Bacterial Cellulose - DOC

					Bacterial Cellulose



        A Thesis Presented for the
    Degree of Master of Engineering in
    Chemical and Process Engineering




                                         David Holmes
    Department of Chemical and Process Engineering
                            University of Canterbury
                         Christchurch, New Zealand
                                                2004
Bacterial Cellulose

Abstract
A strain of Acetobacter xylinum was isolated from the New Zealand environment.
The characteristics affecting cellulose production by A. xylinum were investigated and
compared to previously isolated strains. The effect of various environmental
parameters on the mechanical strength and water holding capacity of the bacterial
cellulose was also investigated.

The viable strain of A. xylinum was tested in two reactor configurations, statically in
beakers and dynamically in a rotating biological contactor (RBC). The static
experiments replicated previous studies to confirm optimum conditions and medium
composition. The main focus of the dynamic experiments was to determine the effect
of rotational velocity on cellulose production in the RBC. Operation at constant
overflow and basic level control added rigor for future results/ Strength of the
cellulose at the differing velocities was analysed .

The results of static experiments closely replicated previous findings. The production
rate was significantly less dependent on the volume of medium present than the
available surface area. The production rate was also enhanced by the addition of
1.4% (v/v) ethanol. The production rate varied from as low as 3 g/m2 day to 21 g/m2
day with values most commonly between 10 – 15 g/m2 day.

The production rate of cellulose on plates in an RBC was between 3.6 - 12.7 g/m2 day.
These were extreme cases with the production rate for most runs falling between 6
g/m2 day and 8 g/m2 day. An optimum rotational velocity for the reactor was found to
be 6 to 7 rpm. The microbial cellulose grown statically consistently had water
holding capacity of about 200 times its dry weight. The freshly harvested, fully
saturated dynamically produced cellulose had water holding capacity (WHC) of
between 150 – 450 times the dry weight. The WHC was found to reach a maximum
at between 12 -16 rpm.

The effect of the air/liquid contact ratio on cellulose production was investigated
using a partially submerged cylinder in the RBC. The diameter of the cylinder and
the reactor vessel limited the maximum ratio to 3.0. The greatest cellulose production



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Bacterial Cellulose
rate on the cylinders, 7.81 g/m2 day, was produced at a ratio of 2.33 and a rotational
velocity of 8.57 rpm.

A mutant strain of A. xylinum was isolated that produced reduced amounts of gluconic
and keto gluconic acid. The production of gluconic and keto gluconic acid reduces
the pH below the region for optimal cellulose production. Although not quite as
prodigious at making cellulose, the mutant provided a tangible product that could be
developed from a single colony. Preliminary experiments demonstrated the benefits
and greater rigor that this strain could potentially provide. In addition, the cellulose
produced also had a WHC twice that previously observed under similar conditions




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Bacterial Cellulose
Acknowledgements
I would like to thank firstly and foremost Dr. Peter Gostomski as mentor and advisor
on this project. He provided a lot of the knowledge and inspiration that kept the
project progressing.


Mechanical support was provided by Paul Tolson, Frank Weerts and Ron Boyce and
Electrical support from Bob Gordon. Each provided input and advice that was
concise, prompt and accurate.


Rewi Thompson (Chemistry Department) and Jackie Healy (PAMS) provided
assistance in determining and carrying out the appropriate assays. Craig Gallilee was
sensational at teaching me the necessary microbial skills at the outset of the project
and continued to be a reliable font of wisdom over many of the microbial concerns
that were raised during the project.


Bruce Woodley very kindly donated a sample of the Kombucha mat. This was
greatly appreciated, as without it, this thesis would be markedly shorter and very
possibly would have suffered terminally very near its beginning.


Thanks must also go to Kelvin Chapman for selflessly and enthusiastically providing
the knowledge and equipment to perform an adequate assessment of the strength of
the cellulose.




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Bacterial Cellulose
Table of Contents
Abstract ..........................................................................................................................ii
Acknowledgements ....................................................................................................... iv
Introduction .................................................................................................................... 1
Literature Review........................................................................................................... 4
   Historical Account ..................................................................................................... 4
   Cellulose Biosynthesis ............................................................................................... 5
   Genetics and Regulatory Enzymes ............................................................................ 9
   Mutations ................................................................................................................. 10
   Cellulose produced by Acetobacter xylinum ........................................................... 11
   Nata De Coco ........................................................................................................... 12
   Other Reactor Configurations for Bacterial Cellulose ............................................. 13
       Ajinomoto ............................................................................................................ 13
       Weyerhauser ........................................................................................................ 13
       ICI ........................................................................................................................ 14
       Patented Products ................................................................................................. 14
       Continuous film harvesting .................................................................................. 15
       Internal Loop Air Lift Reactor and Stirred Tank Fermenter................................ 15
       The Rotating Biological Contactor (RBC) .......................................................... 16
Methods........................................................................................................................ 18
   Organism and Culture Maintenance ........................................................................ 18
   Static experiments .................................................................................................... 19
   The Rotating Biological Contactor Reactor (RBC) ................................................. 20
   RBC Operation......................................................................................................... 20
   Harvesting the Cellulose .......................................................................................... 22
   Glucose Assay.......................................................................................................... 23
   Strength test ............................................................................................................. 23
Discussion .................................................................................................................... 24
   Isolating the Bacteria ............................................................................................... 24
   Mutant Investigations............................................................................................... 26
   Modifying the Reactor ............................................................................................. 27
   Modification Results ................................................................................................ 29
   Static Experiments Results ...................................................................................... 30


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Bacterial Cellulose
    Ethanol Concentration ......................................................................................... 30
       Substrate Concentration ....................................................................................... 31
       Defined Media ..................................................................................................... 31
       Surface Area to Volume Ratio ............................................................................. 32
   RBC Results ............................................................................................................. 32
       Comparison with Static Experiments................................................................... 34
       Swelling ............................................................................................................... 35
       Stalling ................................................................................................................. 36
       Rotational Speed .................................................................................................. 38
       Water Holding Capacity ...................................................................................... 39
       pH......................................................................................................................... 40
       Defined Media ..................................................................................................... 41
       Strength Tests....................................................................................................... 42
       Glucose Assay...................................................................................................... 43
       Mutants in the RBC ............................................................................................. 44
   From Discs to Cylinders .......................................................................................... 44
Conclusions .................................................................................................................. 48
Recommendations ........................................................................................................ 50
References .................................................................................................................... 52
Appendix A .................................................................................................................. 61
   Contamination .......................................................................................................... 61
Appendix B .................................................................................................................. 63
   Isolation.................................................................................................................... 63




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Bacterial Cellulose
Table of Figures
Figure 1: The ,1-4 glucan chain that constitutes cellulose (Kimble 2003)………….1
Figure 2: A simplified model for the biosynthetic pathway of cellulose (Serafica 1998)
                …………………………………………………………………………7
Figure 3: Assembly of microfibrils by Acetobacter xylinum (Iguchi et al., 2000) …8
Figure 4: A schematic of the RBC. The PC cover was for temperature control
           purposes. Constant overflow and pH control were not present for all runs.
           T denotes a temperature probe.      ………………………………………..20
Figure 5: The reactor before any modifications had been made………………….….22
Figure 6: A photograph through an oil immersed 100x lens of A. xylinum after
           chemical mutagenesis…………………………………………………….26
Figure 7: A. xylinum colonies on an agar plate……………………………………….27
Figure 8: The new reactor vessel with the modifications shown (LC = Level Control)
                ………………………………………………………………………..29
Figure 9: An upside down statically produced pellicle. Cellulose would have been
           growing on the bottom (as pictured), producing the solid disc that can be
           seen. The partially collapsed, clear gel can be seen sitting on top of the
           pellicle……………………………………………………………………30
Figure 10: The result of a static investigation into the affect of ethanol on cellulose
           production. The terms in brackets refer to the medium used.        ………..31
Figure 11: The results of static experiments to statically determine the optimal initial
           sucrose concentration       32
Figure 12: At 32 hours, a bio film just forming……………………………………...32
Figure 13: Cellulose pellicles in the RBC at 32 hours……………………………….33
Figure 14: Experiments showing that by increasing the surface area for a constant
           volume cellulose production is also enhanced. Against expectations, the
           Defined media outperformed the generic media in one co-current
           experiment. Also, glucose was superior to sucrose when inoculated from
           the same broth.     33
Figure 15: Cellulose pellicles in the RBC At 48 hours………………………………34
Figure 16: Cellulose pellicles in the RBC at 48 hours……………………………….35
Figure 17: Cellulose pellicles in the RBC at 60 hours………………………….……36




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Bacterial Cellulose
Figure 18 Serafica‟s (1998) evaluation of the comparative growth rates of static and
                 dynamic experiments.                      ……………………………………………..…36
Figure 11: Cellulose pellicles in the RBC at 72 hours……………………...………..37
Figure 20: The cellulose production rate as a function of the rotational velocity of the
                 reactor
                       ………………………………………………………………..Error!
                 Bookmark not defined.39
Figure 2: Cellulose pellicles in the RBC at 84 hours……………………………….39
Figure 22: A graphical representation of how Water Holding Capacity changes with
                 respect to rotational velocity using both SH(e) medium and M medium
                       ………………………………………………………………………..40
Figure 23: Cellulose pellicles in the RBC at 122 hours, just prior to harvesting…….40
Figure 24: A depiction of the typical observed path of pH taken from run 30, run 27
                 and run 33.                   ……………………………………………………......41
Figure 25: The strength of cellulose as a function of rotational velocity
                       ………..Error! Bookmark not defined.43
Figure 26: The glucose concentration mapped over the course of an entire run. The
                 initial glucose concentration was 30 g/L and the increases are reflective of
                 fresh media being added. The final increase was with medium containing
                 60 g/L.           ………………………………………………………………..43
Figure 27: End-on view of disk reactor showing the effect of lowering the reactor
                 volume on ratio of submergence to aeration and percentage of total disk
                 usage.            ………………………………………………………………..45
Figure 28: The production rate of cellulose on cylinders at different air/liquid contact
                 ratios and rotational velocities.                     ………………………………………..46


List of Tables
Table 1: The composition of the generic media derived by Schramm and Hestrin
(1954) ........................................................................................................................... 19
Table 2: The defined media used which was adapted from the Mormino media (2001)
...................................................................................................................................... 21
Table 3: The Trace Element Solution adapted from Mormino (2001) ........................ 22
Table 4: HBH reagent composition ............................................................................. 23


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Bacterial Cellulose




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Bacterial Cellulose

Introduction
Cellulose forms the basic structural foundation of the cell wall of eukaryotic plants
and algae and is also found as a major constituent of the cell wall of fungi (Cannon
and Anderson 1991). It is therefore, the most abundant bio-polymer on the earth with
180 billion tons per year produced in nature (Englehart 1995). A simple straight chain
polymer of glucose molecules linked at the ,1-4 position (Figure 1), cellulose is most
commonly harvested from trees and cotton but is also derived from flax, jute and
hemp. Bacteria also synthesise cellulose, including the genus‟s Agrobacterium,
Rhizobium, Psuedomonas, Sarcina and Acetobacter (Cannon and Anderson 1991).
Acetobacter xylinum, a Gram-negative, rod shaped bacteria and a prodigious producer
of cellulose, has been studied extensively. Initially, the extra-cellular cellulose
produced by the bacteria was seen as a method for elucidating the biosynthetic
pathway of cellulose, but bacterial cellulose has developed into a field of study of its
own.




Figure 1: The ,1-4 glucan chain that constitutes cellulose (Kimble 2003)

As opposed to cotton and paper, where the purification of the cellulose decreases the
chain length, bacterial cellulose does not require remedial processing to remove
unwanted polymers and contaminants (e.g., lignin, hemicellulose) and therefore
retains a greater degree of polymerisation (Nishi et al. 1990). This fact gives bacterial
cellulose superior uni-directional strength. In a native state, bacterial cellulose also
has greater hydration , holding over a hundred times its own weight in water. These
properties, along with the in situ ability to mould the cellulose during production,
have led to innovative uses for bacterial cellulose. Included among the uses are high
quality speaker diaphragms, membranes, a food bulking agent, medicinal bandages
and potentially as replacement blood vessels (Serafica 1998).




Holmes Thesis                                                                              1
Bacterial Cellulose
Traditionally, bacterial cellulose has been batch produced in static trays, but to be
commercially viable, other reactor configurations with improved productivity and
reduced labour requirements have been investigated. Cellulose synthesis has been
found to be mass transfer limited by either the oxygen or carbon source (Klemm
2001). Strains have been found that produce cellulose in shaken cultures or internal-
loop airlift reactors, but the product is a conglomerate of pellets about 10 mm in
diameter. This differs significantly from the pellicles produced in static cultures.
These attempts to enhance the oxygen transfer by agitation have been hindered by the
reversion of a portion of the A. xylinum to a cellulose non-producing mutation.


Another option has been the rotating biological contactor, as used by the wastewater
treatment industry (Edwards 1995). The rotating plates alternate between the liquid
and air, providing gentle agitation and a surface for growing an intact cellulose
pellicle. The rotating biological contactor solved the nutritional limitations of static
cultures allowing fed batch operation. Also, the rotating biological contactor
produced the desired form of a pellicle, very similar to that from static cultures.


In the rotating biological contactor, many variables influence the production of
bacterial cellulose. Unlike static cultures, the measurement and control of pH and
nutrient levels is possible in RBC as the medium is accessible. The synthesis
conditions and constitution of the medium heavily influence the quantity and quality
of cellulose produced. With the rotating biological contactor, investigation and
implementation of the permutations and possibilities have become more realistic.


Besides the manipulation of known variables, the rotating biological contactor also
introduces new operating variables. Varying accounts exist for optimum rotational
velocity (Serafica 1998, Kyrstynowicz et al. 2002). Additionally, the different
rotational velocities alter the properties of bacterial cellulose making this a complex
problem. Another new variable is the air/liquid contact ratio. This has been defined
as the ratio of time the plate, or cylinder, spends in the liquid medium to the time
spent in the reactor head space. With a traditional RBC this ratio is 1:1. Since A.
xylinum is a strict aerobe, this ratio was expected to have a significant impact on the
production rate.



Holmes Thesis                                                                              2
Bacterial Cellulose
The raison d‟etre of this thesis was to isolate a strain of Acetobacter xylinum from
within New Zealand and produce cellulose using a previously designed reactor
configuration (Tiong 2001, da Costa 2000). An effort was first made to characterise
this strain of A. xylinum through static experiments. Experimental runs provided a
foundation for future research and an initial comparison to other published results.
This identified the optimal rotational velocity and preliminary information about the
liquid/air contact ratio. Throughout, the reactor was continuously scrutinised and
improvements made where possible.




