US 20080311640A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0311640 A1 Cox et al. (43) Pub. Date: Dec. 18, 2008

(54) ANAEROBC PRODUCTION OF HYDROGEN Related U.S. Application Data AND OTHER CHEMICAL PRODUCTS (60) Provisional application No. 60/678,101, filed on May 3, 2005, provisional application No. 60/677,856, filed (76) Inventors: Marion E. Cox, Morgan Hill, CA on May 3, 2005, provisional application No. 60/678, (US); Jeremy N. McDonald, San 077, filed on May 3, 2005, provisional application No. 60/678,100, filed on May 3, 2005, provisional appli Jose, CA (US); Laura M. Nondorf, cation No. 60/678,098, filed on May 3, 2005, provi Morgan Hill, CA (US); Steven M. sional application No. 60/677,998, filed on May 3, Cox, Morgan Hill, CA (US) 2005. Publication Classification Correspondence Address: WILSON SONSIN GOODRCH & ROSAT (51) Int. C. 650 PAGE MILL ROAD CI2P3/00 (2006.01) CI2M 3/00 (2006.01) PALO ALTO, CA 94304-1050 (US) CI2M I/36 (2006.01) CI2N L/20 (2006.01) (21) Appl. No.: 11/912,881 (52) U.S. Cl...... 435/168; 435/290.4; 435/286.1: 435/303.2:435/252.1 (22) PCT Fled: Apr. 27, 2006 (57) ABSTRACT Described herein are methods for producing chemical prod (86) PCT NO.: PCT/USO6/16332 ucts by anaerobically fermenting a particular biomass using anaerobic bacteria. Such chemical products includehydrogen S371 (c)(1), and other gases, acetic acid and other volatile organic acids, (2), (4) Date: Jun. 23, 2008 Solvents, Solids, and salts of Volatile organic acids.

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ANAEROBC PRODUCTION OF HYDROGEN fermentation of the biomass have been selected by means of AND OTHER CHEMICAL PRODUCTS a Knowledge Management System, and selected and isolated using an isolation/enrichment system. FIELD OF THE INVENTION 0001. The methods and compositions described herein are Bacterial Strains directed to the production of hydrogen and other chemical 0009. In further or alternative embodiments of all aspects products via the anaerobic bacterial fermentation of biomass, described herein using bacterial strains to anaerobically fer and the production of hydrogen and other chemical products ment biomass and obtain chemical products therefrom, the via bacterial conversion of products obtained from anaerobic bacterial strains are substantially purified anaerobic bacteria fermentation. and such substantially purified bacterial strains are selected to anaerobically ferment the biomass, and in still further or BACKGROUND OF THE INVENTION alternative embodiments of all aspects, the Substantially puri 0002 Hydrogen has high energy content, with water being fied bacterial strains are selected from the group consisting of the product resulting from combustion of hydrogen with oxy Acetivibrio cellulolyticus, Acetivibrio cellulosolvens, Acetiv gen. As such, hydrogen represents a potentially ideal fuel ibrio ethanolgignens, Acetivibrio multivorans, Acetoanaero source. However, the use of hydrogen as a viable alternative bium noterae, Acetofilamentum rigidium, Acetogenium kivui, energy source remains challenging, because many methods Acetomicrobium faecale, Acetomicrobium flavidum, Aceto for producing hydrogen, such as water electrolysis, and petro thermus paucivorans, Acidaminobacter hydrogenoformans, leum reforming, are economically and energetically intense. Anaerobiospirillum succiniciproducens, Anaerobiospirillum ihomasii, Anaerorhabdus fircosa, Anaerovibrio burkinaben SUMMARY OF THE INVENTION sis, Anaerovibrio glycerini, Anaerovibrio lipolyticus, Atopo biunfossor; Atopobium minutum, Atopobium parvulum, Ato 0003. In one aspectare methods of preparing nutrients for pobium rimae, Atopobium vaginae, Bacteroides acidifaciens, use in anaerobic production of hydrogen. In another aspect Bacteroides amylophilus, Bacteroides asaccharolyticus are nutrients for use in the anaerobic production of hydrogen, Bacteroides bivius, Bacteroides buccae, Bacteroides bucca wherein the nutrients are prepared by methods, which lis, Bacteroides caccae Bacteroides