Energy production with hyperthermophilic organisms

Abstract

The present invention relates to the field of degradation with hyperthermophilic organisms, and in particular to the use of hyperthermophilic degradation to produce heat from a biomass. In some embodiments, a biomass is fermented in the presence of hyperthermophilic organisms to produce heat. The heat is used to heat a liquid which is used directly in a heat pump or radiant heat or to produce electricity or drive a steam turbine.

Claims

1. A method comprising: a) providing an algal biomass and a population of hyperthermophilic Thermatoga spp.; b) degrading said biomass in the presence of said population of hyperthermophilic Thermatoga spp. at a temperature of above 80 C. under conditions such that degradation products are produced.

2. The method of claim 1, wherein said degradation products are selected from the group consisting of hydrogen, methane and acetate.

3. The method of claim 2, further comprising using said hydrogen in a fuel cell.

4. The method of claim 2, further comprising using said methane in a combustion unit.

5. The method of claim 1, further comprising the step of converting said degradation products into energy.

6. A method for reducing carbon dioxide emissions comprising: a) providing an algal biomass and a population of hyperthermophilic Thermatoga spp.; b) anaerobically degrading said biomass in the presence of said population of hyperthermophilic Thermatoga spp. at a temperature of above 80 C. to produce substrates for energy production; c) producing energy from said substrates, wherein carbon dioxide emissions are reduced as compared to aerobic degradation of said biomass materials.

7. A method for generating carbon credits comprising: a) providing an algal biomass and a population of hyperthermophilic Thermatoga spp.; b) anaerobically degrading said biomass in the presence of said population of hyperthermophilic Thermatoga spp. at a temperature of above 80 C. to produce substrates for energy production, c) producing energy from said substrates under conditions such that carbon credits are generated.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention relates to the field of biomass degradation with hyperthermophilic organisms, and in particular to the use of hyperthermophilic degradation to produce heat from a biomass. For convenience, the description of the invention is provided in the following section: A. Hyperthermophilic organism; B. Biomass; C. Degradation and energy production; and D. Carbon credit generation.

(2) A. Hyperthermophilic Organisms

(3) The present invention contemplates the use of hyperthermophilic organism for fermenting biomass. Thermophilic bacteria are organisms which are capable of growth at elevated temperatures. Unlike the mesophiles, which grow best at temperatures in the range of 25-40 C., or psychrophiles, which grow best at temperatures in the range of 15-20 C., thermophiles grow best at temperatures greater than 50 C. Indeed, some thermophiles grow best at 65-75 C., and some of the hyperthermophiles grow at temperatures up to 113 C. (See e.g., J. G. Black, Microbiology Principles and Applications, 2d edition, Prentice Hall, New Jersey, [1993] p. 145-146; Dworkin, M., Falkow, S., Rosenberg, E, Schleifer, K-H., Stackebarndt E. (eds) The prokaryotes, third edition, volume 3, p. 3-28296 and p. 797-814 and p. 899-924; Madigan M., Martinko, J. Brock Biology of Microorganisms, eleventh edition, p. 430-441 and 414-415).

(4) The thermophilic bacteria encompass a wide variety of genera and species. There are thermophilic representatives included within the phototrophic bacteria (i.e., the purple bacteria, green bacteria, and cyanobacteria), bacteria (i.e., Bacillus, Clostridium, Thiobacillus, Desulfotomaculum, Thermus, Lactic acid bacteria, Actinomycetes, Spirochetes, and numerous other genera), and many hyperthermophilic orders (i.e., Pyrococcus, Thermococcus, Thermotoga, Sulfolobus, and some methanogens). There are aerobic as well as anaerobic thermophilic organisms. Thus, the environments in which thermophiles may be isolated vary greatly, although all of these organisms are isolated from areas associated with high temperatures. Natural geothermal habitats have a worldwide distribution and are primarily associated with tectonically active zones where major movements of the earth's crust occur. Thermophilic bacteria have been isolated from all of the various geothermal habitats, including boiling springs with neutral pH ranges, sulfur-rich acidic springs, and deep-sea vents. In general, the organisms are optimally adapted to the temperatures at which they are living in these geothermal habitats (T. D. Brock, Introduction: An overview of the thermophiles, in T. D. Brock (ed.), Thermophiles. General, Molecular and Applied Microbiology, John Wiley & Sons, New York [1986], pp. 1-16; Madigan M., Martinko, J. Brock Biology of Microorganisms, eleventh edition, p. 442-446 and p. 299-328). Basic, as well as applied research on thermophiles has provided some insight into the physiology of these organisms, as well as promise for use of these organisms in industry and biotechnology.