Holmes Thesis                                                                           3
Bacterial Cellulose

Literature Review

Historical Account
The cellulose mat associated with the production of vinegar, Kobucha tea and Nata de
(be consistent, capital C on Coco in other places ) coco has been observed and used
for centuries, even though not isolated and formerly recognised as secondary
metabolite of the bacterium, Acetobacter xylinum. In 1886 while working with
Bacterium aceti (acetic acid bacteria) Brown (1886) observed a solid mass that was
not part of his standard work. In reference to its connection with vinegar production,
the solid mass was referred to as „vinegar plant‟ or „mother‟. The material was
observed to be tough to tear, with the touch and feel of animal tissue. The compound
was later identified as cellulose and the bacteria that produced it Bacterium xylinum.

The bacterium was referred to by several names since including Acetobacterium
xylinum (Ludwig 1898) and Bacterium xylinodes (Henneberg 1906). It was later
referred to as Acetobacter xylinum (Bergey et al. 1925), and this has become the
official name according to the International Code of Nomenclature of Bacteria (1990
revision). Within scientific literature, A. xylinum is treated as a species, but for the
purpose of strict classification, it is considered a subspecies of A. aceti (Cannon and
Anderson 1991).

Initial work on bacterial cellulose demonstrated that fructose gave the best yield,
while amongst other substrates evaluated, only glucose, sorbitol and mannitol gave
adequate production (Tarr and Hibbert 1931). Glycerol, galactose, lactose, sucrose
and maltose were adequate substrates, while sorbose, mannose, cellobiose, erythritol,
ethanol and acetate completely failed to form any pellicle (Hestrin, Aschner and
Mager 1947). Other hexoses and pentoses were unsatisfactory for cellulose growth,
while it was discovered that a small addition of ethanol enhanced growth. The
fermentation process was also noted to produce gluconic acid that was inhibitive
because it lowered the pH creating unfavourable conditions.

Unlike the very complex formation in green plants (Farr 1940), A. xylinum produced a
well-defined extra-cellular mesh of cellulose (Aschner and Hestrin 1946). The
advances in microscope capabilities were utilised to discern that microbial cellulose

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Bacterial Cellulose
consisted of a discrete, thin web of fibrils. A standing culture of A. xylinum produced
a mesh of cellulose on the surface of media from a range of substrates. Simultaneous
investigations into respiratory inhibitors demonstrated that different paths were
possible from substrate to cellulose and that oxygen was vital for cellulose production.

Although the cellulose produced was extra-cellular, the mechanism was perceived as
being purely intracellular with the active enzymes being present in the outer bacterial
surface (Hestrin and Schramm 1954). It was estimated that A. xylinum cells could
polymerise up to 200,000 glucose molecules per second into -1,4-glucan chains.
The citric acid cycle and the phosphate cycle were investigated as possible metabolic
pathways. Phosphate esters are intermediates in the phosphate cycle and normally
would be expected to stimulate cellulose production under appropriate conditions.
However, phosphate esters failed to penetrate the outer wall of A. xylinum,
strengthening the argument about the location of a key cellulose synthase enzyme in
the outer wall of the bacteria (Schramm et al. 1957).


Cellulose Biosynthesis
Trying to explicate the biosynthetic pathway of cellulose in plants has motivated most
early microbial cellulose research. As an extra-cellular, over-producer of cellulose, A.
xylinum has been a useful tool in clarifying the pathway but has also contributed to
the problem. A. xylinum metabolises other products beside cellulose. For example,
through the enzyme glycosyltransferase (Ishida et al. 2002), produced the hetero
polysaccharide acetan (Couso et al. 1987, MacCormick et al. 1992, Jansson et al.
1993, Ishida et al. 2003) However, almost all proposed hypothetical pathways have
concentrated on cellulose production and ignored other simultaneous polysaccharide
production.

The secondary-wall cellulose found in plants retains the same degrees of
polymerisation independent of time of synthesis, yield of cellulose and the reaction
conditions (Marx-Figini and Pion 1974). It was postulated therefore that this form of
cellulose was governed by an internal template (similar to protein, DNA and RNA
synthesis) and not by the time of synthesis. Consequently, molecules with the same
molecular properties were always produced. In contrast, the degree of polymerisation
in bacterial cellulose increased linearly with time, which accounted for the difference


Holmes Thesis                                                                             5
Bacterial Cellulose
in molecular properties between plant and bacterial cellulose (Marx-Figini 1982). At
the same time, it was discovered that the yield of cellulose and bacterial population
obeyed the same first-order reaction law with the same rate constants. It was also
noted that other compounds synthesized by A. xylinum obey the same law with the
same rate constants. It was concluded that bacterial cellulose synthesis only occurs in
growing populations.

In an attempt to determine the relationship between polymerisation and crystallisation
of  1,4-glucans into microfibrils of cellulose, Calcofluor White ST and
carboxymethylcellulose (CMC) were added to active cultures of A. xylinum (Haigler
and Benziman 1982). The additives affected ribbon formation at different stages,
stopping bundles of microfibrils from forming highly organised fibrils. The
Calcofluor White ST, an industrial brightener, inhibited crystallisation while
accelerating the polymerisation process. The conclusion was that polymerisation and
crystallisation are coupled, consecutive processes, limited by the rate of crystallisation.
The tiny invaginations in the cell, also seen by Colvin and Leppard (1977), were
observed extruding nascent fibrils that were too small to crystallise into „true
cellulose‟. It was postulated that ordered glucan aggregates from more than one
extrusion site fasciate before final crystallisation. The inconclusive evidence cited
was the oddly grouped invaginations in the cell envelope believed to be synthesis
extrusion sites in the lipopolysaccharide layer (Colvin et al. 1977).

Using transmission electron microscopy Colvin and Leppard (1977) observed both A.
xylinum and A. acetigenus cells encased in a smooth or gently undulating envelope.
Surface irregularities, such as „blisters‟ or „strips‟, appeared peeling away from the
cell wall. It was postulated that these features occurred on healthy cells and may
possibly relate to cellulose synthesis. The authors also derived a diagram of the lipid-
phosphate-carbohydrate intermediate pathway from substrate to cellulose that
occurred in an A xylinum cell. A simplified version of the pathway is shown in Figure
2., while Ross et al. (1991) provided an interpretation of the full pathway.




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Bacterial Cellulose


                                UDP- Glucose                      CELLULOSE


                              Glucose-1-phosphate


     GLUCOSE                  Glucose-6-phosphate            Phosphogluconic acid


    FRUCTOSE                 Fructose-1,6-diphosphate




  Fructose-1-phosphate        Fructose-6-phosphate                    TCA cycle


 Figure 3: A simplified model for the biosynthetic pathway of cellulose (Serafica
                                      1998)

Ross (1991) postulated metabolic pathways for cellulose production utilising the
pentose cycle, the pyruvate cycle and citrate cycle. In the production of cellulose I,
four enzymatic steps were characterised using cell extracts of A. xylinum. These are
the phosphorylation of glucose by glucokinase, the isomerisation of glucose-6-
phosphate to glucose-1-phosphate by phosphoglucomutase, the synthesis of UDP-
glucose by UDPG-pyrophosphorylase, and the cellulose synthase reaction.

Cellulose produced by A. xylinum does not occur randomly.

Don‟t understand the above sentence.

The highly ordered synthesis begins with small glucan chains aggregating by a self
assembly mechanism into 3 to 4 nm microfibrils. This is followed by the banding of
microfibrils into bundles, which then form into the complete ribbon with a width
(parallel to the longitudinal axis of the cell) between 40 and 60 nm (Figure 3)
(Cannon and Anderson 1991, Brown 1992).

Comments have appeared on the genetic analysis of A. xylinum. These included the
interesting observation of the existence of a complex pattern of plasmids and the
utility of chemical and transposon mutagenesis to induce cellulose non producing


Holmes Thesis                                                                            7
Bacterial Cellulose
mutants and the apparent genetic instability of the organism. “Mutants” is a term that
has been used loosely, especially with reference to cellulose non-producing strains of
A. xylinum. It was suggested that these were not true genetic mutants, but rather
phenotypical expressions of the gene sequences involved in cellulose production
(Cannon and Anderson 1991).



                      Cellulose I




                                                               Cellulose II




                                    Cytoplasmic Membrane

                                      LPS envelope


                           Cellulose Synthase          Cellulose Microfibril

                           Cellulose Export            Protofibril
                           Component



 Figure 4: Assembly of microfibrils by Acetobacter xylinum (Iguchi et al, 2000)

To make the subject more confusing, it has been established that there are four
structurally different types of cellulose, with different properties that have been
identified:

     Cellulose I                   consists of -1,4 glucan chains aligned in parallel.
        Typically found in nature. It is the cellulose produced in pellicle form by A.
        xylinum. In an undried state, cellulose I may be referred to as „native‟
        cellulose (Ross et al. 1991).

     Cellulose II                  consists of anti-parallel -1,4 glucan chains, Found in
        shaken cultures of A. xylinum or after re-crystallisation or industrial
        mercerisation of cellulose I (Ross et al. 1991).

     Cellulose III                 consists of chemically treated cellulose I (Haigler and
        Weimer 1991)

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Bacterial Cellulose
    Cellulose IV               found in cell wall of higher plants and can be derived
        from chemically treating cellulose II (Haigler and Weimer 1991).

It has been only in the last two decades that key advances have been made into the
cellular activators that cause A. xylinum to secrete cellulose. The initial breakthrough
was the discovery that cellulose I actually has two allomorphs that had been confusing
crystallography work by causing different diffraction patterns (Atalla & VanderHart
1984,1986). Cellulose I (alpha) was produced predominantly by bacteria and many
algae while cellulose I (beta) was derived from plants. However, all native cellulose
contains quantities of both allomorphs (Sugiyama et al. 1991). This suggested a
model of parallel orientation of the cellulose chains with Iα containing a triclinic unit
cell and I containing a monoclinic unit cell. Furthermore, silver labelling proved
that all the reducing groups were orientated the same way (Hieta et al. 1984).


Genetics and Regulatory Enzymes
It has been only in the last decade that some of the key genes and enzymes that are
inherent to cellulose production in A. xylinum have been isolated. After great
endeavour, in vitro synthesis of cellulose was achieved at rates comparable to those
observed in vivo in A. xylinum. cultures. It was obtained on membranes in the
presence of polyethylene glycol and supplied with glucose tri-phosphate (GTP). The
breakthrough discovery was the establishment that polyethylene glycol was
precipitating a soluble enzyme, diguanylate cyclase, that converted GTP to a unique
activator of cellulose synthase, cyclic diguanylic acid (Ross et al. 1987). A prominent
polypeptide of 83 kD was shown to bind the substrate uridine diphosphate glucose
and was accepted as the catalytic sub unit (Lin et al. 1990).

Complimentation with a cellulose deficient mutant was used to isolate an operon of
four genes (Wong et al. 1990) which are called AxCeSA, AxCeSB, AxCeSC and
AxCeSD (Delmer 1999). The first three all play vital roles in the production of
extracellular cellulose by A. xylinum, while the fourth appears to play a role in the
crystallisation of the cellulose.

     AxCeSA            encodes the catalytic subunit of Cellulose Synthase (Saxena et
        al. 1990)



Holmes Thesis                                                                               9
Bacterial Cellulose
    AxCeSB         may encode the regulatory subunit that binds cyclic di GMP or
       concentrate and channel cyclic di GMP (Mayer et al. 1991)

     AxCeSC            encodes the protein that forms a pore in the outer membrane ,
        hence is unnecessary in plants (Saxena et al. 1994)

     AxCeSD            plays a role in aligning the glucan chains during crystallisation
        and when A. xylinum is agitated produces Cellulose II instead of Cellulose I
        (Saxena et al. 1994)

In addition, three other upstream genes and two enzymes have been identified that
each have important functions. Di-guanylate cyclase catalyses the formation of cyclic
di GMP while phosphodiesterase catalyses the degradation of cyclic di GMP (Ross et
al. 1991). One gene appeared to encode an enzyme with cellulase activity while the
purpose of the second was unknown. However, when either gene was disrupted, A.
xylinum replicated only cellulose negative cells (Standal 1994). Another gene was
isolated that was thought to be partially responsible for the switching mechanism
between cellulose I and cellulose II production (Nakai et al. 2002).


Mutations
The strain of Acetobacter xylinum used in Nata de coco production was found to be a
cellulose overproducing wild type. However, by means of mutagenesis other strains
have been derived that also produce prolific amounts of cellulose. Three methods have
been tried: vigorously shaking, chemical mutagenesis and UV mutagenesis. The
mutants have been used for a variety of experiments. Cellulose negative mutants of A.
xylinum are widely recognised as being present in cellulose producing cultures
propagated from a single cell (Schramm and Hestrin 1954, Cannon and Anderson,
1991). Therefore, investigation into the transition mechanism required the isolation of
cellulose negative strains.

In a vigorously shaken culture, A. xylinum reverted to a cellulose negative form. In
spite of this, cellulose production resumed when the bacteria were returned to static
fermentation conditions. By efficient screening techniques, it was discovered that by
enforcing this transition through the cellulose negative state, new strains were created



Holmes Thesis                                                                              10
Bacterial Cellulose
(Steel and Walker 1957). When returned to suitable conditions, some of these new
strains produced quantities of cellulose as the wild type strains.

After using chemical and UV mutagenesis, strains have been isolated that have unique
properties. An attempt was made using a medium containing NaBr and NaBrO3 in a
ratio of 5:1. The resultant strain produced only minimal amounts of gluconic acid and
produced twice as much cellulose as a wild type strain once returned to ideal
conditions (De Wulf et al. 1996). Also, UV mutagenesis was used to obtain strains
that were isolated and compared with A. xylinum cells that did not produce gluconic
acid (Park et al. 1999). This resulted in a reduced cellulose production rate.

Various chemicals have been used to induce mutagenesis. Antibiotics that block
RNA and protein synthesis were used to screen cellulose negative phenotypes. When
grown with wild type A. xylinum cells, the wild type showed a strong selective
advantage (Valla and Kjosbakken 1981).

(above not clear)

N-methyl-N‟-nitro-N-nitrosguanidine was used to isolate and study a cellulose
negative acetan-producing mutant in attemptattempting to understand the biosynthetic
pathway of acetan (MacCormick et al. 1992). Mutant strains have been created with
5-fluorouridine and sulfaguanidine-resistancethat also show potential for enhanced
yield. P-aminobenzoic acid promotes growth of A. xylinum through increased
intracellular levels of UTP synthesis and hence UDP-glucose levels. Sulfaguanidine is
an analog of p-aminobenzoic acid, hence, the increased production (Ishkawa et al.
1995, 1998).


Cellulose produced by Acetobacter xylinum
The cellulose pellicle produced by A. xylinum has been postulated to have many
purposes. Because the cellulose matrix is less dense than water, it was first postulated
that it provided a floating support with easy access to oxygen for the obligate aerobe
nature of the organism (Hestrin and Schramm 1954). Other possible purposes include
protection for the cells from UV light when growing on rotting fruit, denying
competitors access to the nutrients below the pellicle, enhancing colonisation of solid
or broth, and moisture retention (Williams and Cannon 1989).

