watershed,
target nuisance species for the recovery of these nutrients, and evaluate the use of the
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carbon fraction of the biomass to defray energy costs associated with harvest and
transport of materials (Figure 1b).        This analysis will be conducted using literature
values and findings from previous studies that have evaluated individual components of
the proposed recovery loop. Using this cost-sharing and multi-purpose approach to
evaluating harvest, methane conversion, nutrient recovery, and reuse of nuisance biomass
within a watershed, it is expected that the overall effectiveness and feasibility of biomass
recovery from aquatic systems will be significantly increased.

Objectives

       1) Evaluate the nutrient recovery potential from biomass harvest of nuisance
          species based on literature values of standing crop N and P tissue
          concentrations.
       2) Evaluate the potential energy yield from biomass harvesting of nuisance
          species using literature values of standing crop, C, N, and P tissue
          concentrations, and estimate energy cost reduction if biogas is used to defray
          overall energy requirements.
       3) Evaluate digester residue as a soil amendment from the perspective of
          reducing outside fertilizer inputs to the watershed and increasing internal
          cycling of nutrients that have run off the landscape and become assimilated by
          nuisance biomass.


Procedures to accomplish objectives, Experiments and Results

Present Status

At this stage in our investigation we have developed and are populating three separate
databases; 1) for nuisance aquatic plant biomass and tissue composition characteristics, 2)
for aquatic plant biomass conversion to methane and digester residual characteristics, and
3) for soil enhancement characteristics amended with digester residuals or equivalents.
These databases will provide the fundamental dataset(s) needed to test the feasibility of
our biomass conversion concept (see figure 1 for dataset groupings related to conceptual
diagram). At present, each database is approximately 40% complete and is expected to
be sufficiently complete for purposes of this study by the end of February. Data to
populate these three databases is coming mostly from the primary literature; however,
data from grey literature conference proceedings and project reports is also being utilized.
These databases are being set up in the program Microsoft Access and will be
continuously updated during this project as new data is located.


Near-term Outlook

In March, once these datasets are complete, values and coefficients for standing crop
characteristics, conversion ratio’s, and cost benefit/removal ratio’s for conventional and
alternative methods will be determined. These values will then be integrated into a
spreadsheet model used to test various conventional and alternative scenarios and to
58


determine cost/feasibility-limiting steps. Presently, the spreadsheet model is in an early
stage of development with limited integration between production, conversion, and
residual utilization compartments. We expect the integrated linkages between these
compartments to be completed by the end of March. At that time we will begin to query
the dataset and begin to test our proposal hypothesis, that being the feasibility of biomass
conversion and nutrient recovery to improve water quality and promote ecosystem
sustainability.

Recommendations

None at this time

Deliverables

None at this time

How funds were leveraged or cost shared and amount of additional funds received

No additional funds have been directly leveraged for this project at this time. However,
literature gathered as parts of previous project acquisitions and research efforts have been
used extensively for this project.