230 - Microbes

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					         Module 230
Pure and Applied Microbiology
     Enterprise Resource




       In conjunction with
                                 Module 230
                        Pure and Applied Microbiology
                             Enterprise Resource

INTRODUCTION TO THIS RESOURCE                       ERROR! BOOKMARK NOT DEFINED.4
AGRICULTURAL USES OF MICROBIOLOGY                                               6
 BIOLOGICAL CONTROL                                                             6
   Fungal biocontrol of insects.                                                6
   Bacterial biocontrol                                                         6
   Viral biocontrol                                                             6
   Company Case study:                                                          6
   Other References                                                             6
 FUNGAL INOCULATION                                                             7
   Arbuscular Mycorrhizal Fungi (Glomales)                                      7
   Black Truffles                                                               7
 GM RHIZOBIUM - SINORHIZOBIUM MELILOTI STRAIN RMBPC-2                           7
ENVIRONMENTAL USES OF MICROBIOLOGY                                              9
 SEWAGE TREATMENT AND BIOGAS                                                    9
 BIOFUELS                                                                       9
 BIOREMEDIATION OF HAZARDOUS WASTES OR POLLUTANTS                               9
   Land based bioremediation                                                    9
   Water based bioremediation                                                   9
   Bioremediation Case Studies                                                 10
   Further references and Links                                                11
 MICROBIAL SUPPLIERS                                                           11
 REFERENCES                                                                    11
USES OF MICROBIOLOGY IN FOOD                                                   12
 PROBIOTICS                                                                    12
 CHEESE                                                                        12
 BREAD                                                                         12
 BEER                                                                          12
 SOY SAUCE                                                                     12
INDUSTRIAL SCALE FERMENTATION                                                  13
 FOOD                                                                          13
   Cheese                                                                      13
   Bread                                                                       13
   Beer                                                                        13
   Sugar                                                                       13
   Soy Sauce                                                                   13
 ENZYMES AND CONVERSIONS                                                       14
   Enzymes                                                                     14
   Amino Acids                                                                 14


                                                                                2
   Steroids                                                        14
 CASE STUDIES OF INDUSTRIAL FERMENTATIONS                          14
   Subtilisin                                                      14
   Glycerol (Glycerine)                                            14
   Penicillin                                                      14
   PHB (Poly(3-Hydroxybutyrate)                                    15
   Bovine Somatotrophin                                            15
   Aspartame sweetener                                             15
USES OF MICROBES IN OTHER INDUSTRIES                               16
 FERMENTATIONS                                                     16
 MICROBIAL ENHANCED OIL RECOVERY (MEOR)                            16
   Exo-Polymer floods                                              16
   Well acidising                                                  16
   Oil channel plugging                                            16
   Paraffin wax and scale removal from well heads and pipelines.   16
   Pipe and tanker cleaning                                        17
   Additional MEOR Resources                                       17
 MICROBIAL METAL RECOVERY FROM WASTE MINING ORE                    17
   Additional Microbial Mining Resources                           17
MEDICAL USES OF MICROBES                                           18
 ANTIMICROBIALS                                                    18
   Cephalosporin                                                   18
   Penicillin                                                      18
   Tetracycline                                                    18
   Erythromycin                                                    18
 BACTERIAL DIAGNOSTICS                                             18
 BIOSENSORS                                                        19
 VACCINES                                                          19
POLYMERASE CHAIN REACTION                                          20
 HISTORY OF PCR                                                    20
 USES OF PCR                                                       20
 MARKET FOR PCR                                                    20
 PCR EQUIPMENT                                                     20
   DNA Polymerases                                                 20
   Thermocyclers                                                   20
 DNA SEQUENCERS                                                    20
COMMERCIAL ASPECTS OF THE PROTOZOA AND HELMINTHS                   21
 PROTOZOA                                                          21
   Amoebal Diseases                                                21
   AntiMalarials                                                   21
 ATOVAQUONE/PROGUANIL (PALUDRINE® FROM ASTRAZENECA)                21
   Leishmania                                                      21
   Trypanosomiasis                                                 21
   Vaccines                                                        21


                                                                    3
 HELMINTHS                                                       21
   Anthelmintics                                                 21
   Albendazole (Albenza®)                                        22
   Thiabendazole                                                 22
   Flukicide                                                     22
   Ivermectin (Stromectol®)                                      22
   Others                                                        22
   Vaccines (Dictol)                                             22
COMMERCIALLY IMPORTANT ALGAE                                     23
 RED ALGAE (RHODOPHYTA)                                          23
   Carrageenan                                                   23
   Agar                                                          23
   Nori                                                          23
   Maërl                                                         23
 BROWN ALGAE (PHAEOPHYTA)                                        24
   Alginate                                                      24
 GREEN ALGAE (CHLOROPHYTA)                                       24
   Chlorella                                                     24
 GENERAL REFERENCES FOR ALGAE                                    24
COMMERCIAL USES OF VIRUSES                                       25
 VIRAL BIOCONTROL                                                25
 VIRAL VACCINES                                                  25
 VIRAL CONTROL PRODUCTS                                          25
 VIRAL PROMOTERS                                                 25
 GM CROPS – VIRUS RESISTANT                                      25
   Papaya                                                        26
   Squash (Pumpkins)                                             26
   Potato                                                        26
   Tobacco                                                       26
COMMERCIAL USES OF RECOMBINANT DNA TECHNOLOGY.                   27
 SOME EXAMPLES OF COMMERCIALLY SUCCESSFUL RECOMBINANT PROTEINS   27
   Insulin                                                       27
   DNase I                                                       27
   Factor VIII                                                   27




                                                                  4
Introduction to this resource

This resource is designed with one aim in mind, that being to help the students taking module 520
appreciate the diverse ways in which biological knowledge has been harnessed to create wealth and
resolve problems.

The facts and examples are by no means exhaustive, but they should help illustrate the biological and
economic importance of the topics covered within the module. This is also written for the students rather
than academics, and all of this material, including that for other modules is on the York BioEnterprise
website.




                             http://www.york.ac.uk/depts/biol/bioenterprise/




                                                                                                       5
Agricultural uses of microbiology

Biological control
Where the application of chemical insecticides is not viable, perhaps for environmental reasons, a
biological alternative often exists, though biological control is usually most successful in enclosed
controlled environments such as glasshouses. Global crop protection sales are in excess of $30 billion
annually, biological control takes some 1% of this.

   Fungal biocontrol of insects.
   Various fungi are sold commercially, to be seeded into the soil or area of control. For example
   Metarhizium anisopliae is registered in the U.S. for control of household cockroaches. Beauveria
   bassiana is registered to control grasshoppers, locusts, Paecilomyces fumosoroseus has been approved
   to manage whiteflies, aphids, thrips, and spider mites. Nematode worms also damage crops and
   various nematophagous fungi are now on the market, for example, Verticillium lecanii is used to
   control the soybean cyst nematode Heterodera glycines which cost farmers an estimated $420 million
   a year (1980 figures).
   http://www.emeraldbio.com/ - Mycotrol® and Botaniguard® fungal insecticides.
   http://www.barc.usda.gov/psi/nem/pellets.htm - Soybean cyst nematode control.
   http://www.biological-research.com/philip-jacobs%20BRIC/ - Overview of nematophagous fungi.