Holmes Thesis                                                                         11
Bacterial Cellulose
The cellulose pellicle forms at the air/liquid interface when grown on a broth (Borzani
and de Souza 1995). As the pellicle grows, it is pushed down the medium, since new
cellulose crystallises predominantly on the top surface of the pellicle. For this reason,
while kinetics are initially limited by oxygen (Wantanabe and Yamanaka 1995), as
the pellicle thickens the kinetics become limited by substrate diffusion through the
pellicle.


Nata De Coco
Interest in A. xylinum heightened when it was discovered as being the microorganism
responsible for producing Nata de Coco (Lapuz et al. 1967). Nata de Coco is a
popular food or dessert produced in the Philippines. The method for producing Nata
de Coco involves inoculating a medium containing 5-6% sucrose supplied as cane
sugar, 3% coconut milk from grated coconut, 1.9% glacial acetic acid, 70% water and
20% recycled medium from a previous run. After 10 – 14 days at 30°C, the bottom of
the cellulose pellicles are cleaned of a non-adhering cream, then soaked in water to
remove residual acetic acid. The bacteria are killed by boiling the pellicles before
sugar, food additives and colouring are added. Finally the cellulose is boiled in a
sugar solution and packaged.

Predominantly, the market is supplied by a multitude of backyard operations that
produce microbial cellulose utilising the tailings from fruit processing plants. The
backyard operations were subject to large variations in the quantity and quality of
microbial cellulose produced. Lapuz et al. (1967) compared growth in a nutrient
medium, a basal medium and in coconut milk, an abundant domestic waste product.
The optimal cultural requirements were a pH between 5 – 5.5, temperature at 28°C
with ammonium salts for a nitrogen source and either sucrose or glucose for a carbon
source.

A demand for consistency of product and a range of textures promoted further
research. At least 150 samples were screened for Nata producing organisms. The
sources included rotting fruit and fermented coconut water-vinegar. Cellulose was
produced by 33 organisms, with 14 of them producing cellulose at an encouraging
rate. Specific strains were determined from these 14 isolates to give the desired soft,
medium and tough textures (Gallardo-de-Jesus et al. 1971).


Holmes Thesis                                                                          12
Bacterial Cellulose
Work continues on enhancing production of cellulose by static cultures on natural
media, such as coconut water from the waste stream of the copra process. It was
found that after storing the coconut water for three days, glucose was the only
carbohydrate found in measurable concentrations. In contrast, fructose and sucrose
were very unstable unless sterilised immediately. Also, the addition of carbohydrates
and nitrogen containing compounds above levels of 1% and 0.1% respectively did not
increase the cellulose yield (Budhiono et al., 1999). As with the generic medium, the
optimum pH was 5 and was shown to have no affect on the water holding capacity of
the pellicle. In addition, the results showed that there was no direct correlation
between cellulose formation and oxygen demand. Consequently, other oxygen
consuming processes must be prevalent (Verschuren et al., 2000).


Other Reactor Configurations for Bacterial Cellulose

Ajinomoto
Named after the Japanese company, this method used the surface culture technique
associated with the Nata de Coco process as a guide to try to improve the economics
of growing cellulose. Cells were first propagated in an agitated air lift reactor, before
being placed in static trays. After 3 days in the airlift reactor the cell density reached
2x107cells/ml. At this point the fermentation broth was transferred to aerated trays.
Compared to the Nata de Coco process the cellulose production rate was improved by
up to 140%. The product had a low water holding capacity, and a yield of
approximately 10% of initial sucrose content (Okiyama et al., 1992). By processing
the bacterial cellulose product with sugar alcohol, the texture became similar to that of
a grape.

Weyerhauser
Again named after the American company promoting it, a process was developed that
produced fibres about 1/300 the size of softwood pulp fibres. Using chemical
mutagenesis to change enzyme levels, strains of A. xylinum were isolated that reduced
the amount of by-products being produced. This especially included gluconic acid
which adversely lowers the pH level. More significantly, strains were isolated that
produced cellulose when agitated. Previously, agitating a broth to assist with oxygen
supply, and hence metabolism, caused cellulose production to cease. However, the


Holmes Thesis                                                                            13
Bacterial Cellulose
product from these agitated fermenters was a viscous suspension, given the trade
name Cellulon. Cellulon‟s benefits included having 200 times more surface area than
softwood pulp fibres making it useful in binding, thickening and coating at very low
concentrations (Black, 1990).

ICI
The Imperial Chemical Industry process contained four steps and produced a viscous
slurry similar to the Weyerhauser process. The first step consisted of an accumulation
step where the bacteria were allowed to grow in a stirred batch reactor until the
carbon source neared exhaustion. Supplemental carbon source was then added at a
rate that allowed the cellulose to accumulate. The final steps removed the suspension
from the reactor and then separated the cells from the product. A typical method for
removing the cells was treatment with sodium hydroxide at concentrations between
0.1-5 %. The end uses of the product included as a bulking agent in food and as a
tableting aid (Serafica 1998)

Patented Products
The defining properties of microbial cellulose, namely the product strength and
hydrophilicity, have generated several patents. Most of these revolve around a similar
method as the Ajinomoto process. The appropriate strain of A. xylinum is placed in a
vessel in which it is encouraged to grow. Optimum conditions for the production of
cellulose are then applied to the broth. The processes all use an adapted form of the
generic medium developed by Schramm and Hestrin (1954).

Gengiflex® has intended applications within the dental industry. It was developed to
aid periodontal tissue recovery (Novaes and Novaes 1997). A description was given
of a complete restoration of an osseus defect around an IMZ implant in association
with a Gengiflex® therapy. The benefits included the re-establishment of aesthetics
and function of the mouth and that a reduced number of surgical steps were required.
A recent trial on mongrel dogs, however showed that negligible improvements were
gained from using Gengiflex® (Novaes et al. 2003).

A more successful product, called Biofill®, is used as a bandage that can be applied
to cases of second and third degree burns and ulcers. Biofill® is ideal as a temporary


Holmes Thesis                                                                           14
Bacterial Cellulose
substitute for human skin. The greatest drawback of the product was the limited
elasticity when applied to areas of great mobility. In contrast, the benefits included
immediate pain relief, close adhesion to the wound bed, diminished post surgery
discomfort and reduced infection rate. The transparent cellulose made wound
inspection easy while Biofill® detached when new skin formed. By reducing
treatment time and costs, Biofill® has potentially financial viability (Fontana et al.
1990).

A team of chemists, biologists and surgeons developed a product called BASYC®
(BActerial SYnthesised Cellulose). BASYC® is a statically produced tube to be used
as s replacement blood vessel. Tubes of 1 mm inner diameter and 5mm length were
produced, which could be used as replacement blood vessels. Another benefit was that
the inner surface of the BASYC® tube was smoother than other synthetic materials
used for similar purposes. To test the product, a BASYC® tube was inserted as an
endo-prosthesis into a white rat. A histological exam showed that the micro-vessel
had become covered with well orientated endogenous cells indicating a regular
vascular wall had formed inside the cellulose wall (Klemm et al. 2001).


Continuous film harvesting
In an attempt primarily focused on volumetric productivity a continuous model for
harvesting cellulose was designed and built at laboratory scale. Using a shallow pan
to minimise medium requirements, the bacterial cellulose filament was harvested
using a continuous roller at a rate of 35 mm h-1. After 3 days growth statically, the
pellicle could be picked up and attached to the roller mechanism which included
passing the filament through a bacterial washing bath containing aqueous sodium
dodecyl sulphate. The greatest benefit was that cellulose could be harvested
continuously as long as fresh medium was added every 12 hours (Saikairi et al. 1997).

Internal Loop Air Lift Reactor and Stirred Tank Fermenter
In static cultures, the doubling time for A. xylinum is 8 to 10 hours, while in aerated
(by shaking) cultures the organism doubles every 4 to 6 hours (Cannon and Anderson
1991). By increasing the mixing and oxygen uptake of a fermentation broth, the rate
at which cells grow is also increased. Foreseeing that the potential for large scale
production was possible, agitated and aerated fermenter configurations were


Holmes Thesis                                                                             15
Bacterial Cellulose
investigated, despite the product being cellulose II. The high viscosity of the culture
broth and high agitation power required to meet the high oxygen transfer rate made
agitation difficult and the fibrous bacterial cellulose product exhibited structural
abnormalities (Kouda 1996,1997).

Better performance was gained by using a 50 L internal-circulation loop airlift reactor
to produce bacterial cellulose. The initial attempt had a production rate of 0.116 g/L
hr. This was similar to the agitated tank but allowed for greater accessibility. For
example, using oxygen enriched air increased production and produced an elliptical
pellet with a degree of polymerisation (DP) of 16,000. This compared favourably to
static cultures that have a DP of 17,000, and was superior to agitated cultures that
have a DP of 9,700 (Chao et al. 2000).


The Rotating Biological Contactor (RBC)
Fixed film reactors are used extensive;y in the waste water treatment industry where
microorganisms degrade unwanted compounds. In the case of the RBC, a high degree
of carbonaceous and nitrogenous BOD removal can be obtained. A biofilm housing
the bacteria forms on rotating plates which pass alternately through the wastewater
and the air. The plates have many roles, including mixing, sloughing off unwanted
biofilm and aeration.

Serafica (1997) investigated these fixed film technologies for bacterial cellulose
production. Firstly, a trickling film reactor was investigated, but the pellicle formed
was not uniform, being significantly thicker at the bottom. Flocs of cellulose also
inhibited the reticulations system and the film was subject to rather drastic
channelling of the media. The alternative was a rotating biological contactor (RBC).
Instead of passing the medium over the surface, this configuration involved a
stationary liquid phase with rotating plates passing through the medium. This method
had several distinct advantages. Access to the reactor and the plates was easier,
increasing the ability to access and control the medium. This included being able to
add suspended solids which could then be incorporated into the cellulose matrix while
it was growing.




Holmes Thesis                                                                          16
Bacterial Cellulose
Optimum conditions using the new, dynamic method were found to be the same as for
static cultures. A temperature of 28-30 °C, initial substrate concentrations of not
more than 5.0% w/v and initial pH of 5 all provided ideal growing conditions. With
respect to film thickness, the optimum rotational velocity was found to be 12-16 rpm
(Serafica 1997).

In the next phase, a rigorous experimental control structure was implemented on the
RBC. To enable a study of substrate uptake, a defined media was derived. It was
shown that glucose was consumed linearly and proportional to cellulose production.
An investigation was also made into the uptake of particles. Small, irregular shaped
particles of 40 – 150 µm were incorporated easily into the bacterial cellulose matrix,
as were blended newsprint fibres. However, size, shape and density were important
factors that affected the radial distribution of the particles. In addition, these
experiments demonstrated the possibility of using bacterial cellulose as a method for
recycling paper and as an ion exchange membrane. Each application has the potential
to reduce costs on current methods of production (Mormino 2001).

A separate study using the application of a RBC to bacterial cellulose synthesis gave
further insight into the characteristics of the product. As rotational velocity was
increased, it was observed that the pellicles grew at much faster rates. However, this
was predominantly through greater hydration of the cellulose and the yields were in
fact lower. Using yield as a basis for optimisation, 4 rpm provided the greatest
amount of cellulose, but this was also the lowest rotational velocity studied
(Krystynowicz et al. 2002).




Holmes Thesis                                                                          17
Bacterial Cellulose
Methods

Organism and Culture Maintenance
The organism exploited for the production of cellulose in this work was a strain of
Acetobacter xylinum. Other groups have propagated a cellulose over-producing strain
originally isolated from the Nata de Coco process in the Philippines, ITDI 2.1
(Bernardo et al. 1998), ATCC 53582 (Brown RM Jr. 1990). Ideally, this strain would
have been most suitable for the comparison work, but the Environmental Risk
Management Authority (ERMA) did not recognise that the bacteria was currently in
the environment, and hence importation of a viable culture required laboratory
containment standards not yet available in CAPE. Instead Kombucha tea, an under-
recognised health drink and widely known to contain A. xylinum, was propagated and
used to isolate A. xylinum. The Kombucha tea sample was obtained from a New
Zealand supplier, Mr Bruce Woodley.

The recommended method for propagating Kombucha tea starts with soaking five tea
bags in boiled water for fifteen minutes. Upon removing the tea bags, add 200g of
sugar, ensuring it is very well dissolved. The sweet tea should be cooled to less than
25 °C. At this time, a piece of Kombucha mat and a portion of the transport fluid (or
fluid from a previous batch of tea) can safely be added without endangering the
microbes present. The broth should be stored in clean containers with breathable lids
at 20-30°C (Frank 1995).

A sample of the new mat was placed in a universal tube containing 10 ml of generic
media,Table 1 (Schramm and Hestrin 1954), and a series of serial dilutions performed.
From each universal tube, 1 ml was placed on agar plates using a glass spreader.
After 24 hours in an incubator, the bacteria that had initially been present had formed
colonies while the fungi took up to 72 hours to manifest. A streak was made of each
bacteria colony that showed unique morphology. Individual colonies of the bacteria
were then returned to fresh media to see if a pellicle would form.

The majority of the mat forming on the top of a broth of Kombucha tea was
constituted of cellulose, which also contained living A. xylinum cells. A piece of this
mat was placed in the generic medium (Table 1).


Holmes Thesis                                                                         18
Bacterial Cellulose
   Table 1: The composition of the generic medium derived by Schramm and
                                Hestrin (1954)

                      Compound                           Quantity

                      Sucrose                               20 g
                      Na2HPO4                               2.7 g
                      Bacto Peptone                           5g
                      Yeast Extract (Sigma)                   5g
                      Citric Acid                           1.5 g
                      Deionised Water                        1L


When a fresh pellicle formed, the pellicle and a sample of the medium were taken and
transferred to fresh media. A streak was made from the new pellicle. Again each
bacterium with an unique morphology was streaked and then returned to media to see
if a pellicle would form. Identification of A. xylinum was by phenotype and the
production of the cellulose pellicle, but no genetic analysis of the organism was made.

The A. xylinum was maintained in the generic liquid medium. Some samples were
refrigerated or frozen for use at a latter date should the bacteria become contaminated.
Likewise, fresh medium was refrigerated until it was required. Three days prior to the
start-up of the reactor, the bacteria was used to inoculate a universal tube of fresh
medium. 5 ml of the broth from the young inoculum was introduced to the reactor at
start-up to ensure that the bacteria were healthy and could take advantage of the sterile
conditions provided, preventing any undesired contamination occurring. Aseptic
techniques were used where appropriate, as described by Tiong (2001) and Mormino
(2001).