capillosus, Bacteroides include, but are not limited to concentrating, sterilization and capillus, Bacteroides cellulosolvens, Bacteroides coagulans, deoxygenation. Bacteroides corporis, Bacteroides denticola, Bacteroides 0004. In one aspect are isolation/enrichment systems for disiens, Bacteroides distasonis, Bacteroides eggerthii, selecting bacteria for use in anaerobic hydrogen production, Bacteroides endodontalis, Bacteroides forsythus, Bacteroi wherein the bacterial strains are selected for their ability to des fragilis, Bacteroides fircosus, Bacteroides galacturoni utilize biomass as nutrients and produce hydrogen gas and cus, Bacteroides gingivalis, Bacteroides gracilis, Bacteroi other chemical products. In another aspect is an apparatus for des helcogenes, Bacteroides heparinolyticus, Bacteroides selecting bacteria for use in anaerobic hydrogen production hypermegas, Bacteroides intermedius, Bacteroides levi, comprising a sealed chamber capable of providing an appro Bacteroides loescheii, Bacteroides macacae, Bacteroides priate environment and growing conditions. melaminogenicus, Bacteroides meloninogenicus subsp. 0005. In one aspect is a Knowledge Management System intermedius, Bacteroides melaminogenicus subsp. macaca, used to identify bacteria for use in anaerobic production of Bacteroides melaminogenicus subsp. melaninogenicus, hydrogen from using biomass, wherein bacteria are identified Bacteroides merdae, Bacteroides microfitsus, Bacteroides by collecting appropriate information from various bacterial multiacidus, Bacteroides nodosus, Bacteroides ochraceus, species and deriving a probabilistic model from the informa Bacteroides oralis, Bacteroides Oris, Bacteroides Oulorum, tion. In another aspect is a computer program product to Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides obtain candidate bacterial species for use in the anaerobic pentosaceus, Bacteroides pneumosintes, Bacteroides production of hydrogen and other chemical products from a polypragmatus, Bacteroides praeacutus, Bacteroides putre particular biomass. dinis, Bacteroides pyogenes, Bacteroides ruminicola, 0006. In one aspect are chemical products produced by Bacteroides ruminicola subsp. brevis, Bacteroides rumini anaerobically fermenting biomass with bacteria. In another cola subsp. ruminicola, Bacteroides salivosus, Bacteroides aspectare chemical products formed by admixing hydrogen, splanchnicus, Bacteroides stercoris, Bacteroides succino a starting material and a catalyst, wherein the hydrogen is genes, Bacteroides suis, Bacteroides tectus, Bacteroides ter produced by anaerobically fermenting a biomass with bacte mitidis, Bacteroides thetaiotaOmicron, Bacteroides unifor ria. In another aspectare chemical products formed by admix mis, Bacteroides ureolyticus, Bacteroides veroralis, ing a mineral and a volatile organic acid, wherein the Volatile Bacteroides vulgatus, Bacteroides xylanolyticus, Bacteroides organic acid is produced by anaerobically fermenting a bio zoogleoformans, Bifidobacterium adolescentis, Bifidobacte mass with bacteria. rium angulatum, Bifidobacterium animalis, Bifidobacterium 0007. In one aspect are anaerobic fermentation appara animalis subsp. animalis, Bifidobacterium animalis subsp. tuses which use bacteria for anaerobically fermenting a bio lactis, Bifidobacterium asteroids, Bifidobacterium bifidum, mass into chemical products. In another aspect are assem Bifidobacterium bourn, Bifidobacterium breve, Bifidobacte blages which comprise Sources of Steam; turbines; and rium catenulatum, Bifidobacterium choerinum, Bifidobacte digesters. rium coryneforme, Bifidobacterium cuniculi, Bifidobacte 0008. In one aspect is the use of bacteria for the production rium denticolens, Bifidobacterium dentium, Bifidobacterium of hydrogen and other chemical products. In another aspect gallicum, Bifidobacterium gallinarum, Bifidobacterium glo biomass prepared for use in hydrogen production has been bosum, Bifidobacterium indicum, Bifidobacterium infantis, sterilized, deoxygenated, concentrated, detoxificated, and/or Bifidobacterium inopinatum, Bifidobacterium lactis, Bifido pre-digested while optimal bacterial strains for anaerobic bacterium longum, Bifidobacterium magnum, Bifidobacte US 2008/0311640 A1 Dec. 18, 2008