   Bacterial biocontrol
   Bacillus thuringiensis (bt) – The gene coding for the toxin produced by this bacteria has been
   successfully inserted into seed crops such as cotton, potatoes and maize. In 2004 Monsanto sold over
   1.3 million packets of transgenic cotton seeds in India alone. GM is a highly contentious area of
   bioscience, but supporters of bt transgenic crops highlight increased yields, lower use of chemical
   pesticides and supposedly better returns for the farmers. The 2003 turnover of the big four GM seed
   producers Aventis, Monsanto, Syngenta and DuPont were $17.8, $4.9, $5.5 and $26.9 billion
   respectively. What part of this is due to bt crop sales is not known.

   Viral biocontrol
   Baculoviridae – Insecticidal viruses such as Cydia pomonella granulovirus used to kill larvae of the
   codling moth, Cydia pomonella. Baculoviruses represent 0.2% of the market for biological pesticides,
   which is itself only 1% of the insecticide market. i.e. approximately $600,000 out of a $30 billion (or
   more) market.

   Company Case study:
   http://www.certisusa.com/
   Certis annual sales are approximately $30 million. It is a leading producer many biological control
   products including foliar Bacillus thuringiensis (Bt) bioinsecticides, Neem-based botanical
   biopesticides, and various baculoviruses, nematodes and pheromones. The company’s products are
   sold under the brands Agree®, Deliver®, Javelin®, Thuricide®, and Teknar®. Bt bioinsecticides
   include Azatin® and Neemix®. They also market a botanical insect growth regulator, Trilogy®, and
   an insecticidal nematode, BioVector®.

   Other References
   http://www.ippc.orst.edu/biocontrol/biopesticides/address.cfm - Database of over 60 Biopesticide
   companies.



                                                                                                        6
   http://www.biosupplynet.com/cfdocs/products/prod_supp.cfm?prod_id=1778 - List of over 40
   Baculovirus suppliers
   http://www.nysaes.cornell.edu/ent/biocontrol/ - Biocontrol website by Cornell University. Has links to
   hundreds of other biocontrol related sites.

Fungal Inoculation
Many companies now sell symbiotic or otherwise useful fungi for the purposeful inoculation of land or
plants. Examples include Mycorrhizals and Truffles.
    http://www.world-of-fungi.org/ - And you thought Fungi were boring?

   Arbuscular Mycorrhizal Fungi (Glomales)
   Massively important due to ancient (400my) symbiosis with over 90% of the worlds plant species.
   They invade the plant root system, but rather than just take organic carbon compounds from the plant,
   they act as a source of inorganic carbon and other nutrients for the plant. In effect, the plant gains a
   secondary root system. There is also evidence that Mycorrhizal fungi produce Glomalin, a soil
   glycoprotein important for the structure and nutrient retaining capability of soils worldwide. It is in
   effect a glue capable of binding the inorganic and organic components of the soil together.
   http://www.mycorrhizae.com/ - Sells MycoApply® inoculums to agricultural and forestry sectors.
   Coat seeds or inject fungus into soil.
   http://www.ars.usda.gov/is/AR/archive/sep02/soil0902.htm - Details about glomalin from the USDA

   Black Truffles
   An example of a student enterprise from the University of Sheffield - Paul Thomas won the 2004
   WRCE business plan competition and is now setting up his black truffle farms. Paul developed a
   method of cultivating the valuable black truffle commercially and having won £2500 from the WRCE
   is looking for backing for his first plantation. A 5 hectare plantation could eventually yield over
   10,000kg of truffles per year, worth over £1,000,000 on the wholesale market.
   http://www.mycorrhizalsystems.com


GM Rhizobium - Sinorhizobium meliloti strain RMBPC-2
  Rhizobia are a group of bacteria, encompassing the genera Rhizobium, Sinorhizobium and
                  Bradyrhizobium, normally found in soil, which establish mutually beneficial
                  (symbiotic) relationships with legumes. Rhizobia form growths called nodules
                  (nodulation) on the roots of the legumes, and provide usable nitrogen to the plants. In
                  return, the plants provide a carbon and energy source for the rhizobia. Rhizobia have
                  been used commercially as seed inoculants in the form of seed coatings for over one
                  hundred years. Currently, about 80% of alfalfa grown in the United States is inoculated
                  with rhizobia prior to planting. Traditional strains though have been supplemented by a
  GM version, which has 5 new genes and is sold by Becker-Underwood. Their Dormal PLUS strain
  increases alfalfa production by an estimated 6%.

   However, as with any GM product there has been a long debate over whether a GM bacterium should
   be released at all. The RMBPC-2 strain has an extra copy of the nifA regulatory gene. NifA has a
   positive regulatory role on the expression of the other genes necessary for nitrogen fixation. The nifD
   promoter from Bradyrhizobium japonicum, the rhizobium that nodulates soybeans, was inserted
   upstream of the additional copy of nifA to control and prevent deleterious excess expression. In
   addition it was noticed that the C4-dicarboxylate transport system which is encoded by the dctA gene,


                                                                                                         7
was enhanced by addition of dctB and dctD genes. Thus a dctABD sequence from Rhizobium
leguminosarum (the rhizobium that nodulates peas and beans) was added to the RMBPC-2 genome.
Finally, resistance to streptomycin and spectinomycin was added so the strain could be tracked during
field trials.

The GM debate focuses around the key issues of whether the antibiotic resistance would spread to
other bacteria, especially human pathogens, as well as what other plants the rhizobia could inoculate.
Several scientists submitted evidence that Rhizobium meliloti can inoculate not only alfalfa, but also
other legumes such as sweet clover or mesquite, which are both known as weeds in US agriculture.
Could these become inoculated “superweeds”? The EPA and the manufacturers say there is no
evidence; the environmentalists don’t believe them…
http://www.epa.gov/opptintr/biotech/factdft6.htm - US EPA risk assessment of RMBPC-2
http://www.researchseeds.com/ - Research Seeds Inc.
http://www.alfalfa.org/ - US National Alfalfa Growers Alliance.
http://archive.greenpeace.org/geneng/reports/bio/rhizobium.pdf - Greenpeace arguing against the release of RMBPC-2
http://www.beckerunderwood.com/inoculants/productdisplay.asp?product=dpa – Dormal PLUS and other products
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=201891 – Paper (available free) detailing the results of field
trials for RMBPC-2




                                                                                                                           8
Environmental Uses of MicroBiology

Sewage Treatment and Biogas
Sewage treated with anaerobic and aerobic bacteria in several stages where the sewage is first aerated and
mixed then left in large ponds where solids are precipitated. Globally, waste-water/sewage treatment
accounts for some 15-25% of the $655bn world water market, which in itself accounts for almost 2% of
the annual global GDP.

Methane from landfills and sewage treatment can be collected and used in power stations. There are over
7 million small sewage-powered biogas plants in China alone! Many UK landfills also have methane
powered generators, including the landfill at Garforth, Leeds. Globally, biogas from human waste meets
about 14% of the worlds energy needs. (slightly less than hydro-electric power).
http://www.twm.co.nz/wtrmark.html - World water market figures
http://www.eere.energy.gov/RE/bio_biopower.html - Information on Biopower from the US government

Biofuels
Bioethanol or “gasohol” produced from fermentation of crops
such as sugar-cane or maize. As oil prices soar past $50 per
barrel, biofuels become increasingly more attractive and
commercially sound. Production costs for bioethanol are
currently approx $29 per barrel.

http://www.iogen.ca/index.html - partnered with shell, sells EcoEthanol® on Canadian market
http://www.jxj.com/magsandj/rew/2000_03/bioethanol.html -Review of potential of bioethanol
http://www.novozymes.com/cgi-bin/bvisapi.dll/biotimes/one_article_green.jsp?id=30698&lang=en

Bioremediation of hazardous wastes or pollutants

   Land based bioremediation
   The current UK bioremediation market is worth in the region of £120–£130 million. Contaminated
   land consultancy work has a value of approximately £130 –£140 million per annum.