Static experiments
Medium was sterilised and transferred into sterile beakers. Unless otherwise stated
the beakers were filled to the following volumes: 600 ml beakers to 250 ml, 400 ml
beakers filled to 200 ml, 250 ml beakers filled to 150 ml. Each beaker was then
inoculated with 1ml of broth from a previously prepared universal tube. To minimise
contamination, tinfoil was placed over the mouth of the beaker. The beakers were
incubated at 30°C for a period of thirteen days.


Holmes Thesis                                                                           19
Bacterial Cellulose
The Rotating Biological Contactor Reactor (RBC)
A rotating biological contactor had been designed and built (Tiong 2001) to enable
replication of earlier results (Serafica 1998). It consisted of a 0.25kW ASEA motor,
with variable speed drive, rotating six ø120mm 316 SS plates in a Perspex reactor
vessel 280mm long with a diameter of 130mm. Cylinders were used instead of the
plates for some runs. The length of each cylinder was 130 mm with diameters of 75
mm and 100 mm. The cylinders and plates were covered with a layer of felt to
enhance water retention and provide a surface on which the cellulose pellicle could
grow. The sterilisation process caused the Perspex vessel to craze. A new vessel was
constructed out of 1 mm stainless steel with a length of 300 mm and a diameter of 130
mm. The reactor vessel was placed inside a box to maintain a constant temperature.



                                               Peristaltic Pump
     pH Controller                                                    NaOH

                  PC Cover

                                pH Probe
                                                                         Beaker
  VSD     MOTOR

                                                                          Peristaltic
                                                                          Pump (2)

   T Controller                            T
                                                        Fan


Figure 5: A schematic of the RBC. The PC cover was for temperature control
purposes. Constant overflow and pH control were not present for all runs. T
denotes a temperature probe.

RBC Operation
The generic medium was autoclaved and placed into the Perspex reactor vessel, which
had been cleaned using 70% ethanol and placed under UV light for 30 minutes. From
a universal tube, 5 ml of media containing A. xylinum was used to inoculate 2 L of
media in the reactor (Figure 4). The pH was adjusted with dilute HNO3 to an initial


Holmes Thesis                                                                         20
Bacterial Cellulose
value of 5.0. The pH was allowed to drop naturally over the course of a run because
of the co-current production of gluconic acid. The temperature was maintained at
30°C. The reactor was operated in a fed batch mode, with medium being added to
account for the entrapment of water in the cellulose matrix above the liquid level and
for evaporation. The reactor was run for 6-8 days before harvesting occurred. For
later runs, the generic medium was substituted by a defined medium as found in Table
2 and Table 3.

Control of the pH was implemented during later runs. A pH probe was mounted in
the rector. The signal was sent through a pH isolater/amplifier (complements of the
Dept of Civil engineering, UoC) to a computer-mounted A/D board. The computer
running a custom Turbo Pascal program provided signal recording and controlled the
system at a pH of 4 setpoint using On/Off control addition of 0.5M NaOH. The
control action was a peristaltic pump activated through a relay system connected to
the parallel port.


    Table 2: The defined medium adapted from the Mormino medium (2001)

                     Compound                         Quantity

                     Glucose                              50 g

                     (NH)2SO4                              5g

                     Na2HPO4                              2.7 g

                     Citric Acid                          1.2 g

                     MgSO4                                 1g

                     Yeast Extract (Sigma)                0.5 g

                     C2H5OH                              14 ml

                     Trace Element Solution               2 ml

                     Deionised Water                       1L




Holmes Thesis                                                                         21
Bacterial Cellulose


      Table 3: The Trace Element Solution adapted from Mormino (2001)

                      Compound                        Quantity

                      EDTA Tetrasodium Salt            570 mg

                      FeSO4 . 7 H2O                    200 mg

                      ZnSO4 . 7 H2O                     10 mg

                      MnSO4 . 4 H2O                     34 mg

                      H3BO3                             30 mg

                      Co(NO3)2 . 6 H2O                  30 mg

                      NiCl2 . 6 H2O                    3.2 mg

                      (NH4)6Mo7O14 . 4 H2O             2.4 mg




              Figure 5: The reactor before any modifications
              were made

Harvesting the Cellulose
The bacterial cellulose was harvested in a similar method to that described by
Mormino (2001). Firstly, the pellicles were removed from the surface of the plates in
the RBC or media surface in the beakers, weighed and then boiled in 0.5M NaOH for
15 minutes to flush out A. xylinum cells. To remove the NaOH, the pellicles were



Holmes Thesis                                                                      22
Bacterial Cellulose
soaked in deionised water for 24 hours with the water changed after 12 hours. For
aesthetic reason, the pellicles were then soaked in bleach for 24 hours before
repeating the washing step. The wet cellulose was dried at room temperature on filter
paper, supported by a stainless steel mesh over a drain. Finally, the dry cellulose was
weighed.


Glucose Assay
A 4 ml sample of medium was taken from the reactor and diluted to 100 ml. From the
diluted medium, 20 µl was combined with 3 ml of HBH reagent (see Table 4) and
boiled for 3 minutes after which it was quenched in ice straight away. The sample
was the read on a spectrophotometer at 430 nm absorbance and compared to a
calibration curve generated from standardised glucose samples.

                         Table 4: HBH reagent composition


                     Compound                         Quantity

                     Tri Sodium Citrate                     5g

                     Calcium Chloride                      1.3g

                     Sodium Hydroxide                      12g

                     4 Hydroxy benzoylhydrazin             7.6g

                     Distilled Water                        1L

Strength test
Two metal clamps were placed at either end of a 100 mm by 10 mm strip of dried
cellulose. The clamps were then mounted on an Instron 5566 that measured both
elongation and maximum tensile load before fracture. The cross sectional area was
estimated by weighing the samples prior to testing and dividing by the density and
length of the sample. A constant density of 1760 kg/m3 was approximated for
amorphous cellulose and used for all samples.




Holmes Thesis                                                                        23
Bacterial Cellulose

Discussion

Isolating the Bacteria
Acetobacter xylinum is not recognized by the Environmental Risk Management
Authority (ERMA) as naturally being in the New Zealand environment prior to the
passing of the Hazardous Substance and New Organism Act of 1996. Therefore, any
experimental work with imported strains of A. xylinum must be performed in a
laboratory certified by the Ministry for Agriculture and Fisheries. At the inception of
the project, the Chemical and Process Engineering (CAPE) did not have the appropriate
certification. However, vinegar has been commercially produced in New Zealand, and
Kombucha tea has been propagated. Since both rely on fermentation processes
involving A. xylinum, a submission was made to ERMA requesting that A. xylinum be
reclassified as an organism already present in the New Zealand environment. The
cautious doctrine on the importation of new microorganisms and the potential hazard
they may cause to New Zealand‟s bio security was justification for ERMA to decline
the submission. The alternative was to isolate A. xylinum from the environment, which
while not likely to be an overproducing strain, could at least provide basis for
comparison for various reactor configurations.

Kombucha tea is reputed to have many health benefits and many who have drunk it
regularly testify accordingly. Globally, it has a cult-like following, while in New
Zealand, however, it has yet to gain widespread notoriety. A. xylinum has been
recognised as a vital part of the microbial consortium that constitutes Kombucha tea
(Tietze 1995). A cellulose mat is produced by this bacterium, which provides an
accommodating surface for various yeasts that are also present. The mat is found near
the surface of a Kombucha broth, probably resulting from the enhanced supply of
oxygen for the aerobic nature of A. xylinum and the yeasts that inhabit the cellulose
matrix.

Having obtained a Kombucha tea mat from a New Zealand supplier, an attempt was
made to isolate a pure strain of A. xylinum. The tea was propagated in the
recommended method (Tiong 2001). A mat formed on top of the tea that was much
weaker in appearance and structure than the parent and was readily contaminated by


Holmes Thesis                                                                             24
Bacterial Cellulose
Aspergillus sp., easily noted by the green dust like spores that coated the upper surface
of the mat. An inspection of the mat indicated cellulose was present and composed the
majority of the mat.

Plates streaked with scrapings from the Kombucha „baby‟ showed no signs of A.
xylinum. A Gram stain performed on the plated bacteria produced Gram negative rods,
similar to those expected from A. xylinum. This strongly suggested that the bacterium
present was probably either a non- cellulose producing strain of A. xylinum or A. aceti.
Kombucha tea also contained A. aceti, the bacteria more commonly recognised for the
role it plays in the production of vinegar. Serial dilutions were repeated several times
until it was clearly ascertained that A. xylinum was unlikely to produce an easily
identifiable individual colony that could be used to obtain a pure strain.

Instead of using the serial dilution method (Tiong, 2001), a cutting of the parent
Kombucha mat was used to inoculate sterile generic media in a small tube. Within 24
hours, a distinct pellicle had manifested itself at the air/liquid interface. Samples of the
mat and the medium below it were streaked separately. Three morphologically distinct
colonies appeared randomly on the plates. A non-producer of cellulose formed clusters
of spherical colonies that were each very distinct. One potential producer of cellulose
reproduced so quickly that only for a short period was it possible to view individual
colonies. Quickly a clear gelatinous fluid was secreted, merging neighbouring colonies.
This would eventually become one thick, opaque leathery skin over the entire surface of
the plate. Unfortunately, if either a colony, or a sample of gelatinous fluid or the
leathery skin were reintroduced to fresh media, no pellicle formed. However, the last
observed morphology which had small, structurally strong, white peaks did produce a
pellicle, but compared to the parent culture the pellicle was very weak.

One thing that assisted in attaining the isolates was a modification to the generic media.
Ethanol at a concentration of 70% (v/v) was used to sterilise surfaces. Conversely, at
lower concentrations the effects vary greatly between organisms. A. xylinum was one
organism for which small quantities of ethanol promote growth while inhibiting growth
of other organisms that may be present and competing for the same carbohydrate
source. The addition of 1.4% (v/v) ethanol was the optimum concentration (Mormino
(2001), Son et al. (2001)).



Holmes Thesis                                                                              25
Bacterial Cellulose
Using the isolated bacteria separately proved to be tedious, and the results of both static
cultures and dynamic runs were not encouraging. In contrast using the consortium of
the three bacteria provided strong pellicles, that grew rapidly. The culture containing
the three bacteria was robust enough that inoculation always produced a pellicle. If by
the time a pellicle had formed, no contamination was present, then pellicle could grow
with little likelihood of contamination until the fermentation reached the
death/stationary phase. The purity of the three bacteria cocktail was determined by the
fact that no other colonies were observed when pellicle and medium samples were
plated. In addition, the liquid media below any pellicle remained translucent, which did
not happen upon contamination.


Mutant Investigations
Chemical mutagenesis of A. xylinum by sodium bromide and sodium bromate, has been
shown to reduce the gluconic acid production and increase the production of cellulose
(De Wulf et al. 1996). An attempt was made to replicate the experiment. The
justification stemmed from being unable to isolate a pure strain instead of the three
morphologically different colonies that appeared when streaked from a Kombucha tea
derived sample. These three observed colonies did not form the desired strong, easily
discernible pellicles when individually reintroduced to generic media. It was hoped that
mutagenesis would provide a better strain of A. xylinum for future work.

Nutrient agar plates were prepared using the generic media with the addition of NaBr
and NaBrO3 in a ratio of 5:1 and concentrations of 100 mM and 10 mM total Br. At
100 mM, growth was severely inhibited with colonies forming after about 7 days on
only half the plates. However, on the 10 mmol plates, it took 3 days to form small
translucent spherical colonies, which
later became opaque cellulose pyramids.
When these colonies were then grown on
generic media nutrient plates only one
morphologically distinctive colony was
observed (Figure 7). This was very
promising as it indicated that cellulose-
negative mutants were not present in vast      Figure 6: A photograph through an
                                               oil immersed 100x lens of A.
                                               xylinum after chemical mutagenesis

Holmes Thesis                                                                             26
Bacterial Cellulose
enough quantities to form individual
colonies. A gram stain (Figure 6)
showed Gram negative rods, as was
expected. This gave a strong
indication that the bacteria present
were A. xylinum. Therefore, the
translucent spheres shown in Figure 2
are a good example of what a young
culture of how A. xylinum should
appear when grown on nutrient agar           Figure 7: A. xylinum colonies on an
plates.                                      agar plate.

The greatest benefit of the strain developed through chemical mutagenesis was that it
produced a pellicle when transferred to a liquid medium. Unlike the previous
consortium, where no pellicle formed on reintroduction to a broth, a small single mutant
colony yielded success. The pellicle was less fickle than those developed in tubes
directly from the Kombucha tea. It grew quickly, producing a strong easily discernible
pellicle that quickly adapted to a new environment when used to inoculate a freshly
sterilised broth.


Modifying the Reactor


The initial dilemma was rectifying the inadequate method by which the felt material
was attached to the stainless steel plates. An adhesive, Ados F2, was tried (Tiong,
2001), but was discarded under suspicions that it was poisoning the bacteria. Instead,
two large oversized paper clips made of copper wire were used to adhere the material to
the plates. This proved inadequate as the material collapsed toward the media at all
points except the two small regions held fast by the clips. The solution reached was to
drill four equispaced holes in the plates near the outer radius and sew the fabric for each
back-to-back through the holes. The shearing force created by the rotating disc and the
surface tension of the water were enough hold the fabric fast against the plates. Some
plates had the material sewn around the entire circumference when lower rotational
velocities generated reduced shear forces.



Holmes Thesis                                                                            27
Bacterial Cellulose
The modified plates provided an exemplary surface for the growth of cellulose pellicles.
The pellicles grew so well that the size of them began to affect the mixing
characteristics of the liquid within the reactor vessel. With no pellicles attached to the
plates, it took 15 s for a 2 ml dose of ink to evenly disperse throughout the reactor. A 2
ml dose of ink immediately prior to harvesting the pellicles created a lag of 150 s before
each compartment, separated by individual plates, became coloured. The total mixing
time exceeded 10 minutes.

To improve mixing, a peristaltic pump was added to distribute medium from one end of
the reactor vessel to the other. The pump was operated at 50 rpm with a #16 Masterflex
head giving a flow rate of 50 ml/minute. This was equivalent to a complete recycle
time of 37 min, not taking into account the volume of the pellicles. This reduced the
period required to disperse 2 ml of ink throughout the reactor vessel to 90 s. The pump
was subsequently operated at 80 ml/min or a recycle time of 23 min. This did not affect
the mixing but was beneficial in keeping the pump tubes free from blockages caused by
cellulose that had been dislodged from the plates by the shearing. As a precautionary
measure to minimise contamination, the pump was only used 24 hours after inoculation.