(5) The present invention is not limited to the use any particular hyperthermophilic organism. In some embodiments, mixtures of hyperthermophilic organisms are utilized. In some embodiments, the hyperthermophiles are from the archaeal order Thermococcales, including but not limited to hyperthermophiles of the genera Pyrococcus, Thermococcus, and Palaeococcus. Examples of particular organisms within these genera include, but are not limited to, Pyrococcus furiosus, Thermococcus barophilus, T. aggregans, T. aegaeicus, T. litoralis, T. alcaliphilus, T. sibiricus, T. atlanticus, T. siculi, T. pacificus, T. waiotapuensis, T zilligi, T. guaymasensis, T. fumicolans, T. gorgonarius, T. celer, T. barossii, T. hydrothermalis, T. acidaminovorans, T. prfundus, T. stetteri, T. kodakaraenis, T. peptonophilis. In some embodiments, aerobic hyperthermophilic organisms such as Aeropyrum pernix, Sulfolobus solfataricus, Metallosphaera sedula, Sulfobus tokodaii, Thermoplasma acidophilum and Thermoplasma volcanium are utilized. While in other embodiments, anerobic or facultative aerobic organisms such as Pyrobaculum calidifontis and Pyrobaculum oguniense are utilized. Other useful archaeal organisms include, but are not limited to, Sulfolobus acidocaldarius and Acidianus ambivalens. In some embodiments, the hyperthermophilic organisms are bacteria, such as Thermus aquaticus, Thermus thermophilus, Thermus flavu, Thermus ruber, Bacillus caldotenax, Bacillus stearothermophilus, Anaerocellum thermophilus, Thermoactinomycees vulgaris, and members of the order Thermotogales, including, but not limited to Thermotoga elfeii, Thermotoga hypogea, Thermotoga maritima, Thermotoga neapolitana, Thermotoga subterranean, Thermotoga thermarum, Petrotoga miotherma, Petrotoga mobilis, Thermosipho africanus, Thermosipho melanesiensis, Fervidobacterium islandicum, Fervidobacterium nodosum, Fervidobacterium pennavorans, Fervidobacterium gondwanense, Geotoga petraea, Geotoga subterranea.

(6) In some embodiments, hyperthermophilic strains of the above organisms suitable for fermenting biomass will be selected by screening and selecting for suitable strains. In still further embodiments, suitable strains will be genetically modified to include desirable metabolic enzymes, including, but not limited to hydrolytic enzymes, proteases, alcohol dehydrogenase, and pyruvate decarboxylase. See, e.g., (Bra/u, B., and H. Sahm [1986] Arch. Microbiol. 146:105-110; Bra/u, B. and H. Sahm [1986] Arch. Microbiol. 144:296-301; Conway, T., Y. A. Osman, J. I. Konnan, E. M. Hoffmann, and L. O. Ingram [1987] J. Bacteriol. 169:949-954; Conway, T., G. W. Sewell, Y. A. Osman, and L. O. Ingram [1987] J. Bacteriol. 169:2591-2597; Neale, A. D., R. K. Scopes, R. E. H. Wettenhall, and N. J. Hoogenraad [1987] Nucleic Acid. Res. 15:1753-1761; Ingram, L. O., and T. Conway [1988] Appl. Environ. Microbiol. 54:397-404; Ingram, L. O., T. Conway, D. P. Clark, G. W. Sewell, and J. F. Preston [1987] Appl. Environ. Microbiol. 53:2420-2425). In some embodiments, a PET operon is introduced into the hyperthermophile. See U.S. Pat. No. 5,000,000, incorporated herein by reference in its entirety.