   The variety of sites where bioremediation is required is huge. For example, military land is often
   contaminated with explosive and hydrocarbon residues. NATO has estimated that 217,000 sites in the
   US require remediation, costing approx. $187 billion over the next 20 years. Brownfield sites and sites
   of industrial pollution also often require extensive remediation as they are increasingly targeted for
   housing development and the strict environmental regulations this incurs. The sources of pollution on
   contaminated lands are varied e.g. heavy metals, hydrocarbons or other chemicals, often meaning that
   a combination of approaches to a single site have to be taken.

   Bioreactor landfills also use bioremediation techniques to rapidly transform and degrade organic
   waste. The increase in waste degradation and stabilisation is accomplished through the addition of
   liquid and air to enhance inherent microbial processes. Anaerobic processes release methane which
   can be captured for energy processes. (BioGas, see above.)

   Water based bioremediation
   Oil companies have used bioremediation since the first studies in 1989 after the Exxon Valdez disaster
   at Prince William Sound in Alaska. Environmental legislation and huge negative publicity promoted


                                                                                                        9
industry research into effective and environmentally friendly remediation technology. A large number
of companies market products aimed at cleaning oil spills on the shoreline, but products also exist for
cleaning out oil tankers while in port. Microbes also play a part in cleaning water supplies and aquifers
of heavy metals and excess concentrations of chemicals. One example recently highlighted has been
the link between arsenic concentrations in aquifers and the absence of sulphate-reducing bacteria
which normally reduce sulphate into sulphide which then reacts to precipitate arsenic from the water
http://oil-spill-pollution-control-degreasing-bioremediation.co.uk/home.asp - large list of products for
industrial bioremediation purposes
http://www.checkbiotech.org/root/index.cfm?fuseaction=news&doc_id=8940&start=1&control=229&
page_start=1&page_nr=101&pg=1 - Arsenic in groundwater


Bioremediation Case Studies
Decontamination of housing project Bedford. - Response Bioremediation Contracting had to degrade
1,1,1 trichloroethane at 213 mg/kg to a target value of 5 mg/kg using their Bio-Gel™ product. Bio-Gel
was manually injected under positive pressure into the target areas of contamination to act as a nutrient
substrate for selected microbial cultures, promoting the growth of the culture and metabolizing the
pollutant. Within 11 weeks the maximum concentrations of 1,1,1 trichloroethane were <0.02 mg/kg
http://www.biowise.org.uk/detail.asp?menucode=001000030004&type=industrialexample&id=818&c
urrentPage=1
http://www.response-uk.com/bio_gel.htm

                   Queen Mary II - Orelis membrane bioreactor: Orelis membrane bioreactor chosen
                   to act as the liners sewage plant, filtering and biodegrading the effluent before
                   releasing clean waste water into the ocean enabling the ship to operate in waters
                   protected by stringent wastewater discharge regulations.

                   http://www.ship-technology.com/contractors/separators/rhodia/index.html

Contaminated land in Essex – Cleanaway Ltd: The 64 hectare site contained significant contamination
including volatile organic compounds, oils, polyaromatic hydrocarbons, red-list organic substances
and heavy metals. Cleanaway installed a 200 m3/day-capacity continuous feed treatment plant using a
combination of anoxic and aerobic treatments.
http://www.biowise.org.uk/detail.asp?menucode=001000030004&type=industrialexample&id=819&c
urrentPage=1

              Magnadata International, a Lincolnshire-based printing company, was faced with a
              requirement to abate Volatile Organic Compound (VOC) emissions from its magnetic
              ticket coating process. A Sutcliffe Croftshaw SC Bioscrubber system was installed at
              the factory to treat the 30,000 m3/hour of process exhaust air. The system strips the
              solvents from the air into water and passes them to a biotrickling filter where a selected
              microbial biomass degrades them to carbon dioxide and water. Operating costs for the
              system are less than £4/day.
              http://www.sutcliffespeakman.com/techfrmr.htm

EcoRem won a $20million NATO contract for remediation of the “van Oss” fuel storage depot in
Holland. EcoRem had to reduce concentrations of mineral oil in the groundwater and soil from
5000mg/kg to 900mg/kg They chose to Biovent the area (pump air into the soil and mechanically mix


                                                                                                      10
it in), and within 12 months the mean contamination was at 475ppm exceeding a target value of
900ppm.
http://www.epa.gov/tio/download/partner/2002_annual_report.pdf
http://www.ecorem.be/defaulten.html

Further references and Links
http://www.dynamax.com/ - Bioremediation sensors: Engineering solutions and remote data logging
capability for remediation sites.
http://www.kavlico.com/library/sensorsmag.html - BioSensors
http://www.environment-agency.gov.uk/subjects/landquality/113813/?version=1&lang=_e                -
Environment agency bioremediation and contaminated land page
http://www.clarrc.ed.ac.uk/link/links.htm - Environment agency site covering UK environment laws
and policy.
http://www.ciwm.co.uk/pm/316 - chartered institute of wastes management, details on all regulations
and laws on waste management.
http://www.biowise.org.uk - Many case studies from around UK
http://www.wws.princeton.edu/cgi-bin/byteserv.prl/~ota/disk1/1991/9109/9109.PDF - Study into
bioremediation of oil spills by the US congress office of technology assessment.
http://www.oil-spill-web.com/oilspill/directory/products.asp?query=p2 - Large list of private sector
companies offering bioremediation solutions.
http://clu-in.org/ - The Hazardous Waste Clean-Up Information (CLU-IN) – Plenty of information
about innovative treatment technologies in hazardous waste remediation

Remediation Consultancies
http://www.erm.com/ERM/SVC/Brownfields.NSF
http://www.wspgroup.com/uk/
http://www.vhe.co.uk/
http://www.aeat.com/html/business/environment.htm
http://www.shanks.co.uk/
http://www.micro-bac.com/
http://www.obio.com/
http://www.regenesis.com/ - Look in Resources section for some good case studies
http://www.cytoculture.com/index.html - Academics who started their own company.

Products
BioGel - http://www.response-uk.com/bio_gel.htm
SpillSorb - http://www.spillsorb.com/
SC Bioscrubber - http://www.sutcliffespeakman.com/techfrmr.htm

Microbial Suppliers
http://altivia.com

References
Caplan, J.A. (1993). The worldwide bioremediation industry: Prospects for profit. Trends in
Biotechnology 11 (8) 320-323.




                                                                                                 11
Uses of Microbiology in Food

Probiotics
The Probiotic dairy market was valued at over £3.3 billion in 2004. This includes Marketed Probiotics
themselves as well as Yoghurts and Cheeses. Examples include BioPot, Onken, Yakult, Danone, Actimel.
Products generally contain species of the Lactobacillus and Bifidobacterium genera
http://news.scotsman.com/features.cfm?id=362982004 - Great article on everything Probiotic.