The recirculation system was further adapted to give better medium level control in the
reactor. The maximum medium level when the system was operated with plates was
just below the height of the shaft or the middle of the plates. The level was subject to
variation through a small amount of evaporation and more significantly liquid
entrapment in the cellulose matrix above the water level. The addition of fresh medium
corrected this while simultaneously favourably increasing the pH. Nevertheless, upon
harvesting, easily distinguishable lines were observed on the pellicle indicating the
different operating levels. Furthermore, the pellicle showed distinct signs of being
mechanically weaker but having greater water holding capacity in the regions that had
spent less time regularly submerged. With the addition of a #13 head to the peristaltic
pump, and a beaker containing media, the reactor was converted to constant overflow.
Medium was fed from the beaker to the reactor using the #13 head. The level in the
reactor was maintained at a constant level by the #16 head extracting any excess fluid
away and depositing it back in the beaker. For later runs, the pump was operated in
reverse for short periods. This was to clear the lines of blockages and return small
chunks of cellulose from the beaker to the reactor.


Holmes Thesis                                                                              28
Bacterial Cellulose



                   Motor


                                                                 LC Pump




                                                                Four holes




              LC Beaker



                                                                       pH probe

       Figure 8: The new reactor vessel with the modifications shown
                           (LC = Level Control)

Modification Results
The addition of the recirculation pump prior to rigorous level control had the peculiar
effect of enhancing the growth rate of the pellicles at the end from which the media was
being removed, even when the tube was fully submerged. It was especially noticeable
in the early stages of a run. One possible explanation for this was a consequence of the
affinity that the highly aerobic A. xylinum has for the air water interface and the fluid
mechanics of the reactor (Schramm and Hestrin 1954, Cook and Colvin 1980). Hence,
the cellulose producing bacteria floating at the surface were drawn towards one end of
the reactor but were not pulled under the surface by the submerged pump inlet. These
floating bacteria may have then been incorporated into matrix of cellulose fibrils on the
last plate.

The constant overflow alteration had a surprising side effect. The pump inlets were
prone to becoming blocked at intervals, the reactor feed inlet by submerged chunks of
cellulose in the beaker and the reactor outlet by cellulose floating on the media surface.
By increasing the speed or reversing the direction of the pump, it was possible to
extricate the lodged cellulose. However, the mixture of air and media removed at the

Holmes Thesis                                                                               29
Bacterial Cellulose
reactor outlet for level control provided favourable conditions for the A. xylinum to
continue producing cellulose. Hence, the cellulose being extracted from the reactor was
augmented by further growth. The result was lengths of cellulose being extruded from
the reactor outlet into the beaker. The extruded cellulose product was in the form of
filaments from 30 mm to 1 m and was of varying strength. The filaments appeared to
be similar to those produced by the direct harvest of filaments during continuous
cultivation method developed by Sakairi et al.,(1997). Attempts to reproduce these
cellulose filaments failed as the low pumping rates could not be adequately replicated.


Static Experiments Results
The static cultures of cellulose formed a film on the surface after 24 hours and after a
further 24 hours, a distinct cellulose pellicle was present. The pellicle was initially
transparent, but as it grew the denser cellulose matrix became an opaque white. When
the pellicle became thicker, layers were easily detectable down the side and a
discernible gel layer manifested on the underside of the pellicle (Figure 9). When
harvested and dried the cellulose pellicle was leathery to touch, with a smooth glossy
surface. On further drying the cellulose developed a plastic texture and a cracked and
wrinkled appearance as the long chains of cellulose appeared to contract.


Ethanol Concentration
The addition of small quantities of ethanol not only enhanced the ease with which
aseptic conditions could be maintained within a culture of A. xylinum, it also enhanced
the productivity of the
bacteria. An optimum
value of 1.45% 0.1% (v/v)
ethanol in each batch of
media was attained (Figure
10). This was within
experimental uncertainty of
other research (Son et al.           Figure 9: An upside down statically
2001). Both defined and              produced pellicle. Cellulose would have
                                     been growing on the bottom (as pictured),
undefined media gave                 producing the solid disc that can be seen.
experimental values within           The partially collapsed, clear gel can be
                                     seen sitting on top of the pellicle.


Holmes Thesis                                                                              30
Bacterial Cellulose
this region of uncertainty.


                              Exp 1 (SH)      Exp 2 (SH)         Exp 3 (M)
          20

          16
 (g/m2 day)
 Prod Rate




          12

              8

              4
                  6             10             14               18                 22
                                  Ethanol conc. (ml/L)
     Figure 10: The result of a static investigation into the affect of ethanol on
      cellulose production. The terms in brackets refer to the medium used.

Substrate Concentration
The relatively high cost of producing microbial cellulose compared to the more
traditional methods makes the substrate concentration of vital significance. High
substrate concentrations decrease the yield while not amounting to increased production
and increasing the cost of the feed stock (Krystynowicz et al. 2002). Generally, for A.
xylinum initial sugar concentrations above 5% w/v have not improved either production
rate or yield (Mormino, 2001, Son et al., 2001). This characteristic was demonstrated
by the New Zealand culture (Figure 11). Above 50 g/L, or 5 % (w/v), the production
rate does not significantly improve, hence correspondingly the yield drastically
decreases. Since the beakers were relatively small (250 ml of medium in 400 ml
beakers), this information probably does not translate well to the experiments
performed on the RBC later.


Defined Media
A modified Mormino (2001) defined medium was used to grow cellulose with good
results. The modification was the addition of 14 ml/L ethanol instead of 10 ml/L of
cycloheximide solution. A range of experiments were performed statically, and in all

Holmes Thesis                                                                           31
Bacterial Cellulose
cases the trends emulated those of the generic media, but with an increased production
rate. As can be seen in Figure 14, the defined medium performed better.


                                             Exp 1    Exp 2
                             30

                             25
     Prod. rate (g/m2 day)




                             20

                             15

                             10

                              5

                              0
                                  0   20     40          60           80          100
                                           Sucrose Conc. (g/L)


  Figure 11: The results of static experiments to statically determine the optimal
                            initial sucrose concentration

Surface Area to Volume Ratio
At constant volume, the surface area has been proven to be directly proportional to the
surface area of the medium (Borzani and Desouza 1995). These results were replicated
(Figure 14) by placing 300 ml of media into 400 ml, 600 ml and 1 L glass beakers. All
four experimental runs clearly demonstrated that by doubling the surface area available,
also doubled the cellulose production rate.


RBC Results
Following the success of the static results,
which demonstrated that a viable organism had
been found for cellulose production, it was
decided to investigate the feasibility of a
previously designed and built RBC (Tiong
2001). There were several factors that affected
the production of cellulose including pH,                Figure 12: At 32 hours, a bio
                                                         film just forming

Holmes Thesis                                                                            32
Bacterial Cellulose
rotational velocity, temperature and oxygen
availability. It must be noted that many of the
parameters are interrelated in unknown ways
that change as the pellicle thickens. Some
work has been done previously to optimise the
production of cellulose by A. xylinum using a

rotating biological contactor (Serafica 1998,
                                                        Figure 13: Cellulose pellicles
Mormino 2001, Krystynowicz 2002).                       in the RBC at 32 hours
However, the New Zealand strain of A.
xylinum has not been used previously, so experimental work was performed, emulating
previous work, but also providing a strong basis from which to make further studies.




                                 Glucose    Sucrose     Defined     Generic
                       18%
                       16%
                       14%
   Yeild (gcel/gsub)




                       12%
                       10%
                       8%
                       6%
                       4%
                       2%
                       0%
                             5     10              15               20              25
                                        Surface Area/Vol. (m2/m3)

   Figure 14: Experiments showing that by increasing the surface area for a
constant volume cellulose production is also enhanced. Against expectations, the
 Defined media outperformed the generic media in one co-current experiment.
  Also, glucose was superior to sucrose when inoculated from the same broth.




Holmes Thesis                                                                            33
Bacterial Cellulose
When A. xylinum was used to inoculate the
medium in the rotating biological contactor
(RBC) it took 36-48 hours before a discernible
biofilm formed on the plates. A further 24
hours was required before the film became a
solid pellicle coating the entire surface area of
all the plates. Initially the period from
inoculation to harvesting was as close as was          Figure 15: Cellulose pellicles
                                                       in the RBC At 48 hours
practical to 6 days. Once a further glucose
assays have been performed, a more reliable method of deciding on the start time
should be defined.

(above sentence is clumsy, and I am not sure how to fix it)

This would probably be when the substrate concentration dropped below a percentage
of its initial concentration. As biological processes are inherently variable, this would
eliminate the uncertainty surrounding how long the bacteria used to inoculate the media
take to acclimate to the dynamic conditions.


Comparison with Static Experiments
The RBC had advantages and disadvantages which were expected to influence
cellulose production when compared to statically grown cultures. These included:
Advantages
     Diffusion through the pellicle not limiting substrate and oxygen concentrations
        at the synthesis site
     Increased surface area to medium ratio
     Better for cell growth through the gentle mixing
Disadvantages
     Distance between plates limits pellicle size
     Agitation of medium causing cellulose negative mutations

The desired features of cellulose grown in the RBC was that the product was similar to
that formed on stationary cultures and could also be produced at a higher volumetric
production rate. The greatest difference was that the WHC was higher in the RBC at
rotational velocities above 7 rpm. To ensure distinct pellicles were harvested from the

Holmes Thesis                                                                           34
Bacterial Cellulose
RBC, the cellulose was harvested after 6-7
days. As can be seen from Figure 18 this is
just before a period of sustained, accelerated
growth. The static cultures harvested after 11
– 13 days benefited during this extra period
perhaps accentuated the superior yield. If this
period is as substantial as it appears
graphically, any new reactor configurations           Figure 16: Cellulose pellicles
                                                      in the RBC at 48 hours
should cater for a growth period of at least 10
days.

The production rate was severely reduced in the dynamic system, but was
compensated by the far greater surface area available for the volume of medium.
Operating in fed batch mode meant that the substrate was being continually added to
replace fluid lost through evaporation and entrapment in the pellicles. It may be
pertinent for future investigations to either add nutrient free replacement fluid or to
reduce the substrate concentration. Yields, based on mass of cellulose produced
against mass of substrate supplied, were between 5 to 14 % for most static
experiments. The best yield for the RBC was 8%. These were similar to previous
results. For static experiments and highly agitated aerated systems 12 to 14% was
commonly achieved (Krystyowicz et al. 2002, Yamanaka et al. 1989, Son et al. 2001).
In an RBC the reported yields have varied from 6.6% (Krystynowicz et al. 2002) to
9% (Serafica 1998).

Swelling
One of the characteristics of the dynamically produced cellulose was that when the
plate was fully laden with cellulose, the thickness of the pellicle varied markedly
between the liquid phase and the gas phase. As the pellicle passed through the surface
of the medium, gravity rapidly drained the cellulose matrix of any liquid it could not
support. Simultaneously, on the opposite side of the plate, as it entered the liquid phase,
the cellulose matrix swelled by about 30% in size, encompassing a greater quantity of
medium.




Holmes Thesis                                                                             35
Bacterial Cellulose
Stalling
Stalling was defined as a significant drop in the
rate of change of pH. This phenomenon
predominantly occurred in runs that had
accelerated cellulose growth. Instead of the
typical production rate (Figure 20), in these
few runs the production rates were about 12
g/m2 day. Since A. xylinum produced gluconic
acid simultaneous with cellulose production,          Figure 17: Cellulose pellicles
                                                      in the RBC at 60 hours
this would often indicate contamination.
However, after a period of 24 to 36 hrs, cellulose production resumed at an increased
rate based on the observed pellicle growth. Serafica (1998) did not comment on this
phenomenon, presumably because of shorter experimental duration. Nevertheless,
Figure 18 clearly demonstrates stalling in regards to cellulose production between 5 and
6 days. It appears that even during an experiment, the activity of A. xylinum is prone to
large phenotypical changes. It must also be noted that stalling did not occur after pH
control was implemented.




  Figure 18 Serafica’s (1998) evaluation of the comparative growth rates of static
                            and dynamic experiments.

The best hypothesis explaining the stalling phenomenon after approximately 6 days was
that a proportion of the bacteria changed metabolic pathways. When the initial



Holmes Thesis                                                                            36
Bacterial Cellulose
substrate supply has diminished, and the gluconic acid and 5-keto gluconic acid have
accumulated the bacteria make a phenotypical response and begin consuming the acids.

A related phenomenon was a change in the type of cellulose produced. In the liquid
phase a submerged cellulose matrix formed that is very similar to the translucent gel
that formed on the underside of static pellicles. The overall affect was to significantly
lower the WHC of the pellicle and considerably increase in the cellulose production rate
and therefore the strength of the pellicle.

Previous experiments support this scenario. The gluconic acid producing operon was
removed from the genetic structure of A. xylinum. Gluconic acid reduces the pH of the
fermentation broth to sub-optimal conditions for both cell viability and cellulose
synthesis, therefore by eliminating the production of gluconic acid it was hoped to
increase yield. However, it severely decreased the yield suggesting that gluconic acid
may have an important but as yet undefined role in cellulose production (Park et al.,
1999). In addition, it was observed by Klemm (2001) that the gluconic acid
concentration reached a maximum between Day 5 and Day 6, while the 5-keto gluconic
acid levels reached a maximum after 7 days.

The suggestion that the cellulose was growing using two different pathways implies that
this may be possible in static cultures as well. Further studies would have to be made to
establish whether the cellulose was actually growing at two interfaces instead of only
the air/liquid interface as previously assumed. The cellulose growth at the two
interfaces would be subject to different conditions. In the latter stages, growth beneath
the static pellicle would be governed by oxygen limitation, while growth above the
pellicle has been observed to be substrate limited.

Alternatively, the gel forming with in the reactor vessel could be cellulose II or acetan,
instead of cellulose I. How A. xylinum converts from a cellulose producer to becoming
cellulose non-producer or cellulose II producer
in agitated systems has remained ambiguous.
The transparent gel forming liquid has
similarities to that produced in the stirred tank
and internal air lift reactors. The difference



Holmes Thesis                                                                            37
                                                       Figure 19: Cellulose pellicles
                                                       in the RBC at 72 hours
Bacterial Cellulose
being that the significantly lower levels of agitation produces a cellulose matrix in the
pseudo static liquid phase of the reactor, as opposed to the gelatinous pellets.
Unfortunately, this would fail to explain the observed stalling of the pH.



Rotational Speed

                                         Sh(e)     M(e)     Stalled
                          14

                          12
  Prod. Rate (g/m2 day)




                          10

                           8

                           6

                           4

                           2

                           0
                               0   5   10          15          20            25             30
                                       Rotational Velocity (rpm)


Figure 20: The cellulose production rate as a function of the rotational velocity of
                                   the reactor

The optimum rotational velocity was 6-7 rpm (Figure 20). Values of 4 rpm
(Krystynowicz et al. 2002) and 14 rpm (Serafica 1998) have been reported. However,
Krystynowicz et al. used yield (g/L) as the determining property, which is subject to
discrepancies as more media would be fed to the reactor at a higher WHC to replace
entrapped media. Serafica‟s optimum of 14
rpm was derived considering purely the size of
the pellicle and not the dry weight of cellulose
produced. In all cases the size of the plates
was approximately the same. Since the most
significant parameter for cellulose production
was surface area at constant volume (Masaoka
1992), it was considered that optimisation
                                                        Figure 21: Cellulose pellicles
                                                        in the RBC at 84 hours
Holmes Thesis                                                                            38
Bacterial Cellulose
should be performed on a basis of dry weight per square meter of surface area and unit
time.