(7) In some embodiments, hyperthermophiles that produce ethanol via degradation are selected. In some embodiments, such hyperthermophiles are selected in media containing progressively higher amounts of ethanol to select for strains with increased ethanol tolerance. Accordingly, some embodiments of the present invention provide hyperthermophiles with increased ethanol tolerance or increased ability to produce ethanol. In some preferred embodiments, the hyperthermophiles utilize lignocellulosic biomass. In further preferred embodiments, the hyperthermophile utilize glucose, xylose, arabinose, galactose, and mannose.

(8) B. Biomass

(9) The present invention contemplates the degradation of biomass with hyperthermophilic organisms. The present invention is not limited to the use of any particular biomass. Suitable biomass includes, but is not limited to, sewage, agricultural waste products, brewery grain by-products, food waste, organic industry waste, forestry waste, crops, grass, seaweed, plankton, algae, fish, fish waste, and combinations thereof. In some embodiments, the biomass is harvested particularly for use in hyperthermophilic degradation processes, while in other embodiments waste or by-products materials from a pre-existing industry are utilized.

(10) In some preferred embodiments, the biomass is lignocellulosic. In some embodiments, the biomass is pretreated with cellulases or other enzymes to digest the cellulose. In some embodiments, the biomass is pretreated by heating in the presence of a mineral acid or base catalyst to completely or partially hydrolyze hemicellulose, decrystallize cellulose, and remove lignin. This allows cellulose enzymes to access the cellulose.

(11) In still other preferred embodiments, the biomass is supplemented with minerals, energy sources or other organic substances. Examples of minerals include, but are not limited, to those found in seawater such as NaCl, MgSO.sub.47H.sub.2O, MgCl.sub.26H.sub.2O, CaCl.sub.22H.sub.2O, KCl, NaBr, H.sub.3BO.sub.3 and SrCl.sub.26H.sub.2) and other minerals such as MnSO4H.sub.2O, FeSO.sub.47H.sub.2O, CoSO.sub.47H.sub.2O, ZnSO.sub.47H.sub.2O, CuSO.sub.45H.sub.2O, KAl(SO.sub.4)212H.sub.2O, Na.sub.2MoOSO.sub.42H.sub.2O, (NHSO.sub.4)2Ni(SO.sub.4).sub.26H.sub.2O, Na.sub.2WO.sub.42H.sub.2O and Na.sub.2SeO.sub.4. Examples of energy sources and other substrates include, but are not limited to, purified sucrose, fructose, glucose, starch, peptone, yeast extract, amino acids, nucleotides, nucleosides, and other components commonly included in cell culture media.

(12) C. Degradation and Energy Production

(13) In preferred embodiments of the present invention, one or more populations of hyperthermophilic organisms are utilized to degrade biomass. In some embodiments, the biomass is transferred to a vessel such as a bioreactor and inoculated with one or more strains of hyperthermophilic organisms. In some embodiments, the environment of the vessel is maintained at a temperature, pressure, and pH sufficient to allow the strain(s) to metabolize the feedstock. In some preferred embodiments, the environment has no added sulfur or inorganic sulfide salts or is treated to remove or neutralize such compounds. In some preferred embodiments, the environment is maintained at a temperature above 45 C. In still further embodiments, the environment the environment is maintained at between 55 C. and 90 C. In some preferred embodiments, sugars, starches, xylans, celluloses, oils, petroleums, bitumens, amino acids, long-chain fatty acids, proteins, or combinations thereof, are added to the biomass. In some embodiments, water is added to the biomass to form an at least a partially aqueous medium. In some embodiments, the aqueous medium has a dissolved oxygen gas concentration of between about 0.2 mg/liter and 2.8 mg/liter. In some embodiments, the environment is maintained at a pH of between approximately 4 and 10. In some embodiments, the environment is preconditioned with an inert gas selected from a group consisting of nitrogen, carbon dioxide, helium, neon, argon, krypton, xenon, and combinations thereof. While in other embodiments, oxygen is added to the environment to support aerobic degradation.

(14) In some embodiments, where lignocellulosic material are utilized, the cellulose is pre-treated as described above. The pre-treated cellulose is enzymatically hydrolyzed either prior to degradation in sequential saccharification and degradation or by adding the cellulose and hyperthermophile inoculum together for simultaneous saccharification and degradation.