Cheese
Worldwide sales of cheese topped $22bn in 1980. Various fungal cultures are used eg Penicillium
roqueforti. Biotechnology has made an impact on the market, for example – the use of recombinant
Chymosin for the curdling of milk. The traditional source of Chymosin, also know as Rennin, is calves
stomachs but due to BSE there has been a global fall in cattle production and a resulting shortage of
Chymosin. Therefore Chymosin-encoding DNA has been introduced into three different microorganisms:
the yeast Kluyveromyces lactis, the fungus Aspergillus niger var. awamori, and a strain of the bacterium
Escherichia coli. 50% of US cheddar is now produced using recombinant Chymosin. Companies such as
DSM market recombinant chymosin eg “Maxiren®”
http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/ENZYMES/maxiren.html - Maxiren chymosin
http://www.dsm.com/ - Search for chymosin to see their product.

Bread
Yeast sales were $860m in 1981. The traditional bakers yeast is Saccharomyces cerevisiae but Various
species are used in baking around the world.

Beer
Combined sales of the top 10 global brewers and top 10 distilled spirits companies totalled nearly $200
billion in 1999. Saccharomyces uvarum (Also know as S.carlsbergensis) is vital for lager production.
Different cultures and processing methods lead to different tastes. One report on the components of a lager
identified 96 different substances contributing to aroma and taste. Only 30 of these compounds could be
characterised however.

Biotech is helping the process of beer making. Cetus Corp along with Guinness, fused the membranes of
S. uvarum with a genetically constructed S. diastaticus to introduce a novel glucoamylase into the yeast.
The end result was a stable yeast that retained the taste and characteristics they were looking for, but
increased the fermentation rate and lowered production costs accordingly.
http://www.nal.usda.gov/bic/Biotech_Patents/1995patents/05422267.html - Patent application

Soy Sauce
Aspergillus oryzae is added to a soy bean mash, then aerobically fermented. Several high profile
companies produce soy sauce such as Kikkoman and Wanjashan, and world production is over 1 billion
litres.
http://www.kikkoman.com/
http://www.wanjashan.com




                                                                                                        12
Industrial scale fermentation

Food
Oldest use of fermentations. Current world markets are huge and profitable.

   Cheese
   Worldwide sales of cheese topped $22bn in 1980. Various fungal cultures are used eg Penicillium
   roqueforti. Biotechnology has made an impact on the market, for example – the use of recombinant
   Chymosin for the curdling of milk. The traditional source of Chymosin, also know as Rennin, is calves
   stomachs but due to BSE there has been a global fall in cattle production and a resulting shortage of
   Chymosin. Therefore Chymosin-encoding DNA has been introduced into three different
   microorganisms: the yeast Kluyveromyces lactis, the fungus Aspergillus niger var. awamori, and a
   strain of the bacterium Escherichia coli. 50% of US cheddar is now produced using recombinant
   Chymosin. Companies such as DSM market recombinant chymosin eg “Maxiren®”
   http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/ENZYMES/maxiren.html - Maxiren chymosin
   http://www.dsm.com/ - Search for chymosin to see their product.

   Bread
   Yeast sales were $860m in 1981. The traditional bakers yeast is Saccharomyces cerevisiae but Various
   species are used in baking around the world.

   Beer
   Combined sales of the top 10 global brewers and top 10 distilled spirits companies totalled nearly
   $200 billion in 1999. Saccharomyces uvarum (Also know as S.carlsbergensis) is vital for lager
   production. Different cultures and processing methods lead to subtly different tastes. One report on the
   components of a lager identified 96 different substances contributing to aroma and taste and more
   surprisingly only 30 of these compounds could be characterised!

   Biotech is helping the process of beer making. Cetus Corp along with Guinness, fused the membranes
   of S. uvarum with a genetically constructed S. diastaticus to introduce a novel glucoamylase into the
   yeast. The end result was a stable yeast that retained the taste and characteristics they were looking for,
   but increased the fermentation rate and lowered production costs accordingly.
   http://www.nal.usda.gov/bic/Biotech_Patents/1995patents/05422267.html - Patent application

   Sugar
   Microbial sources of fructose have cut over $1 billion from the world sucrose market. Glucose
   Isomerase from Streptomyces olivaceous produces high-fructose syrup which has a higher perceived
   sweetness and fewer calories than sucrose.

   Soy Sauce
   Aspergillus oryzae is added to a soy bean mash, then aerobically fermented. Several high profile
   companies produce soy sauce such as Kikkoman and Wanjashan, and world production is over 1
   billion litres.
   http://www.kikkoman.com/
   http://www.wanjashan.com




                                                                                                           13
Enzymes and conversions
A huge number of commercial and industrial enzymes exist, taken from various microbes. Over 80% of
these enzymes are hydrolases. Market figure for the commercial worth of industrially produced enzymes
as of 2000 was $1.5bn.

   Enzymes
   Thermostable enzymes such as Subtilisin (See the case study below)

   Amino Acids
   Most are produced from bacterial systems. For example lysine and glutamic acid are produced by
   Corynebacterium glutamicum. Used in food supplements, medicines, or as precursors for industrial
   products. Optically Pure Amino Acids are also produced using microbes, such as L-aminoacyclase
   from Aspergillus oryzae. Microbes can also perform conversions, such as acylating and de-acylating
   various L-amino acids. The products are differentially soluble and can be separated with high
   specificity.

   Steroids
   Huge variety and usually far more specific, efficient and cost-effective than chemical transformations.
   EG – Plant steroids such as diosgenin and stigmasterol are converted into progesterone by Rhizopus
   spp. From progesterone they are transformed into virtually all medically important steroids. The
   human body cannot use stigmasterol, this metabolic capability is unique to bacteria. Steroids are used
   in a variety of applications from Medicine to Farming to Body Building, unfortunately no figures for
   the size of the market were found, but it is presumed to be large.



Case studies of industrial fermentations

   Subtilisin
   Discovered by Centre for Advanced Research in Biotechnology (CARB). Used bisulphite mutagenesis
   of bacterial plasmids. Then plated the mutants, and tested for enzyme product for thermostability.
   They gained a 4 fold increase in stability at 65C. Used in washing powders such as Ace, Bold and
   Ariel. Patent owned by Procter & Gamble. http://householdproducts.nlm.nih.gov/cgi-
   bin/household/brands?tbl=chem&id=2167

   Glycerol (Glycerine)
   Production pioneered by Germans during war. Prior to fermentation methods it was obtained from
   vegetable oil. Then it was found that yeast produces glycerol rather than alcohol if sodium bisulphate
   is added.