rium merycicum, Bifidobacterium minimum, Bifidobacterium Leptotrichia Shahi, Leptotrichia trevisani, Leptotrichia pseudocatenulatum, Bifidobacterium pseudolongum, Bifido wadei, Malonomonas rubra, Megamonas hypermegale, Mit bacterium pseudolongum Subsp. globosum, Bifidobacterium suokella dentalis, Mitsuokella jalaludinii, Mitsuokella mult pseudolongum Subsp. pseudolongum, Bifidobacterium psy acida, Oxalobacter formigenes, Oxalobacter vibrioformis, chraerophilum, Bifidobacterium pullorum, Bifidobacterium Pectinatus cerevisiphilus, Pectinatus frisingensis, Pectinatus ruminantium, Bifidobacterium saeculare, Bifidobacterium portalensis, Pelobacter acetylenicus, Pelobacter acidigallici, scardovii, Bifidobacterium subtile, Bifidobacterium suis, Pelobacter carbinolicus, Pelobacter massiliensis, Pelobacte Bifidobacterium thermacidophilum, Bifidobacterium ther rpropionicus, Pelobacter venetianus, Porphyromonas a sac macidophilum Subsp. porcinum, Bifidobacterium thermaci charolytica, Poiphyromonas Cangingivalis, Porphyromonas dophilum Subsp. thermacidophilum, Bifidobacterium ther canoris, Porphyromonas can sulci, Porphyromonas Catoniae, mophilum, Bilophila wadsworthia, Butyrivibrio crossotus, Porphyromonas circumdentaria, Porphyromonas creviorica Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Campylo nis, Poiphyromonas endodontalis, Porphyromonas gingiva bacter butzleri, Campylobacter cinaedi, Campylobacter coli, lis, Porphyromonas gingivicanis, Porphyromonas guilae, Poi Campylobacter concisus, Campylobacter Cryaerophilus, phyromonas levi, Porphyromonas macacae, Porphyromonas Campylobacter curvus, Campylobacterfennelliae, Campylo salivosa, Porphyromonas uenonis, PrevOtella albensis, Pre bacter fetus, Campylobacter fetus Subsp. fetus, Campylo votella bivia, Prevotella brevis, PrevOtella bryantii, Prevo bacter fetus Subsp. venerealis, Campylobacter gracilis, tella buccae, Prevotella buccalis, PrevOtella corporis, Prevo Campylobacter helveticus, Campylobacter horninis, Campy tella dentalis, Prevotella denticola, Prevotella disiens, lobacter hyoilei, Campylobacter hyointestinalis, Campylo Prevotella enoeca, Prevotella heparinolytica, Prevotella bacter hyointestinalis Subsp. hyointestinalis, Campylobacter intermedia, PrevOtella loescheii, Prevotella melamino hyointestinalis Subsp. lawsonii, Campylobacter insulaemi genica, Prevotella multiformis, Prevotella nigrescens, Prevo grae, Campylobacter jejuni, Campylobacter jejuni Subsp. tella oralis, Prevotella Oris, Prevotella Oulorum, Prevotella doylei, Campylobacter jejuni Subsp. jejuni, Campylobacter pollens, Prevotella ruminicola, Prevotella salivae, Prevotella lanienae, Campylobacter lari, Campylobacter mucosalis, Shahi, Prevotella tannerae, Prevotella veroralis, Prevotella Campylobacter mustelae, Campylobacter nitrofigilis, zoogleoformans, Propionibacterium acidipropionici, Propi Campylobacter pylori, Campylobacter pylori Subsp. muste Onibacterium acnes, Propionibacterium australiense, Propi lae, Campylobacter pylori Subsp. pylori, Campylobacter rec Onibacterium avidum, Propionibacterium cyclohexanicum, tus, Campylobacter Showae, Campylobacter sputorum, Propionibacterium feudenreichii, Propionibacterium Campylobacter sputorum subsp. bubulus, Campylobacter freudenreichii subsp. Freudenreichii, Propionibacterium sputorum Subsp. mucosalis, Campylobactersputorum Subsp. feudenreichi Subsp. Shermanii, Propionibacterium granu sputorum, Campylobacter upsaliensis, Catonella morbi, losum, Propionibacterium innocuum, Propionibacterium Centipeda periodontii, Dialister invisus, Dialister pneumos jensenii, Propionibacterium lymphophilum, Propionibacte intes, Dichelobacter nodosus, Fervidobacterium gondwan rium microaerophilum, Propionibacterium propionicum, ens, Fervidobacterium