(15) It is contemplated that degradation of the biomass will both directly produce energy in the form of heat as well as produce products that can be used in subsequent processes, including the production of energy. In some embodiments, hydrogen, methane, and ethanol are produced by the degradation and utilized for energy production. In preferred embodiments, these products are removed from the vessel. It is contemplated that removal of these materials in the gas phase will be facilitated by the high temperature in the culture vessel. These products may be converted into energy by standard processes including combustion and/or formation of steam to drive steam turbines or generators. In some embodiments, the hydrogen is utilized in fuel cells. In some embodiments, proteins, acids and glycerol are formed which can be purified for other uses or, for, example, used as animal feeds.

(16) In some embodiments, the degradation products are removed from the vessel. It is contemplated that the high temperatures at which the degradation can be conducted facilitate removal of valuable degradation products from the vessel in the gas phase. In some embodiments, methane, hydrogen and/or ethanol are removed from the vessel. In some embodiments, these materials are moved from the vessel via a system of pipes so that the product can be used to generate power or electricity. For example, in some embodiments, methane or ethanol are used in a combustion unit to generate power or electricity. In some embodiments, steam power is generated via a steam turbine or generator. In some embodiments, the products are packages for use. For example, the ethanol, methane or hydrogen can be packaged in tanks or tankers and transported to a site remote from the fermenting vessel. In other embodiments, the products are fed into a pipeline system.

(17) In still other embodiments, heat generated in the vessel is utilized. In some embodiments, the heat generated is utilized in radiant system where a liquid is heated and then circulated via pipes or tubes in an area requiring heating. In some embodiments, the heat is utilized in a heat pump system. In still other embodiments, the heat is utilized to produce electricity via a thermocouple. In some embodiments, the electricity produced is used to generate hydrogen via an electrolysis reaction.

(18) D. Carbon Credit Trading

(19) In some embodiments, the present invention provides methods for generating carbon credits for trading in established carbon credit trading programs such as those established under the Kyoto protocol. The European Union Emission Trading System (EU ETS), which began operation in January 2005, is the largest multi-national, multi-sector greenhouse gas emissions trading scheme in the world. The system was set up as the EU's response to the Kyoto Protocol to the United Nations Framework Convention on Climate Change which was negotiated in 1997 and ratified in 2005. It is a commitment among participating industrialised nations to curb the rise in global temperature by abating their emissions of six greenhouse gases including carbon dioxide, methane, nitrous oxide, sulfur hexafluoride, perfluorocarbons (PFCs) and hydrofluorocarbons (HFCs). To date, 162 nations have ratified the agreement. Notable exceptions are the United States and Australia. Furthermore, two of the fastest growing economies, India and China, are not required to reduce their carbon emissions under the current agreement.

(20) The Kyoto Protocol provides three implementation mechanisms to regulate greenhouse gas emissions. The first, International Emissions Trading (IET), permits countries below their current emissions limits to sell their excess allowances to other countries on the open market. The second, Joint Implementation (JI), allows investors from industrialised countries financing greenhouse gas emissions reduction projects in other industrialised countries to receive emission credits called emissions reduction units (ERUs). The third, Clean Development Mechanism (CDM), lets investors from industrialised countries accumulate certified emission reduction units (CERs) for helping finance carbon reduction projects in developing countries.

(21) The EU ETS exists in two phases and encompasses all of the high use energy and power sectors. The first phase, which started in 2005 and will end in 2007, allows for the trade of CO.sub.2 allowances with the potential to expand into the other five greenhouse gasses. So far, it has set caps on the emissions of 12,000 to 15,000 industrial installations across Europe. It covers 45% of emission activities including power, concrete, pulp, paper, and ferrous metals. The second phase, from 2008 to 2012, could possibly cover all greenhouse gases and installations, and will include JI and CDM credits in the market. It is important to note that in the first phase an amendment called the Linking Directive was implemented which enabled installations to use CERs and ERUs from JI and CDM to meet their emission targets.