   Penicillin
   DSM fermented a modified strain of Penicillium chrysogenum, providing large quantities of high
   quality penicillin. Process of modification patented and used on other products. Process called
   PlugBugtm by DSM. Company made profits of E194m on sales of E1.24bn in 2003. Proportion of this
   solely due to penicillin is not known. http://www.dsm.com/




                                                                                                       14
PHB (Poly(3-Hydroxybutyrate)
An energy storage medium in many bacteria, eg Alcaligenes eutrophus. Accumulated and stored as
granules in intracellular compartments. Cultures are permitted to grow, then a essential nutrient such
as N, O, P is withheld. The bacteria then switch to PHP production, with eventual concentrations of up
to 100g per litre. Applications include: biodegradable plastic, scaffold for tissue re-growth, coating on
surgical devices, disposable packaging, coatings for paper and boards, blow and injection moulded
containers. http://www.fmcbiopolymer.com/

Bovine Somatotrophin
BST gene expressed in E.coli (Developed by Monsanto and Gentech 1980) Recombinant product is
virtually identical to the natural hormone, and increases milk production in dairy herds by 10-25%
with only 10% increase in feed intake. Currently produced by Monsanto as Posilac®
http://www.monsantodairy.com/

Aspartame sweetener
(L-aspartyl-L-phenylalanine) or Nutrasweet® used in huge number of common food products. The
product’s profitability is suffering though, due to cultural changes away from “unhealthy” soft drinks
and sugars. Also recently linked to health problems! http://www.nutrasweet.com/ (Market leader in the
$1.1bn high-intensity sweetener market)




                                                                                                      15
Uses of Microbes in Other Industries

Fermentations
See above

Microbial Enhanced Oil Recovery (MEOR)
It has been estimated that more than 300 billion barrels of oil within the U.S. cannot be recovered by
conventional technology but may be accessible through enhanced oil production. This is about 2.5 times
the amount of oil produced in the United States since 1983. At a figure of $30 per barrel, this equates to
$9trillion of untapped oil. Many non-microbial methods of enhancing oil production exist, but
increasingly microbial methods are being favoured as a cheaper and more effective alternative.

   Exo-Polymer floods
   Once the primary flow of oil stops, water is pumped into the oil field at another location to supply
   pressure and force the oil to the extraction head. Problems occur when water seeps through porous
   rocks at a greater rate than the general water:oil interface. These thief zones reduce field productivity
   as the pressure is reduced. Biotechnology however has provided an answer in the form of microbially
   produced polymers which increase the density of the injected water and plug the thief zones allowing
   the extraction of more oil from the field. Microbes and nutrients are injected with the water, and they
   produce exo-polymers, plugging the areas of high flow, forcing the water through lower density (oil
   filled) channels. This can increase field production by significant amounts, equating into large oil
   revenues otherwise not achievable. The process and equipment adds approx $1-4 to each barrel of oil
   extracted but releases thousands/millions of extra barrels of oil (depending on the field size). An
   example of an exo-polymer is XC polymer from Xanthomonas campestris. (Xanthan gum)
   http://www.titanoilrecovery.com/

   Well acidising
   In initial production it is often beneficial to pump water into the field at high pressure to physically
   crack the rocks and open channels for the oil to flow through. These channels are kept open using
   injected “proppants” such as sand particles which hold the cracks open once the water pressure is
   relieved. Acids are also injected to dissolve the rock round the fractures. This however is not ideal, as
   the effectiveness of the acid declines with distance from the point of injection, and it can also damage
   the oil extraction equipment. Therefore acid-generating bacteria are sometimes used in the place of
   inorganic acids. These bacteria can be pumped deep into the fractures, where they release organic
   acids slowly and over time. Problems occur however if the bacterial culture starts to plug the fractures
   and pores, stopping the extraction of oil.

   Oil channel plugging
   Similar to exo-polymer floods – see above

   Paraffin wax and scale removal from well heads and pipelines.
   A problem in the drilling and pumping equipment. Can lead to blockages or jamming. Conventional
   treatment is to flush hot oil through the pipes, or use chemicals. Bacterial solutions are much cheaper
   and environmentally sound. Estimated annual cost per well head of standard chemical treatment is
   $12,300 vs. a microbial solution costing approx. $7,000.
   http://www.custombio.com/products/pf_conc.html




                                                                                                         16
   Pipe and tanker cleaning
   More environmentally acceptable way of cleaning out tankers and storage facilities.
   http://www.storesonline.com/site/508377/page/96817
   http://www.ecochem.com/t_cbpa2.html
   http://www.ship-technology.com/contractors/separators/marine_enviromental/


   Additional MEOR Resources
   http://www.oilfield.slb.com/media/resources/oilfieldreview/ors97/spr97/bad_guys.pdf - Overview of
   oilfield microbiology
   http://www.glossary.oilfield.slb.com/Display.cfm?Term=XC%20polymer - Xanthan gum
   http://www.wws.princeton.edu/cgi-bin/byteserv.prl/~ota/disk1/1991/9110/911011.PDF               -
   Environmental uses for biotechnology in MEOR

Microbial metal recovery from waste mining ore
Copper has been leached out of waste ore using micro organisms since roman times, the technique only
gained commercial recognition once it was adapted and used for the production of gold. Billiton made
huge profits from such technology, contributing to their $10billion worth.

   Thiobacillus ferrooxidans concentrations can reach up to 106 per g of rock in waste copper ore heaps.
   They leach the remaining copper from the low-grade ore by oxidising sulphur and iron compounds
   from the rock. The bacterial solution and soluble copper is collected in a large pond at the base of the
   heap, where the copper precipitates and is collected. The waste water is then recycled to the top of the
   heap along with its bacterial load. Approx 20% of worldwide copper production is due to leachate
   technology. The process is also applied to Gold, Nickel and Zinc.
   www.BHPBilliton.com - BiOX technology for gold recovery took company to leading international
   position with 2003 market capitalisation in excess of $10billion. BiOX later used for copper nickel and
   zinc. Enter company website and search for BiOx, or click the next link for just one report.
   http://www.bactech.com/s/Projects.asp - Four case studies and further information.
   http://www.bhpbilliton.com/bbContentRepository/News/RelatedContent/NR_chile1.pdf


   Additional Microbial Mining Resources
   http://www.imm.org.uk/gilbertsonpaper.htm
   - Useful resource with good detail on biochemistry of microbial mining and its history.




                                                                                                        17
Medical uses of microbes

Antimicrobials
   The global antimicrobial market in 2000, was estimated at almost $40 billion with the majority of the
   market ($31 billion) in pharmaceutical antimicrobials. The rest of the market comprises antimicrobial
   agents used in industrial applications ($3 billion) and the plant health industry ($6 billion). It has been
   forecasted that the pharmaceutical antimicrobial market will more than double in less than a decade,
   growing to $69 billion in 2008. Antimicrobials are traditionally produced via the fermentation of
   microbial cultures, but some are now produced synthetically from chemical precursors. Over 800
   antibiotics have been discovered, and many of these are marketed. A few examples include:

   Cephalosporin
   The cephalosporin class of antibiotics is currently the largest by sales value, accounting for $7bn of
   sales in 2002. Various products exists including Cefaclor® from Zenith, CEDAX® from Biovail and
   Suprax® by Wyeth-Ayerst.