The rotational velocity was a vital parameter as it affected many of the observed
phenomena. The swelling and the stalling were probably highly affected by the rate at
which cellulose was sloughed off the plates. This in turn is directly attributable to the
shear forces generated by the rotating plate. There also appears to be a correlation
between rotational speed and water holding capacity.

At the lowest rotational velocity, 2 rpm, the agitation provided by the plates passing
through the medium became ineffectual. Instead of the cellulose forming primarily
on the plates, solid masses of cellulose were able to grow on the liquid surface. This
probably reduced the doubling time of the bacteria and allowed the outer edge of the
plates to become starved of substrate as the media drained away during the aerated
phase. Ultimately, this caused a significant reduction in the production rate.

Water Holding Capacity
The greatest Water Holding Capacity (WHC) was found to be between 12 and 16 rpm
(Figure 22). The bacterial cellulose produced in the RBC could hold water at almost
450 times the weight of the dried product. Since the WHC shows that water was
making up at least 99% of the pellicle, it had a greater affect on the size of a pellicle
than cellulose production. Hence the observed maximum pellicle growth between 12
and 16 rpm (Serafica 1998) correlated to a maximum in WHC, but unfortunately not to
cellulose production. The WHC was one example of changing properties and the
complex nature of ascertaining the optimum parameters for bacterial cellulose growth.

The operating conditions are recognised as having a significant affect on the properties
of bacterial cellulose (Chao et al. 1999). The two key parameters for WHC are the
distance between discs and stalling. The smaller the distance between the discs, reduces
the maximum size to which the pellicle can grow. Qualitatively, it has been observed
that the bacteria apparently recognise that the boundary conditions existed and rather
than confront shear forces, began to reinforce the previously constructed cellulose
matrix. For this reason, time becomes a factor also. The longer a pellicle is left
growing, the bigger it will be and the more likely it would „interact‟ with another
surface apart from the plate on which it was growing. Stalling, and the accelerated rates

Holmes Thesis                                                                               39
Bacterial Cellulose
of growth associated with it, reduced the WHC. This too, was probably reflective of
greater cellulose density within the pellicle restricting the maximum size and possibly
reducing the size of water bloated fibrils within the pellicle.


                                               SH(e)        M(e)

                         500
                         450
                         400
       WHC (gwet/gdry)




                         350
                         300
                         250
                         200
                         150
                         100
                          50
                           0
                               0   5     10          15            20          25             30
                                       Rotational Velocity (rpm)

 Figure 22: A graphical representation of how Water Holding Capacity changes
  with respect to rotational velocity using both SH(e) medium and M medium

pH
The affects of pH are well documented (Serafica 1998, Hwang et al. 1999). The
production of gluconic acid co-currently by A. xylinum decreased the pH. Initially,
without capability for applying active pH control, the di-sodium hydrogen phosphate
component of the media was relied on to buffer the fermentation broth. Static
experiments had proven that the pH usually
drops to 3.5 (Klemm et al. 2001). The reactor
operated predominantly at a pH between 3.2
and 3.4 (Figure 24), once the growth of A.
xylinum had dropped the pH from 5.0 by the
production of gluconic acid and 5-keto
gluconic acid (Klemm et al. 2001) along with
the cellulose. The addition of fresh medium,
also at a pH of 5.2, in volumes of 200-400 ml             Figure 23: Cellulose pellicles in
                                                          the RBC at 122 hours, just
                                                          prior to harvesting
Holmes Thesis                                                                                 40
Bacterial Cellulose
to maintain the level in the reactor had a negligible affect on the overall pH of the
reactor. The pH was raised approximately 0.2, but was quickly lowered by the
buffering action of the NaH2PO4 and by further production of gluconic acid.

For later runs, pH measurement and control were implemented. As can be seen from
Figure 24, the pH begins to drop significantly after a short lag phase. In calculating the
period for growth of cellulose for experiments in the future, this phenomenon will be
extremely valuable. It gives a method of eliminating the uncertainty surrounding the
ability of A. xylinum to adapt to a new environment. The implementation of pH control
suffered from a noisy signal, but still provided adequate manipulation of pH. A
constant pH is a necessity when other variables are being investigated to ensure
rigorous results for the manipulated variable such as the air/liquid ratio.




                                   Mutant      Typical      Controlled
       5.5

        5

       4.5

        4
  pH




       3.5

        3

       2.5

        2
             0     25       50       75      100      125      150      175     200     225
                                            Time (hours)

Figure 24: A depiction of the typical observed path of pH taken from run 30, run
                                  27 and run 33.

Defined Media
The defined or adapted Mormino (2001) medium became part of standard operating
procedure for several reasons. The negative affects were minimal. The production rate
was reduced insignificantly and the buffered pH dropped to 3.1-3.2, slightly lower than
the generic media at 3.2-3.4. The greatest difference between the two media was the

Holmes Thesis                                                                           41
Bacterial Cellulose
WHC, where the defined media appeared to shift the maximum to a slightly higher
rotational velocity. The defined media was clearer, allowing easier observation of what
was happening inside the reactor vessel. It also gave a more accurate depiction of the
glucose present when an assay was performed and allowed for more accurate
calculations of yield. Both of these had previously been affected by the peptone and
yeast extract containing unknown of carbon sources and contributing over 20% of
solids added to the media.


Strength Tests

                                      Dried       Def Media     Gen Media       Print Paper      Cyls
                           90
                           80
  Tensile Strength (MPa)




                           70
                           60
                           50
                           40
                           30
                           20
                           10
                            0
                                0             5            10             15             20              25
                                                      Rotational Velocity (rpm)

                           Figure 25: The strength of cellulose as a function of rotational velocity

Basic tensile strength tests were performed to confirm the expected trend that increasing
the rotational velocity adversely affects the strength of the product. The increased shear
forces applied to the pellicle at higher rotational velocity were expected to cause a
weakening in the bonds between the cellulose fibrils. The results are shown in Figure
25. The cellulose produced on the cylinders were almost twice as strong as any other
sample tested. It was surprising that almost identical values were gained for both
directions, circumferential and lengthwise. The generic medium produced a stronger
product than printer paper at all rotational velocities tested. The defined media
produced weaker cellulose than the generic medium. Cellulose produced by the defined
medium was weaker than paper at high rotational velocities. It must be emphasised that

Holmes Thesis                                                                                           42
Bacterial Cellulose
these values were generated for a quick qualitative analysis. Greater care in sample
preparation and increased accuracy in determining the cross sectional area would be
required to attain accurate figures.

Glucose Assay

                                           Glucose conc (Run 34)
                        20
                        18
                        16
  Glucose Conc. (g/L)




                        14
                        12
                        10
                         8
                         6
                         4
                         2
                         0
                             0   25   50   75       100    125     150     175     200
                                                Time (hours)

             Figure 26: The glucose concentration mapped over the course of an entire
             run. The initial glucose concentration was 30 g/L and the increases are
             reflective of fresh medium being added. The final increase was with
             medium containing 60 g/L.

A glucose assay established whether glucose was a limiting factor. Although the
glucose concentration reached very low levels (Figure 26), cellulose continued to be
produced and the pellicles became visibly larger. Cellulose was also produced in the
period between 25 and 75 hours. This was strong indication that A. xylinum has the
capability to switch metabolic pathways, possibly consuming gluconic acid, before
continuing to consume both glucose and gluconic acid. This would also explain
Klemm et al. (2001) establishing that gluconic acid and keto gluconic acid reach a
maximum after 5-6 days. It would also explain the irregular growth rates observed
(Serafica 1997) with respect to time and may indicate that there are highly specific
conditions which may further stimulate even an over-producing strain of A. xylinum.




Holmes Thesis                                                                           43
Bacterial Cellulose

Mutants in the RBC
Having developed healthy plates containing the bromine/bromide induced mutants, a
single colony was used to inoculate a tube of defined medium. When a single colony
was placed in a tube of generic medium, a strong, easily distinguished pellicle quickly
grew. When these it turn were used to inoculate the reactor, a growth rate of 3.1 g/ m2
day was obtained at 8.7 rpm. This compares to 8.0 g/m2 day obtained using the three
bacteria consortium at a similar rotational velocity. A unique characteristic was that the
WHC was 462 which was almost double the WHC obtained with the consortium of 225
at a similar rotational velocity.

During the run, pH control was not required as 125 hours to reach a pH that most runs
would reach after 36 hours. Throughout this time the pH smoothly and slowly dropped.
No stalling occurred, but a greater mass of cellulose was observed in the liquid phase
than for normal runs. In addition, the pellicle that formed on the plates had an
undulating appearance as opposed to the smooth constant surface from a normal run.


From Discs to Cylinders
Initial experimental runs showed indications that one of the primary parameters that
affected optimal growth of microbial cellulose was the ratio of time the surface spent
submerged to the time that the surface spent aerated, the liquid air contact ratio. This
was derived from the different growth characteristics exhibited by the cellulose in the
middle of the plates. This area, before the implementation of level control, was subject
to a varying amount of time spent submerged due to the evaporation and entrapment of
the medium. The pellicles were fatter in this region because of either the cellulose
growing more prodigiously or the cellulose matrix holding a greater amount of water.
With respect to both the quality of cellulose being produced and the rate at which it was
being produced, an investigation into this phenomenon was deemed worthy.

One method to vary the ratio of time submerged to time aerated with the same
apparatus was to physically lower the medium level in the reactor below half full. This
was not ideal as each radial position on the plate would experience a different ratio.
The disparity would become greater at lower medium levels. Also, the available
surface area for the cellulose to grow on became reduced as some of the plate would not


Holmes Thesis                                                                              44
Bacterial Cellulose
be submerged at all. Originally, the plates were chosen as they provided the greatest
surface area to media ratio possible in a rotating biological contactor (Serafica 1998).
This, however, was based on the assumption that the reactor vessel was filled to the
level of the shaft and that the diameter of the vessel and the plates be large (greater than
0.3m).

Plates being more favourable, cylinders were initially overlooked as being useful
surfaces on which to grow cellulose in a rotating biological contactor. When
considering scale-up for commercial production, the larger reactor vessel and surface
area required, plates provide a better surface area to media ratio than a single cylinder
and were easier to harvest and clean than concentric cylinders (Da Costa 2000). While
the surface area/media ratio remains relatively constant for plates, it rapidly decreases
for cylinders as the diameter of the vessel and the cylinder increase. Indeed, at smaller
diameters, such as the original reactor vessel, a hollow cylinder configuration has about
80% of the surface area available of the plates. A solid cylinder would be even better
having about 90% of the surface area of the plates due to the reduced media volume.
Additionally, reducing the media level to 90% left both plates and the cylinder with the
same surface area contacting the medium for the current reactor configuration. Below
this level, the cylinder became more favourable. The benefits of the cylinder included
producing two rectangular pellicles of cellulose that were very easy to harvest.
Additionally, the potential size of any individual sheet of product was far greater using
a cylinder.




Figure 27: End-on view of disk reactor showing the effect of lowering the reactor
volume on ratio of submergence to aeration and percentage of total disk usage.

The main reason for revisiting the feasibility of a cylinder is that it provided a simplistic
method of evaluating the effect of changing the ratio of time spent submerged to the



Holmes Thesis                                                                               45
Bacterial Cellulose
time spent aerated of the cellulose (Figure 27). As opposed to the discs, which would
require an awkward averaging to attain the ratio, as well as producing a non-uniform
product, every point on the cylinder would have exactly the same ratio. The drawback
of using a cylinder was the possibility of concentration gradients of substrate or pH
being generated along the length of the cylinder. This could be especially prevalent in a
large scale reactor being operated at constant overflow, with the reactor being fed at one
end and depleted at the other end.




                                                                               8
                                                                               7
                                                                              6
                                                                              5
                                                                                   Prod Rate
                                                                              4
                                                                                   (g/m2 day)
                                                                              3
                                                                              2
                                                                              1
          3.0
                                                                              0
                 2.0
      Air/                                                       10    11
                       1.2                           8      9
    Liquid                                    7
                                5      6
     ratio                                               Rotational Velocity (rpm)



Figure 2824: The production rate of cellulose on cylinders at different air/liquid
                   contact ratios and rotational velocities.

In trying to find benefits by encouraging the aerobic nature of A. xylinum, other positive
attributes were also derived from the cylinder configuration. The outer surface of the
cylinder provided structural support to the cellulose when it was not submerged. By
assisting the cellulose to negate the effects of gravity, the initial pellicle was able to
bind quickly to the fabric and form a strong pellicle. The medium was also more likely
to be entrained in the fabric and cellulose matrix providing substrate to bacteria also
present there.




Holmes Thesis                                                                                46
Bacterial Cellulose
The results from experiments with the cylinders (Figure 28) showed a positive trend
towards improved cellulose production. Early experiments were often interrupted by a
contamination problem (See Appendix A), therefore some circumspection must be
reserved for the rigor with which arguments can be made. However, the observed trend
was towards a higher air liquid contact ratio. The maximum ratio tested was 3.0. The
maximum production rate of 7.8 g/m2 day occurred at a lower ratio of 2.33, but the
rotational velocity was also lower. The rotational velocity still had a noticeable affect.
The trend was towards a similar rotational velocity as the half-submerged plates of
between 6-7 rpm. Although not conclusive, the promising results including the higher
strength should encourage further work in this direction.

Two significant boundaries were reached. At a higher air/liquid ratio, the cellulose on
the inside of the cylinders grew significantly altering the ratio. More importantly, this
surface became starved of fresh medium as it got closer to the same level as the media.
To improve this characteristic a larger reactor vessel and larger cylinders would be
required to ensure that this factor was not interfering with cellulose growth at higher
ratios. In addition, at lower rotational velocity and higher air/liquid ratios, it was
observed that the felt was having difficulty maintaining a moist enough surface. With
this low wetting rate, the bacteria apparently were not willing to remain on the surface
as cellulose production was almost non-existent. These runs were also prone to
contamination .




Holmes Thesis                                                                             47
Bacterial Cellulose
Conclusions
The strain of A. xylinum isolated from the New Zealand environment provided an
adequate (as a) basis for comparison to previous work. The data will provide a
substantial grounding for any continuation of the project. Having initially isolated a
usable strain, experiments have been performed that compare and contrast to previous
work and identify specific regions for further research. Furthermore, through
chemical mutagenesis a strain was isolated that will provide rigor to future work that
was not possible for this project. The ability to promote cellulose growth from a
single colony inoculation of a medium limits the possibility that other organism are
present. In addition, the strain showed increased capacity to generate the desired
cellulose product and less gluconic and keto gluconic acid with the adverse side
affects associated with them.