islandicum, Fervidobacterium Propionibacterium thoenii, Propionigenium maris, Propi nodosum, Fervidobacterium pennivorans, Fibrobacter intes Onigenium modestum, Propionispira arboris, Rikenella tinalis, Fibrobacter succinogenes, Fibrobacter succinogenes microfitsus, Roseburia Cecicola, Roseburia intestinalis, Subsp. Elongatus, Fibrobacter succinogenes Subsp. succino Ruminobacter amylophilus, Sebaldella termitidis, Selenomo genes, Fusobacterium alocis, Fusobacterium canifelinum, nas acidaminovorans, Selenomonas artemidis, Selenomonas Fusobacterium equinum, Fusobacterium gonidiaformans, dianae, Selenomonas flueggei, Selenomonas infelix, Seleno Fusobacterium mortiferum, Fusobacterium naviforme, monas lacticifex, Selenomonas lipolytica, Selenomonas Fusobacterium necrogenes, Fusobacterium necrophorum, noxia, Setenomonas ruminantium, Selenormonas ruminan Fusobacterium necrophorum Subsp. funduliforme, Fusobac tium Subsp. lactilytica, Selenomonas ruminantium Subsp. terium necrophorum Subsp. necrophorum, Fusobacterium ruminantium, Selenomonas sputigena, Sporomusa aci nucleatum, Fusobacterium nucleaturn Subsp. animalis, dovorans, Sporomusa aerivorans, Sporomusa malonica, Fusobacterium nucleatum Subsp. fusiforme, Fusobacterium Sporomusa ovata, Sporomusa paucivorans, Sporomusa sil nucleatum Subsp. nucleatum, Fusobacterium nucleatum vacetica, Sporomusa sphaeroides, Sporomusa termitida, Suc Subsp. polymorphum, Fusobacterium nucleatum Subsp. vin cinimonas amylolytica, Sitccinivibrio dextrinosolvens, Syn centii, Fusobacterium perfoetens, Fusobacterium periodon trophobacterfiumaroxidans, Syntrophobacter pfennigii, ticum, Fusobacterium plauti, Fusobacterium polysaccharo Syntrophobacter wolinii, Syntrophomonas curvata, Syntro lyticum, Fusobacterium prausnitzii, Fusobacterium phomonas erecta, Syntrophomonas Sapovorans, Syntroph pseudonecrophorum, Fusobacterium russii, Fusobacterium Omonas wolfei, Syntrophomonas wolfei, Sutterella Stercori simiae, Fusobacterium sulci, Fusobacterium ulcerans, Fuso Canis, Sutterella Wadsworthensi, Saponavida, bacterium varium, Halanaerobacter chitinivorans, Hala Thermobacteroides acetoethylicus, Thermobacteroides lep naerobacter lacunarum, Halanaerobacter salinarius, Hala to spartum, Thermobacteroides proteolyticus, Thermosipho naerobium acetethylicum, Halanaerobium alcaliphilum, africanus, Thermosipho atlanticus, Thermosipho geolei, Halanaerobium congolense, Halanaerobium fermentans, Thermosiphojaponicus, Thermosipho melanesiensis, Ther Halanaerobium kushneri, Halanaerobium lacusrosei, Hala motoga elfi, Thermotoga hypogea, Thermotoga lettingae, naerobium praevalens, Halanaerobium saccharolyticum, Thermotoga maritima, Thermotoga naphthophila, Thermo Halanaerobium saccharolyticum Subsp. Saccharolyticum, toga neapolitana, Thermotoga petrophila, Thermotoga Sub Halanaerobium saccharolyticum Subsp. Senegalense, Hala terranea, Thermotoga thermarum, Tissierella creatinini, Tis naerobium salsuginis, Ilyobacter delafieldii, Ilvobacter sierella creatinophila, Tissierella praeacuta, Wolinella curva, insuetus, Ilvobacter polytropus, Ilvobacter tartaricus, Wolinella recta, Wolinella succinogenes, Zymophilus pau Johnsonella ignava, Lachnobacterium bovis, Leptotrichia civorans, Zymophilus raffinosivorans, Desulfobacter curva buccalis, Leptotrichia goodfellowii, Leptotrichia hofstadii, tus, Desulfobacter halotolerans, Desulfobacter hydrogeno US 2008/0311640 A1 Dec. 18, 2008 philus, Desulfobacter latus, Desulfobacterpostgatei, scens Subsp. Criceti, Veillonella alcalescens Subsp. dispar, Desulfobacter vibrioformis, Desulfobacterium anilini, Des Veillonella alcalescens Subsp. ratti, Veillonella atypica, Veil ulfobacterium autotrophicum, Desulfobacterium catecholi lonella caviae, Veillonella Criceti, Veillonella dispar, Veil cum, Desulfobacterium cetonicum, Desulfobacterium indoli lonella montpellierensis, Veillonella parvula, Veillonella par cum, Desulfobacterium macestii, Desulfobacterium vula Subsp. atypical, Veillonella parvula Subsp. parvula, phenolicum, Desulfobulbus