   Penicillin
   First commercial antibiotic, and second largest market sector behind the Cephalosporins. Penicillin
   acts by interfering with the production of cell wall during cell division. DSM ferment a modified strain
   of Penicillium chrysogenum, providing large quantities of high quality penicillin. Process of
   modification patented and used on other products. Process called PlugBugtm by DSM who made
   profits of €194m on sales of €1.24bn in 2003. Proportion of this solely due to penicillin is not known.
   Augmentin produced by Glaxo-SmithKline is also one of the worlds top antibiotics with sales of $1.2
   billion in 2002 alone.
   http://www.dsm.com/
   http://www.augmentin.com/

   Tetracycline
   Originally from Streptomyces aureofaciens, now generally synthetically produced by hydrogenolysis
   of chlortetracycline. Traded under the names Achromycin, and doxycycline, which has recently been
   linked to a possible cancer treatment.
   http://news.bbc.co.uk/2/hi/health/3726124.stm

   Erythromycin
   Produced from Saccaropolyspora erythraea (formerly classified as Streptomyces erythraeus). Used as
   penicillin alternative for those who have an allergy to penicillins. Used in respiratory tract infections,
   as well as chlamydia, syphilis, and gonorrhoea. Interferes with bacterial protein expression.
   http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Antibiotic_Explorer/Antibiotics_A_Z.ht
   ml - List of antibiotics marketed by Sigma-Aldrich (hundreds)

Bacterial Diagnostics
The bacterial and biosensor diagnostic market covers a wide range of applications including clinical
diagnostics, food testing, veterinary medicine and bioterrorism. The market for general diagnostic kits
covering all these areas was $6 billion in 2000. Of this the clinical diagnostic and research segments are
worth about $2 billion and seem to be experiencing growth rate of almost 25% per year.

Some examples of some diagnostic machinery are:



                                                                                                           18
http://www.accelr8.com/ - In development, advanced identification, counting and antibiotic susceptibility
screening in one package.
http://www.bacbarcodes.com/press_release_021203.htm - Bacterial biosensors linking bacterial strain
information to bioinformatics for quick identification.

Biosensors
Biosensors are machines that utilise biochemical reactions to conduct a test, and
then transduce the signal into a suitable display (i.e. digital). They usually
consist of enzymes within a selectively permeable membrane, with a further
membrane for the product “test” molecule to cross before detection at the
transducer. They provide on the spot tests that previously would have been sent
to the lab, and as such have found applications in medical science, agriculture,
food and environmental monitoring. They are also being developed for use as
sensors for biological and chemical weapons such as Anthrax. The biosensor
market is currently predicted to be in the region of $1-2 billion
http://www.sensornetworks.net.au/biosens.html - Good overview of the technology
http://www.the-scientist.com/yr2002/mar/profile_020318.html - Review by the easy to read journal.
http://www.army.mod.uk/equipment/nbcds/nbcds_nai.htm - British army nerve agent detection using an
immobilised cholinesterase. (NIAD - Nerve Agent Immobilised Enzyme Alarm and Detector).
http://www.analox.com/ - Biosensors for molecules such as glucose, Lactate etc
http://www.ambri.com/ - Medical biosensors
http://biacore.com/products/ - Medical and general bioscience sensors
http://www.medisense.com/ - Market leaders with sales of $170m (mostly medical)
http://news.bbc.co.uk/2/hi/health/1857730.stm - Biosensors in the news
http://news.bbc.co.uk/2/hi/uk_news/wales/2779581.stm - Biosensors in the news

Vaccines
http://www.vaxgen.com/products/index.html - VaxGen won an $878m contract with the US government
to supply anthrax vaccine for civil defence.




                                                                                                      19
Polymerase Chain Reaction

History of PCR
Invented in a moment of inspiration by Kary Mullis from a number of established techniques in 1983. He
has since been most notably been awarded the Nobel prize and a $450,000 Japan award for his work, and
retired to the guest speaker circuit. Mullis worked for CetusCorp, who developed the technique and later
sold the rights to the PCR process to Hoffman-La-Roche for $300m in 1991
Uses of PCR
Anything from criminal forensics to amplifying the genetic material from amber-entrapped flies. The uses
of the process are limited only by the creativity of the scientists using it.
http://www.proteinlogic.com - Mapping the key markers contained in body fluids for human diseases.
http://www.wdnas.com/ - Tracking illegal killing of protected species. The company started at the
University of Bangor and is now set up on its own.
http://news.bbc.co.uk/2/hi/science/nature/4050691.stm - News story about badger DNA tracking
Market for PCR
A single figure for the total worth of the process was not found. However, plenty of data exists to illustrate
the potential size of the market. The PCR process is also licensed depending on the use. Projects such as
the human genome project were able to use the process for a negligible cost, whereas large companies are
made to pay more. Licensed equipment is also sold, negating the need to apply for a paper license.
PCR Equipment
PCR requires several pieces of equipment such as Thermal Cyclers, and the consumables the process
requires, IE buffers, nucleotides, and various enzymes. The PCR equipment market for the EU alone was
estimated at being $623.7 million in 2004
   DNA Polymerases
   These enzymes are one of the vital components of the PCR process, and a multitude of companies
   supply raw enzymes or enzyme “kits” that include the various buffers and reagents. The market is
   huge, with multinational corporations such as Novagen, Promega and Sigma marketing hundreds of
   products. The world market for DNA diagnostic equipment (ie PCR) was $556.3 million in 2003. The
   Nucleic Acids lab at the University of York alone spends approx £17,000 per year on PCR reaction
   kits. New thermostable polymerases are always being sought, for example Pfu and Vent polymerases
   from the thermophilic hydrothermal vent bacteria. One example of a commercial DNA polymerase is
   TAQbead® from Promega. A pack costs £67 per 100 reactions. The polymerase is encased in a stable
   paraffin bead, enabling hot-start PCR http://www.promega.com/
   Thermocyclers
   Thermocyclers can cost anything from £2000 to £10,000 for the latest real-time PCR technology
   http://www.the-scientist.com/yr2001/dec/profile_011210.html - Very good review of the major
   thermocyler manufacturers and the market.
DNA Sequencers
The Applied Biosystems division of Applera Corporation has sold over 10,000 automated sequencers in
the past decade and has over 70% of the market. By mid-2003 the company had over 1700 orders for its
$300,000 ABI Prism3700 DNA analyzer. Total sales of this revolutionary machine = over $510m. The
machines are fully automatic and can sequence 96 samples of DNA at once, 2 or 3 times a day.
http://www.appliedbiosystems.com/ - DNA sequencers
http://www.beckman.com - DNA Sequencers used at the University of York


                                                                                                           20
Commercial aspects of the Protozoa and Helminths

Protozoa

   Amoebal Diseases
   Amoebal diseases such as Giardia and Hepatic Amoebiasis account for hundreds of thousands of
   deaths per year. Various drugs are prescribed to treat the various diseases but the 2 most common are
   Metronidazole and Tinidazole. Tinidazole is produced by Pfizer Pharmaceuticals under the brand
   name Fasigyn. http://www.netdoctor.co.uk/medicines/100001007.html

   AntiMalarials
   The antimalarial drug market is small when compared to other pharmaceutical products, due to few
   developed countries suffering high infection rates and the subsequent lack of money to encourage
   research and drug development. The largest market is for travellers health products, which is worth
   about $200-300 million. However, there is growing political demand for a effective vaccine or better
   drugs, and the WHO’s “Roll Back Malaria” campaign aims to halve malaria deaths by 2010.

   Marketed antimalarials include:
   Atovaquone/proguanil (Paludrine® from AstraZeneca)
   Doxycycline (Antibiotic tetracycline)
   Mefloquine (Lariam® from Roche)
   Primaquine® from AstraZeneca
   Chloroquine (Aralen from Sanofi Pharmaceuticals)

   Leishmania
   Pentamidine and occasionally Amphotericin
   http://www.drugdigest.org/DD/DVH/Uses/0,3915,526|Pentamidine%2BInhalation,00.html
   http://www.health.xq23.com/conditions/part_1/Pentamidine.html

   Trypanosomiasis
   Nifurtimox, Melarsoprol, Eflornithine (and in cattle: Ethidium, Samorin and bereni)

   Vaccines
   No successful commercial vaccines as yet.