(22) The EU ETS is monitored and regulated by the EU Commission (EUC). In both phases, the EUC places limitations on GHG which are satisfied through the trading of EU emission allowances (EUAs). The goal is to force companies to find the lowest cost of abatement by decreasing their GHG internally and selling any unused EUAs into the market. During the first phase, the EUC imposes a penalty of 40 per ton of CO2 for installations that emit more than their target limit. In addition, these installations must acquire their excess emissions in the market. This penalty will go to 100 per ton of CO2 in the second phase.

(23) Participating countries in the EU ETS submit their target GHG reductions through National Allocation Plans (NAPs) which then are approved by the EUC. According to the Norwegian consultant Point Carbon, during the first phase of the EU ETS, the EUC approved circa 6.3 billion allowances and allowed for another 2.1 billion to be distributed each year.

(24) As one example of an established system, the European Bank for Reconstruction and Development (EBRD) and the European Investment Bank (EIB) established the Multilateral Carbon Credit Fund (MCCF) for countries from Central Europe to Central Asia.

(25) By joining the MCCF, private and public companies as well as EBRD and EIB shareholder countries can purchase carbon credits from emission reduction projects financed by the EIB or EBRD to meet their mandatory or voluntary greenhouse gas (GHG) emission reduction targets.

(26) In addition to the project credits, countries can also participate via the MCCF in green investment schemes. This is an innovative way to facilitate government-to-government trade in carbon credits, whereby the selling country uses the revenue from the sale of carbon credits to support investments in climate-friendly projects. Carbon credits can be generated from a large variety of project types, all of which reduce or avoid GHG emissions. These include credits produced from renewable energy such as wind, hydro, biogas (from landfills/wastewater) and biomass.

(27) In some embodiments, the present invention generates carbon credits for trading by utilizing biomass. In other embodiments, the present invention generates carbon credits for trading by utilizing materials that would otherwise create methane that is subsequently released into the atmosphere, such as manure, sewage, waste water, landfilled materials and the like. The present invention is not limited to any particular mechanism of action. Indeed, an understanding of the mechanism of action is not needed to practice the present invention. Nevertheless, it is contemplates that the use of hyperthermophilic organisms in an anaerobic degradation process is highly efficient for reducing carbon emissions, and in particular emissions of carbon dioxide. In particular, the use of anaerobic degradation reduces the amount carbon dioxide released from biomass by about six-fold as compared to aerobic degradation or fermentation processes.

(28) In some embodiments, the present invention provides a system wherein energy is produced by degradation of biomass with hyperthermophilic organisms, and resulting carbon credits generated through the use of the system are used to offset greenhouse gas emissions by conventional energy production systems such as combustion of coal, natural gas, and oil. In some embodiments, the energy production systems are under the control of a single entity, while in other embodiments, the energy production systems are under the control of separate entities and the carbon credits are purchased by or traded to the entity generating power by conventional means with fossil fuels.

EXPERIMENTAL

(29) 1. Selection of Hyperthermophilic Organisms for Degradation Processes

(30) In this example, strains of hyperthermophilic organisms from the genera Pyrococcus, Thermococcus, Palaeococcus, Aeropyrum pernix, Sulfolobus, Pyrobaculum, Pyrolobus, Pyrodictium, Thermus, Bacillus stearothermophilus, Metallosphaera, Anaerocellum, Thermoactinomyces, Thermotoga, Fervidobacterium and Geotoga are selected and screened for the ability to produce fermentation byproducts ethanol, methanol and hydrogen. Briefly, seed inoculums are prepared by culturing the cells in YT medium (yeast extract [2.0 g/liter], tryptone [4.0 g/liter], Na.sub.2S.sub.2O.sub.3 [0.61 g/liter], and ASN-III salts) for 48 h. Flasks containing base medium (tryptone (4.0 g/liter), Na.sub.2S.sub.2O.sub.3 (0.61 g/liter), and ASN-III salts (artificial seawater salts containing NaCl [29.8 g/liter], MgCl.sub.2 [1.1 g/liter], MgSO.sub.4 [2.0 g/liter], CaCl.sub.2 [0.45 g/liter], KCl [0.6 g/liter], and Na.sub.2CO.sub.3 [0.024 g/liter])(pH 7.0)) supplemented with specific carbohydrates (glucose, xylose, arabinose, galactose, and/or mannose) (3.0 g/liter) are inoculated with 10% seed inoculums. The flasks are then purged with prepurified N.sub.2 and the incubation is carried out at 90 C.-110 C. in a rotary shaker at 150 rpm. Cell growth is observed by monitoring optical density at 660 nm (OD.sub.660). Samples are collected from the headspace and culture medium and analyzed by GC for fermentation products.