elongatus, Desulfobulbus medi Veillonella parvula subsp. rodentium, Veillonella ratti, Veil terraneus, Desulfobulbus propionicus, Desulfobulbus lonella rodentium, Coprococcus catu, Coprococcus comes, rhabdoformis, Desulfococcus biacutus, Desulfococcus mul Coprococcus eutactus, Peptococcus asaccharolyticus, Pepto tivorans, Desulfomicrobium apsheronium, Desulfomicrobium coccus glycinophilus, Peptococcus heliotrinreducens, Pepto baculatum, Desulfomicrobium escambiense, Desulfomicro coccus indolicus, Peptococcus magnus, Peptococcus niger, bium macestii, Desulfomicrobium norvegicum, Desulfomi Peptococcus prevotii, Peptococcus saccharolyticus, Pep crobium oralem, Desulfomonas pigra, Desulfomonile limi to streptococcus anaerobius, Peptostreptococcus asaccharo maris, Desulfomonile tiedjei, Desulfonema ishimotonii, lyticus, Peptostreptococcus barnesae, Peptostreptococcus Desulfonema limicola, Desulfonema magnum, Desulfosa harei, Peptostreptococcus heliotrinreducens, Peptostrepto rcina variabilis, Desulfotomaculum acetoxidans, Desulfoto coccus hydrogenalis, Peptostreptococcus indolicus, Pep maculum aeronauticum, Desulfotomaculum alkaliphilum, to streptococcus ivorii, Peptostreptococcus lacrimalis, Pep Desulfotomaculum antarcticum, Desulfotomaculum auripig to streptococcus lactolyticus, Peptostreptococcus magnus, mentum, Desulfotomaculum australicum, Desulfotomaculum Peptostreptococcus micros, Peptostreptococcus octavius, geothermicum, Desulfotomaculum gibsoniae, Desulfoto Peptostreptococcus parvulus, Peptostreptococcus previotii, maculum guttoideum, Desulfotomaculum halophilum, Des Peptostreptococcus productus, Peptostreptococcus tetradius, ulfotomaculum kuznetsovii, Desulfotomaculum luciae, Des Peptostreptococcus vaginalis, Ruminococcus albus, Rumino ulfotomaculum nigrificans, Desulfotomaculum Orientis, coccus bromii, Ruminococcus callidus, Ruminococcus flave Desulfotomaculum putei, Desulfotomaculum ruminis, Des faciens, Ruminococcus gnavus, Ruminococcus hansenii, ulfotomaculum sapomandens, Desulfotomaculum solfatari Ruminococcus hydrogenotrophicus, Ruminococcus lactaris, cum, Desulfotomaculum thermoacetoxidans, Desulfoto Ruminococcus luti, Ruminococcus obeum, Ruminococcus maculum thermobenzoicum, Desulfotomaculum palustris, Ruminococcus pasteurii, Ruminococcus produc thermobenzoicum Subsp. thermobenzoicum, Desulfotomacu tus, Ruminococcus schinki, Ruminococcus torques, Sarcina lum thermobenzoicum Subsp. thermosyntrophicum, Desulfo maxima, Sarcina ventriculi, Clostridium absonium, tomaculum thermocisternum, Desulfotomaculum thermosa Clostridium aceticum, Clostridium acetireducens, povorans, Desulfovibrio acrylicus, Desulfovibrio Clostridium acetobutylicum, Clostridium acidisoli, aespoeensis, Desulfo vibrio africanus, Desulfovibrio Clostridium acidurici, Clostridium aerotolerans, alaskensis, Desulfo vibrio alcoholivorans, Desulfovibrio ami Clostridium akagii, Clostridium aldrichii, Clostridium nophilus, Desulfovibrio baarsii, Desidfovibrio baculatus, algidicarnis, Clostridium algidiyylanolyticum, Clostridium Desulfovibrio bastinii, Desulfovibrio burkinensis, Des aminophilum, Clostridium aminovalericum, Clostridium ulfovibrio carbinolicus, Desulfovibrio cuneatus, Des amygdalinum, Clostridium arcticum, Clostridium argentin ulfovibrio dechloracetivorans, Desulfovibrio desulfuricans, ense, Clostridium aurantibutyricum, Clostridium baratii, Desulfovibrio desulfuricans Subsp. aestuarii, Desulfovibrio Clostridium barkeri, Clostridium bartlettii, Clostridium desulfuricans Subsp. desulfuricans, Desulfovibrio fructo beijerinckii, Clostridium bifermentans, Clostridium bolteae, Sivorans, Desulfo vibriofurfuralis, Desulfovibriogabonensis, Clostridium botulinum, Clostridium bowmanii, Clostridium Desulfovibrio giganteus, Desulfo vibrio gigas, Desidfovibrio bryantii, Clostridium butyricum, Clostridium cadaveris, gracilis, Desulfovibrio halophilus, Desulfo vibrio hydrother Clostridium caninithermale, Clostridium carnis, malis, Desulfovibrio indonesiensis, Desulfovibrio inopina Clostridium