Helminths
   Anthelmintics
   Anthelmintics are drugs for the treatment of parasitic worms (helminths). Antiparasitics and
   antibacterials generate global sales with a worth of over $1 billion in the cattle market alone. An
   accurate figure for the global anthelmintic market is not know, but is likely to be approaching the
   billion dollar mark.
   http://www.pjbpubs.com/pop_report_download. asp?type=toc&subid=206&reportid=283
   http://www.marvistavet.com/html/pharmacy_center.html - List of veterinary drugs, including all the
   anthelmintics.




                                                                                                     21
Albendazole (Albenza®)
GlaxoSmithKline are giving Albendazole to the WHO for free to treat elephantitis. By 2020 GSK
expect to have shipped 6 billion treatments worth $1 billion.
http://www.gsk.com/financial/reps03/annual_report2003.pdf - GSK 2003 Report
http://news.bbc.co.uk/1/hi/health/3512384.stm - Albendazole donated by GSK for treatment of
elephantitis.

Thiabendazole
Thiabendazole animal health products include Mintezol® and Tresaderm®, produced by Merck, who
had 1996 sales of nearly $200 million from abamectin and thiabendazole based products.
http://petplace.netscape.com/articles/artShow.asp?artID=1947 - Thiabendazole products

Flukicide
Off the shelf products such as Fasinex, Fasicare, Fasimec, Flukare, Tremacide and Farm Direct
Flukicide. The active ingredient is Triclabendazole.
http://www.endoparasite.net/products/fasinex.php
http://www.proagri.co.za/pebble.asp?relid=492
http://www.tumble.com/demo_area/Novartis/internet/agri/animalhealth/sh_liverflukes.html

Ivermectin (Stromectol®)
Also known as the antibiotic Cerulenin, Ivermectin is produced by Merck and has been so profitable
that the company started giving the product away to developing countries to treat river blindness
(onchocerciasis). In all over $80 million of Ivermectin has been donated to the WHO to help eradicate
the disease.
http://www.marvistavet.com/html/body_ivermectin.html - Ivermectin facts and info.
http://www.pfizerah.com/product_overview.asp?drug=EQ&country=US&lang=EN&species=EQ                  -
Equimax® from Pfizer

Others
Mebendazole (Vermox®) Oxamniquine (Vansil®) Praziquantel (Biltricide®) Pyrantel (Antiminth®,
Pin-Rid®) Diethylcarbamazine (Hetrazan®)

Vaccines (Dictol)
Dictol - Attenuated L3 stage vaccine against Dictyocaulus viviparus lungworm in cattle. Causes
disease known as Husk. Dictol developed in the 1960's and has since sold millions of units worldwide.
Products such as HuskVac are currently marketed.
http://www.intervet.co.uk/products_public/bovilis_huskvac/010_overview.asp - HuskVac
http://www.abbey-vetgroup.co.uk/parasitic_bronchitis_%28cattle%29.htm - Overview of disease




                                                                                                  22
Commercially Important Algae
Over 2 million tons of seaweed are harvested each year with a estimated worth of over $8 billion.

Red algae (Rhodophyta)
   Carrageenan
   A family of linear sulphated food grade polysaccharides obtained from the red seaweeds. The family
   can form a huge variety of gels at room temperature - rigid or compliant, tough or tender with high or
   low melting point. The gelation requires no refrigeration and the gels can be made stable through
   repeated freeze-thaw cycles. Used in a huge variety of products including gelled milk desserts,
   toothpaste, processed meats and even flame retardant foams. Approx 140,000 tons of Carrageenan are
   produced per annum, fetching up to $2,000 a tonne on the world market, the total size of which is
   valued at over $300 million. One company, Gelymar, posted a turnover of $16.9 million in 2003.
   http://www.fmc.com/Biopolymer/V2/PopProd/0,1421,Sel%253DIntroduction%2526Key%253D1308
   %2526ppID%253D34,00.html - Large international Carrageenan and biopolymer company. Excellent
   resource detailing the structure, biology and uses of Carrageenan.
   http://www.gelymar.com/nu2.htm - Large Chilean exporter of Carrageenan
   http://philexport.org/members/siap/intro.htm - Large resource detailing plenty of info about
   Carrageenan and its uses
   http://www.foodproductdesign.com/archive/1993/0193CS.html - Food gelling agents

   Agar
   Another colloidal agent used for thickening, suspending, and stabilising. However, it is best noted for
   its unique ability to form thermally reversible gels at low temperatures. The greatest use of agar is in
   association with food preparation and in the pharmaceutical industry (as a laxative, or as an inert
   carrier for drug products where slow release of the drug is required). Agar also obviously is used
   worldwide in bacteriology and mycology as a thickening agent in growth media. Some 18-25 thousand
   tons of seaweed are harvested each year for agar production, producing a little under 10,000 tons of
   agar, the market value of which is some $137 million. One Kg of the highest grade Agarose gel can
   cost nearly £2000.

   Nori
   A red seaweed used in sushi, usually grown commercially and has a world-wide market
   http://www.mountfuji.co.uk/seaweed.htm

   Maërl
   Coralline algae such as Phymatolithon calcareum and Lithothamnion corallioides are harvested and
   used as soil conditioners, trace element and mineral sources. They can also replace bone meal in some
   feed applications. Ground maërl is used in water filtration in some industries. These algae contain
   calcium and magnesium carbonates that comprise up to 80% of the wet weight. Maërl is dredged off
   the coasts of France (Brittany), England, and Ireland. Over 600,000 tons are harvested each year from
   live and dead deposits. The dried, ground product is favoured by organic farmers and horticulturists.




                                                                                                        23
Brown algae (Phaeophyta)
   Alginate
   The term usually used for the salts of alginic acid, which is derived from the cell walls of brown algae.
   Alginate forms gels and is used in food products much like Carrageenan and is often used as an
   alternative to pectin. Alginates also find uses in the field of medical dressings. Over 30,000 tons
   alginate are produced each year, from a variety of brown seaweeds.
   http://www.worldwidewounds.com/1998/june/Alginates-FAQ/alginates-questions.html - Alginate uses
   in medical dressings
   http://www.fmcbiopolymer.com/Biopolymer/V2/PopProd/0,1421,Sel%253DIntroduction%2526Key%
   253D1294%2526ppID%253D33,00.html Large international Alginate and biopolymer company.
   Excellent resource detailing the structure, biology and uses of Alginate.


Green algae (Chlorophyta)
   Chlorella
   A single-celled, fresh-water green algae harvested and used as nutritional supplement. The alga is
   credited with aiding the immune system, digestion, joint and muscle condition as well as blood
   pressure and cholesterol levels. In 2000, Sun Chlorella USA posted Japanese sales figures of $200
   million.
   http://www.sunchlorellausa.com/
   http://www.discount-vitamins-herbs.net/chlorella.htm
   http://www.nutraceuticalsworld.com/April021.htm


General References for Algae
http://www.nmnh.si.edu/botany/projects/algae/AlgIntro.htm - Algae and their economic uses
http://www.fao.org/documents/show_cdr.asp?url_file=/DOCREP/006/Y4765E/y4765e06.htm                -   FAO
guide to the seaweed industry
http://www.surialink.com/HANDBOOK/INDEX.ASP - excellent resource




                                                                                                         24
Commercial uses of viruses

Viral biocontrol
Baculoviridae – Baculoviruses represent 0.2% of the market for biological pesticides, which is itself only
1% of the insecticide market. i.e. approximately $600,000 out of a $30 billion (or more) market.