The optimum rotational velocity was 6-7 rpm. This contradicted the optimum
rotational velocity found in previous work of 12-16 rpm. However, this was based on
the physical size of the pellicle. Due to the large amount of water entrapped within
the pellicle this was found to be more indicative of the optimum water holding
capacity which was demonstrated to be between 12 – 16 rpm. At this point mixing
helps promote cell growth while the agitation has limited effect on causing a reversion
by A. xylinum to a cellulose non producing organism. The interaction between the
statically produced cellulose on the surface of the media in the vessel, cellulose
produced on the surface of the plates and the surface of the plates may at this point
become important. The type of material used to cover the plates may possibly need to
be revisited.

The results generated through both static and dynamic experimentation demonstrated
that the organism isolated showed similar trends to those studied elsewhere and
revealed some ways in which growth could be enhanced. The addition of 1.4% (v/v)
ethanol was confirmed. Likewise, exceeding 5% (w/v) sucrose or glucose did not
produce any benefit, which confirmed existing results. It was observed that the ratio
between time aerated and time submerged appeared to have a large affect and this was
subsequently investigated. The most significant difference was that the New Zealand
strain of A. xylinum had a WHC twice that of previously reported strains.



Holmes Thesis                                                                           48
Bacterial Cellulose
The pH measurement showed that there was a sharp decrease near the start of the runs
that could be used in the future to give a more accurate estimation of growth time.
This would be helpful to eliminate the different lag phases occurring on different runs.
The glucose assay showed a sharp decrease initially. The glucose concentration then
stayed constant at approximately 10 g/L for 36 hours before continuing to decline to
minimal levels.

Cylinders (were) used to investigate the ratio of time aerated to time submerged (They)
provided an ideal mechanism, as all points on the inner or outer surfaces of the
cylinder were subject to the same ratio and ideally, exactly the same conditions.
Since A. xylinum was a strict aerobe, and oxygen was perceived to be limiting,
especially at the beginning of an experimental run, the optimum ratio of 2.33 that
resulted was not unexpected. This was below the greatest ratio possible due to the
effects of boundary conditions and a larger reactor with an increased ratio may
provide even better results.




Holmes Thesis                                                                         49
Bacterial Cellulose
Recommendations
The reactor needs to be remounted. Through spillages, the mdf has swelled and
become buckled. At the same time, the gearing of the motor should be reduced
preferably so that its full range is between 0 and 15 rpm. Greater clearance is also
necessary above the top of the reactor to allow ease of installing and access to the pH
probe.


For most future runs the pH should probably held at 4. This was not optimum, but for
the present reactor configuration it was more practical. Higher pH potentially favored
contamination. Alternatively, give the reactor a predetermined seed time (about 1-2
days) before implementing control. This would allow the A. xylinum to adapt
sufficiently to the reactor environment before any competitive organisms are able to
interfere.


By preventing the initial pH drop, and hence, keeping the bacteria in the optimum
cellulose producing conditions, using the mutant culture instead would reduce or
eliminate the need for pH control. Unless an overseas variant of A. xylinum can be
imported this seems to be the best alternative. Although an inferior cellulose producer,
it easily developed pellicles from a single colony, which of course provides greater
rigor for future experiments. There is also the chance that a cellulose over producer
may have been isolated (See Appendix B). While reintroducing the pH problem, it
would be more practical to use a strain that shows greater similarity to those
experimented with previously.


The cylinders provide the greatest interest and key to the most information in the short
term. The optimum air to liquid ratio was not reached, which leads to the suggestion
that a bigger vessel may be required in the future. There is a maximum the ratio can
be set to for a given reactor due to the necessity of maintaining wetting on the inner
surface. A bigger reactor would also reduce the altering of the ratio by the growing
film. The other experiment that would provide insight would be to take
circumferential strips off the cylinder at set intervals. This would give direct
quantitative analysis of how fast the cellulose was growing during different periods.




Holmes Thesis                                                                            50
Bacterial Cellulose
For the discs, every experiment has used 6 days or the time just prior to breaching of
the gap between discs to terminate the run. This does not seem to be the most
rigorous method. In addition, the time of inoculation is often referred to as the
starting point. For both of these, the glucose assay and the pH measurement should
provide a more accurate indication. The ability of a culture to adapt to the reactor
after inoculation can drastically affect the results. By using a pH reading or glucose
concentration, determination that the culture in the reactor is in a replicable state for
each run at the starting time should be more reliable. Likewise, optimum termination
time should be investigated . Usually this would be some form of nutrient limitation,
but may also be the point where a side reaction causes unfavourable conditions. With
the reactor running in fed batch mode, there may not actually be an obvious endpoint.
This would suggest that finding a method to continuously harvest and grow the
cellulose would be better, perhaps along the lines of a conveyor belt system.


The big advantage of using a dynamic method such as the rotating biological
contactor is that the conditions and composition of the media can be changed with
relative ease. With static cultures, gaining access to the media requires damaging the
pellicle to make an addition with no guarantee of adequate mixing. In contrast, the
rotating biological contactor has had pH control applied with relative ease. As yet,
this fact has not been exploited. Cell growth has been shown to peak at a pH of 4,
while cellulose production was optimal at a pH of 5. Therefore, obtaining the
maximum cellulose production may require shifting the pH from an initial value of 4
for the first 2 -3 days, to a pH of 5 once reactor has become well populated. Similarly,
while increasing the substrate concentration above 5% (w/v) shows no benefits in
static cultures, this value may actually be lower in the rotating biological contactor.
Since the substrate was continually added in fed batch mode, there should be no
reason for the reactor to become substrate-limited as was the case in static cultures. It
may be the case that different concentrations of substrate are needed at different
periods during a run to satisfy the different pathways and rates of metabolism. Both
these hypotheses need to be verified by experimental data.




Holmes Thesis                                                                               51
Bacterial Cellulose

References

Aschner, M., Hestrin, S., (1946) “Fibrillar Structure Of Cellulose Of Bacterial And
Animal Origin”, Nature 157 659


Bernardo EB, Neilan BA, Couperwhite I., (1998) “Characterization, differentiation
and identification of wild-type cellulose-synthesizing Acetobacter strains involved in
Nata de Coco production” Systematic And Applied Microbiology 21 (4): 599-608


Black, N., (1990) “Weyerhaeuser And Cetus Develop Bacterial Cellulose Fiber 300
Times Narrower Than Softwoo Pulp Fibers”, Tappi Journal 73 (9-12) 46


Borzani W, Desouza SJ, (1995) “Mechanism Of The Film Thickness Increasing
During The Bacterial Production Of Cellulose On Non-Agitated Liquid-Media”,
Biotechnology Letters 17 (11) p1271-1272


Brown, A. J., (1886). “On Acetic Ferment Which Forms Cellulose”, Journal of
Chemical Society, 49, 432-439.


Brown, R. M. Jr., Microbial cellulose modified during synthesis. US Patent 4,942,128
dated Jul 17 1990


Brown, R. M. Jr., (1991). “Advances in Cellulose Biosynthesis” in “Polymers from
Biobased Materials”, Ed. Chum, H. L., Doyes Data Corp., New Jersey.


Budhiono A, Rosidi B, Taher H, Iguchi M (1999) “Kinetic Aspects Of Bacterial
Cellulose Formation In Nata-De-Coco Culture System”, Carbohydrate Polymers
40 (2) p137-143


Cannon, R. E., Anderson, S. M., (1991). “Biogenesis of Bacterial Cellulose”, Critical
Reviews in Microbiology 17 (6) 435-447.




Holmes Thesis                                                                         52
Bacterial Cellulose
Chao, Y., Ishida, T., Sugano, Y. and Shoda, M., (2000). “Bacterial Cellulose
Production by Acetobacter xylinum in a 50-L Internal Loop Airlift Reactor”,
Biotechnology and Bioengineering, 68 (3), 345-352.


Colvin J, Leppard G., (1977) “Biosynthesis Of Cellulose By Acetobacter-Xylinum
And Acetobacter-Acetigenus,” Canadian Journal Of Microbiology 23 (6): 701-709


Colvin J, Sowden L, Leppard G., (1977) “Structure Of Cellulose-Producing Bacteria,
Acetobacter-Xylinum And Acetobacter-Acetigenus,” Canadian Journal Of
Microbiology 23 (6): 790-797


Cook, K., Colvin, J., 1980 “Evidence for a beneficial Influence of Cellulose
Production on Growth of Acetobacter xylinum in liquid medium”, Current
Microbiology, 55 2448.


Couso, R.O., Ielpi, L, Dankert, M.A. (1987) “A Xanthan-Gum-Like Polysaccharide
From Acetobacter-Xylinum”, Journal Of General Microbiolog, 133 (8) 2123-2135


da Costa, B., (2000). “Design of a Reactor for Microbial Cellulose Production”,
CAPE, University of Canterbury.


Delmer DP (1999) “Cellulose Biosynthesis: Exciting Times For A Difficult Field Of
Study”, Annual Review Of Plant Physiology And Plant Molecular Biology 50 p245-
276 1999


DeWulf P, Joris K, Vandamme EJ., (1996) “Improved Cellulose Formation By An
Acetobacter Xylinum Mutant Limited In (Keto)Gluconate Synthesis,” Journal Of
Chemical Technology And Biotechnology 67 (4): 376-380


Edwards JD., (1995) “Industrial Wastewater Treatment: a Guidebook,” Boca Raton :
Lewis Publishers


Englehardt, J. (1995). “Sources, industrial derivatives, and commercial applications of
cellulose”, Carbohydr. Eur.12 514

Holmes Thesis                                                                       53
Bacterial Cellulose


Farr, W.K., 1940 Nature. 146 153,


Fontana J.D., Desouza A.M., Fontana C.K., Torriani I.L., Moreschi J.C., Gallotti B.J.,
Desouza S.J., Narcisco G.P., Bichara J.A., Farah L.F.X. “Acetobacter Cellulose
Pellicle As A Temporary Skin Substitute”, Applied Biochemistry And Biotechnology
24/5 253-264.


Frank, G. (1995) www.kombu.de/anleit-e.htm


Gostomski P, Bungay H, Mormino R (2002) “Plate and disk bioreactors for making
bacterial cellulose”, Biological Systems Engineering Acs Symposium Series 830 69-78


Haigler, C. H., Benziman, M. (1982) “Biogenesis of Cellulose I Microfibrils Occurs
by Cell-Directed Self-Assembly in Acetobacter xylinum”, Cellulose and Other
Polymer Systems, Ed. Brown Jr, R. M., Plenum press, New York.


Haigler CH, Weimer PJ. 1991. Biosynthesis and biodegradation of cellulose. Marcel
Dekker, Inc p.5-23, p.71-98, p.219-243


Hestrin, S., Aschner, M., Mager, J., (1947) “Synthesis of cellulose by resting Cells of
Acetobacter xylinum”, Nature 159 64-65


Hestrin, S., Schramm, M., (1954) “Synthesis of Cellulose by Acetobacter xylinum: 2
Preparation of freeze Dried Cells Capable of Polymerising Glucose to Cellulose”,
Biochemical Journal 58 345-352


Hieta K, Kuga S, Usuda M. (1984). “Electron staining of reducing ends evidences a
parallel-chain structure in Valonia cellulose”. Biopolymers 23:1807-10


Hwang JW, Yang YK, Hwang JK, Pyun YR, Kim YS (1999) “Effects Of pH And
Dissolved Oxygen On Cellulose Production By Acetobacter Xylinum BRC5 In
Agitated Culture”, Journal Of Bioscience And Bioengineering 88 (2) 183-188



Holmes Thesis                                                                        54
Bacterial Cellulose
Iguchi M, Yamanaka S, Budhiono A (2000) “Bacterial cellulose - a masterpiece of
nature's arts,” Journal Of Materials Science 35 (2): 261-270


Ishida T, Sugano Y, Shoda M, (2002) “Novel Glycosyltransferase Genes Involved In
The Acetan Biosynthesis Of Acetobacter Xylinum”, Biochemical And Biophysical
Research Communications 295 (2): 230-235


Ishida T, Mitarai M, Sugano Y, Shoda M, (2003) “Role Of Water-Soluble
Polysaccharides In Bacterial Cellulose Production”, Biotechnology And
Bioengineering
83 (4): 474-478


Ishkawa A, Matsuoka M, Tsuchida T, Yoshinaga F, (1995) “Increase In Cellulose
Production By Sulfaguanidine-Resistant Mutants Derived From Acetobacter Xylinum
Subsp Sucrofermentans”, Bioscience Biotechnology And Biochemistry 59 (12): 2259-
2262


Ishikawa A, Tonouchi N, Tsuchida T, Yoshinaga F, (1998) “Breeding Of A 5-
Fluorouridine-Resistant Mutant With Increased Cellulose Production From
Acetobacter Xylinum Subsp. Nonacetoxidans”, Bioscience Biotechnology And
Biochemistry 62 (7): 1388-1391


Jansson PE, Lindberg J, Wimalasiri KMS, Dankert MA (1993) “Structural Studies Of
Acetan, An Exopolysaccharide Elaborated By Acetobacter-Xylinum”, Carbohydrate
Research 254 (2) 303-310


Kimball, J.W. (2003)
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Carbohydrates.html


Klemm, D., Schumann, D., Udhart, U., Marsch, S. (2001) “Bacterial synthesized
cellulose – artificial blood vessels for microsurgery”, Progress in Polymer Science 26
pp 1561-1603.




Holmes Thesis                                                                      55
Bacterial Cellulose
Kouda T, Yano H, Yoshinaga F, Kaminoyama M, Kamiwano M, (1996)
“Characterization Of Non-Newtonian Behavior During Mixing Of Bacterial Cellulose
In A Bioreactor”, Journal Of Fermentation And Bioengineering 82 (4) pp382-386


Krystynowicz A, Czaja W, Wiktorowska-Jezierska A, Goncalves-Miskiewicz M,
Turkiewicz M, Bielecki S, (2002) “Factors Affecting The Yield And Properties Of
Bacterial Cellulose”, Journal Of Industrial Microbiology & Biotechnology 29 (4)
pp189-195


Lapuz, M., Gallardo, E., and Palo, M. (1967) “The Nata Organism – Cultural
Requirements, Characteristics, and Identity”, The Philippine Journal of Science, 96.
pp91-109.


Lin FC, Brown RM Jr, Drake RR Jr, Haley BE, (1990). “Identification of the uridine-
5'-diphosphoglucose (UDP-- glc) binding subunit of cellulose synthase in Acetobacter
tylinum using the photoaffinity probe 5-azido-UDP-glc”, Journal of Biological
Chemistry. 265 pp4782-84


MacCormick, C.A., Harris, J.E., Gunning, A.P., Morris, V.J. (1992) “Characterization
of a variant of the Polysaccharide Acetan Produced by a Mutant of Acetobacter
xylinum strain C1/4”, Journal of Applied Biotechnology 74 (2) 196-199.