(31) 2. Growth of Pyrococcus furiosus and Thermotoga maritima on Waste Materials and Biomass Substrates

(32) The hyperthermophilic archaeon Pyrococcus furiosus (growth range 67-103 C., optimal growth at 100 C.) uses simple and complex carbohydrates and converts them to acetate, to C0.sub.2 and to H.sub.2. Only in the presence of elemental sulphur (S), H.sub.2 is used to reduce sulphur to H.sub.2S. An exponentially growing culture produces 1 mol ml.sup.1h.sup.1 H.sub.2 (Schut et al., 2007, J. Bacteriol 189, 4431-4441). Growth experiments in the laboratory have shown that the strain requires peptone and yeast extract (as protein and vitamin source) in addition for good growth (2.210.sup.8 cells/ml). On starch as sole carbon source only poor growth was observed (510.sup.7 cells/ml).

(33) Thermotoga maritima is an obligately anaerobic hyperthermophilic bacterium growing between 55-90 C. (growth optimum at 80 C.). Like Pyrococcus it is of marine origin and is cultivated in media resembling seawater. Thermotoga is an obligate heterotroph preferentially fermenting carbohydrates or complex organic matter. Fermentation of glucose by cell suspensions of Thermotoga yielded 118 mol L-(+) lactate, 47 mol acetate, 54 mol C0.sub.2 and 9 mol H.sub.2 (Huber et al., 1986, Arch. Microbiol. 144, 324-333). Some of the members of the Thermotogales like Fervidobacterium nodosum (Patel et al., 1985 Arch. Microbiol. 141, 63-69) and Fervidobacterium islandicum (Huber et al., Arch. Microbiol. 1990, 154, 105-111 have been described to produce also ethanol. F. nodosum forms after 13 h growth on glucose 25 mol ethanol per 10 ml culture broth (Patel et al., 1985). A quantitative analysis of fermentation products (micromole of product formed per 10 ml culture) of T. nodosum grown on glucose revealed: Ethanol 10, acetate 115, lactate 162, CO.sub.2 120 and H.sub.2 160 per 133 micromol glucose consumed.

(34) Both organisms do not completely oxidize organic matter to CO.sub.2. The carbon of the substrate is in part converted to soluble compounds like acetate and lactate. Both organism produce low amounts of hydrogen and soluble compounds like acetate. Some members of the Thermotogales have been described to produce ethanol in addition (Fervidobacterium). Thus these anaerobic organisms have the potential to synthesize energy rich compounds like H.sub.2 and ethanol. The amount of CO.sub.2 produced during anaerobic degradation of biomass is significantly lower than CO.sub.2 release during aerobic processes which lead to complete oxidation of organic matter to CO.sub.2. Methane formation will not occur during this process when pure cultures are used or when the waste substrate is sterilized. Otherwise methane might be formed from the end products formed by degradation of organic matter from Thermotoga and Pyrococcus (H.sub.2/CO.sub.2 and acetate). Acetate can be also converted to methane but no hyperthermophilic methanogen growing on acetate has been described. Therefore, it is unlikely that methane is formed from acetate when the fermentation will be conducted at temperatures between 80 and 100 C.

(35) The objective was to investigate the potential of P. furiosus and T. maritima as model systems for the degradation of waste products and to investigate their ability to produce and to release heat during growth. The degradation of various waste products was studied in 100 l batch cultures. The energy release during growth was measured in a 10 l glass fermentor. The heating system of this fermentor was modified to lower the input of energy. The fermentor was isolated by the use of an aluminium containing shell and further isolated by styrene. As a control, heat release by a 10 l culture of Saccharomyces cerevisiae was also measured using this system.