cellatum, Clostridium celerecrescens, tus, Desidfovibrio intestinalis, Desulfovibrio litoralis, Des Clostridium cellobioparum, Clostridium cellulofermentans, ulfovibrio longreachensis, Desulfovibrio longus, Clostridium cellulolyticum, Clostridium cellulose, Desulfovibrio magneticus, Desulfovibrio mexicanus, Des Clostridium cellulovorans, Clostridium chartatabidum, ulfovibrio Oxyclinae, Desulfovibrio piger; Desulfovibrio pro Clostridium chauvoei, Clostridium clostridioforme, findus, Desulfo vibrio putealis, Desulfovibrio salexigens, Clostridium coccoides, Clostridium cochlearium, Desulfovibrio Sapovorans, Desulfo vibrio Senezii, Des Clostridium cocleatum, Clostridium colicanis, Clostridium ulfovibrio simplex, Desulfovibrio sulfadismutans, Des colinum, Clostridium collagenovorans, Clostridium cylin ulfovibrio termitidis, Desulfovibrio thermophilus, Des drosporum, Clostridium difficile, Clostridium dioli, ulfovibrio vietnamensis, Desulfovibrio vulgaris, Clostridium disporicum, Clostridium durum, Clostridium Desulfovibrio vulgaris Subsp. oxamicus, Desulfovibrio vul estertheticum, Clostridium estertheticum subsp. estertheti garis Subsp. vulgaris, Desulfovibrio Zosterae, Desulfurella cum, Clostridium estertheticum Subsp. laramiense, acetivorans, Desulfitrella kamchalkensis, Desulfurella multi Clostridium fallax, Clostridium felsineum, Clostridium fervi potens, Desulfurella propionica, Desulfuromonas acetexi dum, Clostridium fimetarium, Clostridium formicaceticum, gens, Desulfuromonas acetoxidans, Desulfuromonas chloro Clostridium frigidicarnis, Clostridium frigoris, Clostridium ethenica, Desulfuromonas paimitatis, Desulfuromonas gasigenes, Clostridium ghonii, Clostridium glycolicum, tkiophila, Thermodesulfobacterium commune, Thermodes Clostridium grantii, Clostridium haemolyticum, Clostridium ulfobacterium hyeragerdense, Thermodesulfobacterium halophilum, Clostridium hastiforme, Clostridium hathewayi, hydrogeniphilum, Thermodesulfobacterium thermophilum, Clostridium herbivorans, Clostridium hiranonis, Acidaminococcus fermentans, Megasphaera cerevisiae, Clostridium histolyticum, Clostridium homopropionicum, Megasphaera elsdenii, Megasphaera micronuciformis Syn Clostridium hungatei, Clostridium hydroxybenzoicum, trophococcus sucromutans, Veillonella alcalescens, Veil Clostridium hylemonae, Clostridium jejuense, Clostridium lonella alcalescens Subsp. alcalescens, Veillonella alcale indolis, Clostridium innocuum, Clostridium intestinale, US 2008/0311640 A1 Dec. 18, 2008

Clostridium irregulare, Clostridium isatidis, Clostridium marismortui, Ectothiorhodospira mobilis, Ectothiorho josui, Clostidium kluyveri, Clostridium lactatifermentans, dospira Shaposhnikovii, Ectothiorhodospira vacuolata, Clostridium lacusfiyxellense, Clostridium laramiense, Rhodobacter adriaticus, Rhodobacter azotoformans, Rhodo Clostridium lentocellum, Clostridium lentoputrescen, bacter blasticus, Rhodobacter capsulatus, Rhodobacter Clostridium leptum, Clostridium limosum, Clostridium lito euryhalinus, Rhodobacter sphaeroides, Rhodobacter sidfido rale, Clostridium lituseburense, Clostridium ljungdahli, philus, Rhodobacter veldkampii, Rhodocyclus gelatinosus, Clostridium lortetii, Clostridium magnum, Clostridium Rhodocyclus purpureus, Rhodocyclus tenuis, Rhodomicro malenominatum, Clostridium mangenotii, Clostridium may bium vannieli, Rhodopilla globiformis, Rhodopseudomonas ombei, Clostridium methoxybenzovorans, Clostridium meth acidophila, Rhodopseudomonas adriatica, Rhodopseudomo ylpentosum, Clostridium neopropionicum, Clostridium next FCS blastica, Rhodopseudomonas capsulata, ille, Clostridium novyi, Clostridium oceanicum, Clostridium Rhodopseudomonas faecalis, Rhodopseudomonas gelati Orbiscindens, Clostridium Oroticum, Clostridium oxalicum, nosa, Rhodopseudomonas globiformis, Rhodopseudomonas Clostridium papyrosolvens, Clostridium paradoxum, Julia, Rhodopseudomonas marina, Rhodopseudomonas Clostridium paraperfringens, Clostridium paraputrificum, palustris, Rhodopseudomonas rhenobacensis, Clostridium