Marx-Figini, M. (1982) he control of Molecular Weight and Molecular-Weight
Distribution in the Biogenesis of Cellulose”, Cellulose and Other Polymer Systems,
Ed. Brown Jr, R. M., Plenum press, New York.


Mayer R, Ross P, Weinhouse H, Amikam D, Volman G, Ohana P, et al. (1991)
“Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose
synthase and the occurrence of immunologically cross reacting proteins in higher
plants”, Proc. Natl. Acad. Sci. USA 88 pp5472-76


Mormino, R., (2001). “Cellulose production in a RBC”, PhD Thesis, Rensselaer
Polytechnic Institute.



Holmes Thesis                                                                          56
Bacterial Cellulose
Nakai T, Nishiyama Y, Kuga S, Sugano Y, Shoda M, (2002) “ORF2 Gene Involves In
The Construction Of High-Order Structure Of Bacterial Cellulose”, Biochemical And
Biophysical Research Communications 295 (2) pp458-462


Nishi Y, Uryu M, Yamanaka S, Watanabe K, Kitamura N, Iguchi M, Mitsuhashi S
(1990) “The Structure And Mechanical-Properties Of Sheets Prepared From Bacterial
Cellulose .2. Improvement Of The Mechanical-Properties Of Sheets And Their
Applicability To Diaphragms Of Electroacoustic Transducers,” Journal Of Materials
Science 25 (6): 2997-3001


Novaes AB, Novaes AB (1997) “Soft Tissue Management For Primary Closure In
Guided Bone Regeneration: Surgical Technique And Case Report”, International
Journal Of Oral & Maxillofacial Implants 12 (1) pp84-87


Novaes AB, Marcaccini AM, Souza SLS, Taba M, Grisi MFM (2003) “Immediate
Placement Of Implants Into Periodontally Infected Sites In Dogs: A
Histomorphometric Study Of Bone-Implant Contact” International Journal Of Oral
& Maxillofacial Implants 18 (3) pp391-398


Okiyama A, Shirae H, Kano H, Yamanaka S (1992) “Bacterial Cellulose .1. 2-Stage
Fermentation Process For Cellulose Production By Acetobacter-Aceti” Food
Hydrocolloids 6 (5) pp471-477


Park ST, Song T, Kim YM, (1999) “Effect Of Gluconic Acid On The Production Of
Cellulose In Acetobacter Xylinum BRC5”, Journal Of Microbiology And
Biotechnology 9 (5) pp683-686


Ross P, Weinhouse H, Aloni Y, Michaeli D, Weinbergerohana P, Mayer R, Braun S,
Devroom E, Vandermarel Ga, Vanboom Jh, Benziman M, (1987) “Regulation Of
Cellulose Synthesis In Acetobacter-Xylinum By Cyclic Diguanylic Acid”, Nature
325 (6101) pp279-281


Ross, P., Mayer, R., Benziman, M., (1991). “Cellulose biosynthesis and Function in
bacteria”, Microbiological Reviews, 55 (1) 35-58

Holmes Thesis                                                                    57
Bacterial Cellulose


Sakairi, N., Asano, H., Ogawa, M., Nishi, N. and Tokura, S., (1998). “A Method for
Direct Harvest of Bacterial Cellulose Filaments During Continuous Cultivation of
Acetobacter xylinum”, Carbohydrate Polymers, 35, 233-237.


Saxena IM, Lin IFC, Brown RM Jr., (1990) “Cloning and sequencing of the cellulose
synthase catalytic subunit gene of Acetobacterxylinum”. Plant Mol. Biol. 15 pp673-
83


Saxena IM, Kudlicka K, Okuda K, Brown RM Jr. (1994) “Characterization of genes
in the cellulose-synthesizing operon (acs Operon) of Acetobacter xylinum: implicaton
for cellulose crystallization”, J. Bacteriol. 176 pp5735-52


Schramm, M., Hestrin, S. (1954). “Factors Affecting Production of Cellulose at the
Air/Liquid Interface of a Culture of Acetobacter xylinum”, Journal of General
Microbiology, 11, 123-129.


Schramm, M., Grommet, Z., and Hestrin, S., (1957) “Synthesis of Cellulose by
Acetobacter xylinum: 3 Substrates and Inhibitors”, Biochemical Journal 67 669-679


Serafica, G. C., (1997). “Production of Bacterial Cellulose Using a Rotating Disk
Film Bioreactor by Acetobacter xylinum”, PhD Thesis, Rensselaer Polytechnic
Institute.


Son, H., Heo, M., Kim, Y., Lee, S., (2001) “Optimization of fermentation Conditions
for the Production of Bacterial Cellulose by a Newly Isolated Acetobacter sp.A9 in
Shaking Cultures”. Biotechnology and Applied Biochemistry 33, 1-5


Standal R, Iversen TG, Coucheron DH, Fjaervik E, Blatny JM, Valla S, (1994) “A
New Gene Required For Cellulose Production And A Gene Encoding Cellulolyt IC
Activity In Acetobacter Xylinum Are Co-Localized With The Bcs Operon”, J.
Bacteriol. 176 pp665-72




Holmes Thesis                                                                        58
Bacterial Cellulose
Sugiyama J, Vuong R, Chanzy H, (1991) “Electron Diffraction Study Of Two
Crystalline Phases Occurring In Native Cellulose From An Algal Cell Wall”
Macromolecules 24 pp4168-75


Tietze, H., (1995). Kombucha: The Miracle Fungus p45-49,80-83 Gateway Books


Tiong, B.H., (2001) “Microbial Cellulose” 3rd Pro Report, CAPE, University of
Canterbury


Vandamme EJ, De Baets S, Vanbaelen A, Joris K, De Wulf P (1998) “Improved
production of bacterial cellulose and its application potential,” Polymer Degradation
And Stability 59 (1-3): 93-99


VanderHart DL, Atalla RH, (1984) “Native Cellulose: A Composite Of Two Distinct
Crystalline Forms”, Science 223 pp283-85


VanderHart DL, Atalla RH., (1986) Cellulose: Structure, Modification and
Hydrolysis, ed. RA Young, RM Rowell, pp. 88-118. New York: Wiley-Intersci


Verschuren PG, Cardona TD, Nout MJR, De Gooijer KD, Van den Heuvel JC, (2000)
“Location And Limitation Of Cellulose Production By Acetobacter Xylinum
Established From Oxygen Profiles”, Journal Of Bioscience And Bioengineering 89 (5)
pp414-419


Watanabe K, Yamanaka S., (1995) “Effects Of Oxygen-Tension In The Gaseous-
Phase On Production And Physical-Properties Of Bacterial Cellulose Formed Under
Static Culture Conditions,” Bioscience Biotechnology And Biochemistry 59 (1): 65-68


Williams WS, Cannon RE, (1989) “Alternative Environmental-Roles For Cellulose
Produced By Acetobacter-Xylinum”, Applied And Environmental Microbiology 55
(10) pp2448-2452




Holmes Thesis                                                                       59
Bacterial Cellulose
Wong HC, Fear AL, Calhoon RD, Eichinger GH, Mayer R, et al., (1990) “Genetic
organization of the cellulose synthase operon in Acetobacter xylinum”, Proc. Natl.
Acad. Sci. USA 87 pp8130-34


Yamanaka S, Watanabe K, Kitamura N, Iguchi M, Mitsuhashi S, Nishi Y, Uryu M
(1989) “The Structure And Mechanical-Properties Of Sheets Prepared From Bacterial
Cellulose,” Journal Of Materials Science 24 (9): 3141-3145




Holmes Thesis                                                                        60
Bacterial Cellulose

Appendix A

Contamination
At stages contamination proved a major obstacle to running the reactor. The main
culprit was a white yeasty looking and smelling microbe. Although it was present in
almost all the runs in minute quantities, suggesting that it is common, it had the ability
to destroy runs. Usually consecutive runs were wiped out, while immense effort was
placed into trying to isolate the source and site of initial contamination and eradicate it.
It was only visibly present on the media surface in the reactor and on the surface of
the disc near the centre where it was not subjected to being submersed at any stage.
This suggested that it was an airborne microbe.


The possible sites of contamination were:
    Behind the felt. It was found that after extensive autoclaving the felt removed
     from the cylinders would still produced a biofilm of the yeasty substance when
     placed statically in media. This was probably not the cause as many of the
     static cultures inoculated and placed in the incubation box also became
     contaminated. Autoclaving the discs, cylinders and reactor vessel for 30
     minutes at least appeared to eliminate this. Also, the felt was replaced. While
     not seen as vital, the pains which contamination caused, deemed it worthy of
     spending the $5 to do this.
    Behind the grub screws. The grub screws, with the thread and hollow end
     were recognised as another potential site. Because they were not open
     surfaces to the steam in the autoclave and to a certain extent were insulated by
     the felt on the discs, autoclaving of the shaft intact with discs may have caused
     the contamination. The extended autoclaving period and/or autoclaving the
     assembly in pieces solved this.
    The pipette. As one of those pieces of equipment used on every run to
     inoculate the reactor it was a vital instrument, however one easily overlooked.
     Autoclaving the pipette and the rubber
     valve used to operate the pipette became
     part of standard operating procedure.
     Subsequently, using the pipette to
     inoculate the reactor was completely
     eliminated from the procedure.
    The pH probe. Although cleaned after
     each run, it was not sterilised. This was
     fine until the reactor was stood down for
     an upgrade. A quick rinse in 70% ethanol
     was the technique used rectify this, along
     with regular exchanging of the fluid in         Picture A 1: Caramelized sugar as substrate
     which the probe was stored while no in          source for A. xylinum



Holmes Thesis                                                                           61
Bacterial Cellulose
       use.

As an aside, it must be noted also that autoclaving the medium for too long also has
detrimental affects. Instead of smooth pellicles, solid clumps formed on the plates (see
Picture A1).


It has been observed that for runs that suffer from mild contamination the best way to
establish the extent of the effect is to look at the slopes and the WHC. The slopes will
be greater as the contamination seems to reduce the chain length of the cellulose
making it more prone to sloughing off the plates. The WHC was also far greater for
these contaminated runs.




Holmes Thesis                                                                          62
Bacterial Cellulose

Appendix B

Isolation
In response to rereading the article by Krystynowicz (2002), it became clear that by
transferring pellicles or transport medium containing A. xylinum from test tube to test
tube, the number of cellulose non-producing mutants increases rapidly. A postulate
would be that when the cell density reaches a certain level, the cellulose non-
producing mutants begin to be created. When they are transferred to fresh medium,
they are capable of multiplying as quickly as cellulose producers (Valla and
Kjosbakken 1982). Of course, the creation of cellulose non-producers could be a
phenotypical response. But that raises the question of how to get back to a broth or
culture dominated by cellulose producers.


The two runs that stalled had the peculiarity that they were inoculated with a freshly
isolated consortium. It is guesstimated that the solution to the cellulose producer
problem may lay in two key facts. The first is that by inoculating a large vessel with a
small sample, the abundance of all trace elements and required substrates encouraged
the bacteria to preferentially multiply cellulose producers. Or alternatively for the
dormant cellulose non-producers to revert to cellulose positive. The second part of
the solution is that after inoculating a tube with a single colony, do not transfer it to
another tube.


That stated, what is believed to be a cellulose over producing strain was isolated in the
following manner.


     Cut off a small piece of the Kombucha mat or Kombucha baby mat (both are
      stored in the refrigerator in the Environmental Laboratory).
     Place in a tube containing either medium (with the ethanol enrichment). It
      may pay to do 2 or 3 at one time, especially if the Kombucha mats are in
      anyway contaminated. Contamination should not matter as there are plenty of
      other organism already present in the Kombucha mat.
     Inoculate the tubes at 30°C.
     After 3-4 days (it may be longer), a pellicle should form on the top of the
      broth in the tube.




Holmes Thesis                                                                               63
Bacterial Cellulose
    As soon as a pellicle can be observed, flip the pellicle into a fresh tube. The
       ethanol enrichment favours Acetobacter genus over the other organism present.
       Also the bacteria have a superior doubling time to most of the fungi present.
    Allow a second pellicle to form in the new tube.
    From beneath the second pellicle, extract 1ml and inoculate a beaker. A 1 L
       beaker with 300-400 ml of medium is good. A 2 L beaker may be better as
       cellulose production is improved by increasing the surface area, but a 600 ml
       or 400 ml beaker have also proved more than adequate. Cover the beaker with
       tin foil to keep the ambient organisms at bay.
    After 7 – 10 days, a solid pellicle should be present. Picking up the pellicle by
       one edge, trim a small amount into fresh tubes of media.
    When pellicles form on the broth in these tubes, create a streak by grabbing
       some of the pellicle with a sterile loop and plating on nutrient agar plates
       containing the same medium as the broth.
    With the luck of the gods, this plate should contain only cellulose producers.
       With these, pellicles will form from single colonies and the broth will contain
       predominantly cellulose producers. Superior performance is expected.

This may be cumbersome, but falls more broadly into three phases. The first stage in
the tubes isolates the Acetobacter organisms. The second stage in the beakers
provides ideal growing conditions for the cellulose producing strain of A. xylinum.
Thirdly, retuning the A. xylinum to tubes and then plating the bacterium helps ensure
purity, and obtains the pure culture in a good state for later propagation.




Holmes Thesis                                                                         64
Bacterial Cellulose

Appendix C

Raw Data
The following is a summary of raw data – the collated information that was used
to lot the graphs presented in this thesis. For futher information consult the
electronic information.
DaBomb                              Runs 1-10
Dabombii                            Runs 11-20
DaBombiii                           Runs 21-
SumDaBomb                           For a summary of all runs and some graphs
Graphs                              For all other graphs.


For Plates
                               2 * (-448985* (0.065- L)^5 + 59609 * (0.065- L)^4 
                                                                                   
                       0.065
SubmergedArea                      - 2806.5* (0.065- L)^3 + 44.507 * (0.065- L)^2dL
                        h                                                          
                                     - 0.3087 * (0.065- L) + 0.065)                
MaxArea   plate  d 2
                   2


     MaxArea  SubmergedArea
R
         SubmergedArea
The submerged area is a bad approximation near the values of 0.000 and 0.065m
The term in the brackets represents x2 + y2 = 0.0652 in an easily integratable form.
The two makes one quarter of the plate into a half. (ie. Only the half of the plate
below the shaft can ever be submerged.
MaxArea is only the area on the plate that is subjected to wetting.


For Cylinders

           0.065  h 
  cos 1           
           0.065 
        2
R
     2  2
(os) in the data refers to the outside of the cylinder
(is) in the data refers to the inside of the cylinder
The cellulose from each cylinder and plate was split into a pellicle from each side.



Holmes Thesis                                                                             65

				
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