An example of a company using Baculoviridae in pest control is CertisUSA who market CYD-X, an
aqueous suspension of Cydia pomonella granulovirus or CpGV which kills the commercially important
pest the Codling moth. http://www.certisusa.com/products/cyd-x/index.html

Viral Vaccines
The world vaccine market, which was valued at approximately €8 billion in 2002 is predicted to triple in
value by 2012. Aventis, one of the 4 major companies selling human vaccines, reported 2002 sales of viral
vaccines worth €1.58 billion. Pfizer, AstraZeneca, Glaxo-SmithKline are the other 3 of the top 4 largest
pharmaceutical companies that sell vaccines.

The hunt for a vaccine for HIV continues, and the amount of money earmarked for HIV/AIDS is massive.
In the United States alone, the National Institutes of Health (NIH) is dedicating more than $2 billion to the
disease next year, and a five-year congressional initiative is adding $15 billion. The World Health
Organization (WHO) will distribute $5.5 billion, the Global Fund to Fight AIDS, Tuberculosis and
Malaria will provide $4.7 billion, and more than $500 million will come from the Bill & Melinda Gates
Foundation.

Viral control products
Creams to control herpes simple virus (HSV-1 and HSV-2) exist, to treat cold sores and genital herpes
respectively. The market leading product is Zovirax® (Acyclovir) with 2003 sales of over £10 million.
(Made by GlaxoSmithKline) http://www.gsk.com/products/zovirax_uk.htm

Viral Promoters
When virus’s assume control of their host cell’s transcription and translation processes they do so using
powerful promoter sequences that are used in preference to the hosts own promoters. Viral promoter DNA
is now used in recombinant DNA technology to enhance the expression of the target protein product. The
first virus promoter developed and used for this purpose was simian virus 40 (SV40), But similar elements
from other viruses such as Cytomegalovirus (CMV), Rous sarcoma virus (RSV), and Semlili Forest virus
(SFV) were also developed. An example of a commercially successful promoter is pTriEx™-1 from
Novagen.

GM Crops – Virus Resistant
Plant viruses cost the agricultural and horticultural sectors millions each year. Biotech companies have
been researching natural resistance as well as trying to develop new strains of GM crops with engineered
traits for resistance. Three commercial products are currently marketed; Papaya, Squash and Potatoes.
http://www.apsnet.org/online/feature/BioTechnology/Engineering.html - Overview of plant virus research.
http://www.whybiotech.com/index.asp?id=1262 - Potential benefits for another crop – strawberries




                                                                                                          25
Papaya
Papaya (Carica papaya L.) is grown commercially in over 48 countries with a
combined harvest of 7 million metric tonnes. It is valued as a nutritional tropical
fruit, grown throughout the Caribbean and south America. Strains were developed
to resist the papaya ringspot virus (PRSV). The plants express viral coat proteins,
and although the exact mechanism is not know, it is thought that this interferes
with one of the first steps in viral replication, that of uncoating (removal of CP
from the incoming virus). The license is held by the Papaya Administration
Committee of Hawaii, and the seeds are marketed as Rainbow and SunUp. The
Hawaiian market is estimated at around $100 million annually.
http://www.whybiotech.com/index.asp?id=1646 - What where who and why of transgenic Papaya
http://www.agbios.com/dbase.php?action=Submit&evidx=37 - Patent history and background

Squash (Pumpkins)
Another important crop in the US, Squash and Pumpkins suffer from infection by cucumber mosaic
virus (CMV), watermelon mosaic virus (WMV) 2, and zucchini yellow mosaic virus (ZYMV). Yellow
crookneck squash (Cucurbita pepo L.), together with pumpkins, gourds, and other squash, are grown
in over 83 countries with a combined harvest of 15 million metric tonnes in 2000. Other than removal
of virus-infected plants, there are no other effective control measures for these crops once infected.
The CZW-3 squash line was developed using recombinant DNA techniques to resist infection by
CMV, ZYMV, and WMV2 by inserting virus-derived sequences that encode the coat proteins (CPs)
from each of these viruses. The mode of action is thought to be the same as for virus resistant Papaya.
http://www.agbios.com/dbase.php?action=ShowProd&data=CZW-3 - Virus resistant squash info.

Potato
Potato (Solanum tuberosum L.) is grown commercially in over 150 countries with a combined harvest
of 311 million metric tons and is the fourth most important food crop in the world, providing more
edible food than the combined world output of fish and meat. Potato crops suffer from viral and insect
attack.
 The Colorado potato beetle is the most destructive insect pest of potatoes in North America and can
completely defoliate potato plants. Traditional control is with insecticides. Viral attack from Potato
virus Y (PVY) is known to infect over 342 plant species in 69 genera and 27 families, causing loss of
yield and crop quality. Both problems were solved in one step by Monsanto who used existing
successful commercial potato strains and added virus and beetle resistance genes. The transgenic
NewLeaf® potato contain two novel genes, the cry3A gene from Bacillus thuringiensis, which
encodes an insecticidal endotoxin protein and the coat protein gene from PVY-O. The exact
mechanism of resistance to the virus is not fully understood.
Monsanto marketed the NewLeaf brands successfully during 2000-2002 but decided to halt production
after McDonalds cancelled orders for the potatoes and introduced a “no-GM” policy for its
restaurants.
http://www.monsanto.co.uk/achievements/newleaf_potatoes.html - Monsanto NewLeaf plus page
http://www.agbios.com/dbase.php?action=ShowProd&data=RBMT15-101%2C+SEMT15-
02%2C+SEMT15-15 - Virus resistant potato info.

Tobacco
Tobacco Mosaic Virus is heavily studied and well characterised. Genes for the virus coat protein have
been inserted into several strains of the plant, conferring resistance to the virus. At the moment though
no GM strains resistant to TMV are available commercially.


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Commercial uses of recombinant DNA technology.

Some examples of commercially successful recombinant proteins

   Insulin
   One of the first recombinant products, now used worldwide. Several companies make or plan to make
   it, but Aventis have the largest market share as yet. In 2003, Aventis generated sales of €16.79 billion,
   and have just opened a new plant in Frankfurt to produce their rDNA insulin products Lantus® and
   Exubris®
   http://www.pharmaceutical-technology.com/projects/aventis/
   http://www.aventis.com/ search for insulin or their products Lantus and Exubris.

   DNase I
   Used to treat patients with cystic fibrosis. Modified CHO cells express a human DNAse I, which
   cleaves bacterial DNA in the lungs, helping reduce the inflammatory response and clear mucus. Sold
   as Pulmozyme by Genetech. Annual sales of over $110 million
   http://www.pulmozyme.com - Product information, info on CF.

   Factor VIII
   Blood clotting factor produced and used to treat patients with haemophilia. It is currently produced in
   CHO cells by Bayer. Very expensive to synthesise and purify but the product has the benefits of
   increased efficiency of activation, increased resistance to inactivation and decrease antigenicity.
   http://www.biological.com/abouthaemophilia_recombinant.cfm - Bayer recombinant Factor VIII




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