pasculi, Clostridium pasteurianum, Clostridium Rhodopseudomonas rosea, Rhodopseudomonas rutila, peptidivorans, Clostridium perenne, Clostridium perfirin Rhodopseudomonas sphaeroides, Rhodopseudomonas sulfi gens, Clostridium pfennigii, Clostridium phytofermentans, dophila, Rhodopseudomonas sulfoviridis, Rhodopseudomo Clostridium piliforme, Clostridium polysaccharolyticum, nas viridis, Rhodospirillum centenum, Rhodospirillum full Clostridium populeti, Clostridium propionicum, Clostridium vum, Rhodospirillum molischianum, Rhodospirillum proteoclasticum, Clostidium proteolyticum, Clostridium photometricum, Rhodospirillum rubrum, Rhodospirillum psychrophilum, Clostridium puniceum, Clostridium purini salexigens, Rhodospirillum salinarum, Rhodospirillum Sodo lyticum, Clostridium putrefaciens, Clostridium putrificum, mense, Rhodospirillum tenue, Erythrobacter aquimaris, Clostridium quercicolum, Clostridium quinii, Clostridium Erythrobacter citreus, Erythrobacterflavus, Erythrobacter ramosum, Clostridium rectum, Clostridium roseum, gaetbuli, Erythrobacter litoralis, Erythrobacter longus, Clostridium saccharobutylicum, Clostidium saccharolyti Erythrobacter Seohaensis, Methanobacterium aarhusense, cum, Clostridium saccharoperbutylacetonicum, Clostridium Methanobacterium alcaliphilum, Methanobacterium arbo Sardiniense, Clostridium Sartagoforme, Clostridium scatolo philicum, Methanobacterium beijingense, Methanebacte genes, Clostridium scindens, Clostridium septicum, rium bryantii, Methanobacterium Congolense, Methanobac Clostridium sordellii, Clostridium sphenoides, Clostridium terium defluvii, Methanobacterium espanolae, spiroforme, Clostridium sporogenes, Clostridium Methanobacterium formicicum, Methanobacterium ivanovii, sporosphaeroides, Clostridium Stercorarium, Clostridium Methanobacterium mobile, Methanobacterium Oryzae, Stercorarium Subsp. leptospartum, Clostridium Stercorarium Methanobacterium ruminantium, Methanobacterium subter Subsp. Stercorarium, Clostridium stercorarium Subsp. ther raneum, Methanobacterium thermaggregains, Methanobac molacticum, Clostridium Sticklandii, Clostridium Stramini terium thermalcaliphilum, Methanobacterium thermau solvens, Clostridium subterminale, Clostridium symbiosum, totrophicum, Methanobacterium thermoflexum, Clostridium termitidis, Clostridium tertium, Clostridium Methanobacterium thermoformicicum, Methanobacterium tetani, Clostridium tetanomorphum, Clostridium thermace thermophilum, Methanobacterium uliginosum, Methanobac ticum, Clostridium thermautotrophicum, Clostridium ther terium wolfei, Methanobrevibacter acididurans, Methano moalcaliphilum, Clostridium thermobutyricum, Clostridium brevibacter arboriphilus, Methanobrevibacter curvatus, thermocellum, Clostridium thermocopriae, Clostridium ther Methanobrevibacter cuticularis, Methanobrevibacter filifor mohydrosulfuricum, Clostridium thermolacticum, Closti mis, Methanobrevibacter gottschalkii, Methanobrevibacter idium thermopalmarium, Clostridium thermopapyrolyticum, oralis, Methanobrevibacter ruminantium, Methanobrevi Clostridium thermosuccinogenes, Clostidium thermosulfil bacter Smithii, Methanobrevibacter thaueri, Methanobrevi rigenes, Clostridium thiosulfatireducens, Clostridium bacter woesei, Methanobrevibacter wolinii, Methanococcus tyrobutyricum, Clostridium uliginosum, Clostridium ultun delta, Methanococcus fervens, Methanococcus frisius, ense, Clostridium villosum, Clostridium vincentii, Methanococcus halophilus, Methanococcus igneus, Metha Clostridium viride, Clostridium Xylanolyticum, Clostridium nococcus infernus, Methanococcus jannaschii, Methanococ xylanovorans, Amoebobacterpedioformis, Amoebobacter cus maripaludis, Methanococcus mazei, Methanococcus pendens, Amoebobacterpurpureus, Amoebobacter roseus, thermolithotrophicus, Methanococcus vannieli, Methano Chromatium buderi, Chromatium glycolicum, Chromatium coccus voltae, Methanococcus vulcanius, Methanococcoides gracile, Chromatium minus, Chromatium minutissimum, burtonii, Methanococcoides