(36) TABLE-US-00001 Utilization of waste substrates Pyrococcus Thermotoga Grain residues no growth poor growth (from a brewery) (8 10.sup.6 ml.sup.1) Mixture of grain residues no growth good growth and whey 1.4 10.sup.8 no pH control 3.2 10.sup.8 w/ pH control Mixture of grain residues 1 10.sup.8 2 10.sup.8 and fish innards Mixture of soluble starch ~1 10.sup.8 not analyzed and whey (final cell density was not determined)
Detailed formulations of the culture media are provided below.

(37) Since ethanol production has been described for some members of the Thermotogales we assayed also ethanol formation during growth on several substrates. We could not detect significant ethanol formation. For ethanol production, Fervidobacterium strains (F. nodosum and F. islandicum) may be utilized.

(38) Heat Production During Growth

(39) The measurement of energy release using a standard fermentor was difficult. When Pyrococcus was growing in the fermentor an input of 1060 Wh was required during an incubation time of 30 h to keep the temperature of the 10 l fermentor constantly at 90 C. In the absence of growing Pyrococcus cells the energy input in 30 h was 1140 Wh. This indicates an energy input of 35.5 W per hour in the absence of growing cells and 32.5 W per hour in the presence of growing cells. When the heat production was measured during growth of Thermotoga no energy release by growing cells could be detected, although the microorganisms grew quite well up within 13.5 hours to a cell density of 410.sup.8 cells/ml.

(40) It is known that large fermentors used for biotechnological processes like ethanol fermentation by yeast require cooling due to the energy released by growing yeast. To control the system for the detection of heat production we grew yeast anaerobically at 30 C. During 95 h after inoculation of the medium no external energy input was required to keep the growth temperature at 30 C. and the temperature of the culture medium was even increased by 0.5 C. This finding suggests that the detecting system is suitable to measure energy release by microorganisms. To confirm the validity of our measurement it is advisable to repeat the experiment in an air conditioned room (room temperature fixed at 20 C.).

(41) 3. Pyrococcus Furiosus SME Medium

(42) TABLE-US-00002 SME Component Amount SME 500.0 ml KH.sub.2PO.sub.4 0.5 g Wolfe's mineral elixir/ 1.0 ml 10/pH 6.5/new + T Resazurin, 0.1% solution 1.0 ml Na.sub.2S 7-9H.sub.2O 0.5 g H.sub.2O 2 distilled, add to a final volume 1000.0 ml of

(43) TABLE-US-00003 Synthetic Seawater - SME Component Amount concentration NaCl 27.7 g 473.99 mM MgSO.sub.4 7H.sub.2O 7.0 g 28.4 mM MgCl.sub.2 6H.sub.2O 5.5 g 27.1 mM CaCl.sub.2 2H.sub.2O 0.75 g 5.1 mM KCl 0.65 g 8.7 mM NaBr 0.1 g 0.97 mM H.sub.3BO.sub.3 0.03 g 0.49 mM SrCl.sub.2 6H.sub.2O 0.015 g 0.056 mM KJ-Lsg., 0.05% ig 0.1 ml 0.30 M H.sub.2O 2 distilled, add to a fnal volume 1000.0 ml of

(44) TABLE-US-00004 Wolfe's mineral elixir 10/pH 6.5/new + Titriplex Component amount concentration Titriplex 1 (Nitrilotriacetic acid) 15.0 g 78.50 mM MgSO.sub.4 7H.sub.2O 30.0 g 121.70 mM MnSO.sub.4 H.sub.2O 5.0 g 29.60 mM NaCl 10.0 g 171.10 mM FeSO.sub.4 7H.sub.2O 1.0 g 3.60 mM CoSO.sub.4 7H.sub.2O 1.8 g 6.40 mM CaCl.sub.2 2H.sub.2O 1.0 g 6.80 mM ZnSO.sub.4 7H.sub.2O 1.8 g 6.30 mM CuSO.sub.4 5H.sub.2O 0.1 g 0.40 mM KAl (SO.sub.4).sub.2 12H.sub.2O 0.18 g 0.38 mM H.sub.3BO.sub.3 0.1 g 1.62 mM Na.sub.2MoO.sub.4 2H.sub.2O 0.1 g 0.41 mM (NH.sub.4).sub.2Ni(SO.sub.4).sub.2 6H.sub.2O 2.80 g 7.09 mM Na.sub.2WO.sub.4 2H.sub.2O 0.1 g 0.30 mM Na.sub.2SeO.sub.4 0.1 g 0.53 mM H.sub.2O add to a final volume of 1000.0 ml
In standard medium, the following organic substrates were added:

(45) TABLE-US-00005 Component Amount Yeast extract (Difco) 0.1% Pepton from casein (Difco) 0.1% Starch (Merck) 0.1%
For Pyrococcus furiosus: pH: 7.0
Headspace: N.sub.2/CO.sub.2

(46) To study utilization of waste products we replaced the organic components of the medium by various waste materials: grain residues: 5%; whey 10%; fish innards 0.95%

(47) 4. Thermotoga MM-I-Medium

(48) TABLE-US-00006 MM-I-medium Compound Amount SME 250.0 ml KH.sub.2PO.sub.4 0.5 g (NH.sub.4).sub.2SO.sub.4 0.5 g NaHCO.sub.3 0.1 g Wolfe's mineral elixir, 1.5 ml 10/pH 6.5/new + T Resazurin, 0.1% solution 1.0 ml Na.sub.2S 7-9H.sub.2O 0.5 g H.sub.2O 2 distilled, add to a final volume 1000.0 ml of

(49) TABLE-US-00007 Synthetic Seawater - SME Compound Amount Concentration NaCl 27.7 g 473.99 mM MgSO.sub.4 7H.sub.2O 7.0 g 28.4 mM MgCl.sub.2 6H.sub.2O 5.5 g 27.1 mM CaCl.sub.2 2H.sub.2O 0.75 g 5.1 mM KCl 0.65 g 8.7 mM NaBr 0.1 g 0.97 mM H.sub.3BO.sub.3 0.03 g 0.49 mM SrCl.sub.2 6H.sub.2O 0.015 g 0.056 mM KJ-solution., 0.05% (w/v) 0.1 ml 0.30 M H.sub.2O 2 distilled, add to a final volume 1000.0 ml of

(50) TABLE-US-00008 Wolfe's mineral elixir 10/pH 6.5/new + Titriplex Compound amount concentration Titriplex 1 (Nitrilotriacetic acid) 15.0 g 78.50 mM MgSO.sub.4 7H.sub.2O 30.0 g 121.70 mM MnSO.sub.4 H.sub.2O 5.0 g 29.60 mM NaCl 10.0 g 171.10 mM FeSO.sub.4 7H.sub.2O 1.0 g 3.0 mM CoSO.sub.4 7H.sub.2O 1.8 g 6.40 mM CaCl.sub.2 2H.sub.2O 1.0 g 6.80 mM ZnSO.sub.4 7H.sub.2O 1.8 g 6.30 mM CuSO.sub.4 5H.sub.2O 0.1 g 0.40 mM KAl(SO.sub.4).sub.2 12H.sub.2O 0.18 g 0.38 mM H.sub.3BO.sub.3 0.1 g 1.62 mM Na.sub.2MoO.sub.4 2H.sub.2O 0.1 g 0.41 mM (NH.sub.4).sub.2Ni(SO.sub.4).sub.2 6H.sub.2O 2.80 g 7.09 mM Na.sub.2WO.sub.4 2H.sub.2O 0.1 g 0.30 mM Na.sub.2SeO.sub.4 0.1 g 0.53 mM H.sub.2O 2 distilled, add to a final volume 1000.0 ml of
For growth of Thermotoga maritima the following organic substrates were added:

(51) TABLE-US-00009 Compound amount Starch (Merck 101252.1000) 0.05% Yeast extract (Difco) 0.05%
To study growth on waste products the organic substrates were replaced by: grain residues (5% w/w), whey 10% (v/v) and homogenized fish innards 0.9% (950 g/100 l).
pH: 7.0
headspace: N.sub.2

(52) In some experiments first growth of Pyrococcus was studied at 90 C., if Pyrococcus failed to grow or after growth of Pyrococcus to 110.sup.8 cells/ml the medium was cooled down to 80 C. and then the same medium was inoculated with Thermotoga. On the substrate mixture grain residues and fish innards good growth of Thermotoga was observed under these conditions; this indicates that Thermotoga grows well in Pyrococcus medium.