methylutens, Methanolobus Chromatium Okenii, Chromatium purpuratum, Chromatium bombayensis, Methanolobus Oregonensis, Methanolobus salexigens, Chromatium tepidum, Chromatium vinosum, Siciliae, Methanolobus taylorii, Methanolobus tindarius, Chromatium violascens, Chromatium warmingii, Chroma Methanolobus vulcani, Methanolacinia paynteri, Metha tium weissei, Lamprobacter modestohalophilus, Lamprocys nomicrobium mobile, Methanomicrobium paynteri, Metha tis purpurea, Lamprocystis roseopersicina, Thiocapsa halo nogenium aggregains, Methanogenium bourgense, Meihano phila, Thiocapsa litoralis, Thiocapsa marina, Thiocapsa genium cariaci, Methanogenium frigidium, Methanogenium penden, Thiocapsa rosea, Thiocapsa roseopersicina, Thio frittonii, Methanogenium liminatans, Methanogenium mari cystis gelatinosa, Thiocystis minor. Thiocystis violacea, Thio num, Methanogenium marisnigri, Methanogenium Olen cystis violascens, Thiodictyon bacillosum, Thiodictyon tangyi, Methanogenium Organophilum, Methanogenium elegans, Thiopedia rosea, Thiospirillum jenense, Ectothior tationis, Methanogenium thermophilicum, Methanospirillum hodospira abdelmalekii, Ectothiorhodospira haloalka hungatei, Methanoplanus endosymbiosus, Methanoplanus liphila, Ectothiorhodospira halochloris, Ectothiorhodospira limicola, Methanoplanus petrolearius, Methanothrix con halophila, Ectothiorhodospira marina, Ectothiorhodospira cilii, Methanothrix soehngenii, Methanothrix thermoaceto US 2008/0311640 A1 Dec. 18, 2008

phila, Methanothrix thermophila, Methanothermus fervidus, cellobiosus, Lactobacillus coleohominis, Lactobacillus col Methanothermus sociabilis, Methanocorpusculum linoide, Lactobacillus confuses, Lactobacillus coryniformis, aggregains, Methanocorpusculum bavaricum, Methanocor Lactobacillus coivniformis Subsp. coryniformis, Lactobacil pusculum labreanum, Methanocorpusculum parvum, Metha lus coryniformis Subsp. torquens, Lactobacillus crispatus, nocorpusculum sinense, Methanoculleus bourgensis, Metha Lactobacillus curvatus, Lactobacillus curvatus Subsp. curva noculleus chikugoensis, Methanoculleus marisnigri, tus, Lactobacillus curvatus Subsp. melibiosus, Lactobacillus Methanoculleus oldenburgensis, Methanoculleus Olentangyi, Cypricasei, Lactobacillus delbrueckii, Lactobacillus del Methanoculleus palmolei, Methanoculleus submarinus, brueckii Subsp. bulgaricus, Lactobacillus delbrueckii Subsp. Methanoculleus thermophilus, Methanohalobium evestiga delbrueckii, Lactobacillus delbrueckii Subsp. indicus, Lacto tum, Methanohalophilus halophilus, Methanohalophilus bacillus delbrueckii Subsp. lactis, Lactobacillus diolivorans, mahi, Methanohalophilus Oregonensis, Methanohalophilus Lactobacillus divergens, Lactobacillus durianis, Lactobacil portucalensis, Methanohalophilus Zhilinae, Methanosarcina lus equi, Lactobacillus farciminis, Lactobacillus ferintoshen acetivorans, Methanosarcina baltica, Methanosarcina bark sis, Lactobacillus fermentuni, Lactobacillus formicalis, Lac eri, Methanosarcina frisia, Methanosarcina lacustris, tobacillus fructivorans, Lactobacillus fructosus, Methanosarcina methanica, Methanosarcina semesia, Lactobacillus frumenti, Lactobacillus fitchuensis, Lactoba Methanosarcina Siciliae, Methanosarcina thermophila, cillus gallinarum, Lactobacillus gasseri, Lactobacillus gas Methanosarcina vacuolata, Methanosphaera cuniculi, tricus, Lactobacillus gramini, Lactobacillus halotolerans, Methanosphaera stadtmanae, Eubacterium acidaminophi Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacil lum, Eubacterium aerofaciens, Eubacterium aggregans, lus helveticus, Lactobacillus heterohiochi, Lactobacillus hil Eubacterium alactolyticum, Eubacterium angustum, Eubac gardii, Lactobacillus homohiochii, Lactobacillus iners, Lac terium barkeri, Eubacterium biforme, Eubacterium brachy, tobacillusinglu viei, Lactobacillus intestinalis, Lactobacillus Eubacterium budayi, Eubacterium callanderi, Eubacterium jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, cellulosolvens, Eubacterium combesii, Eubacterium contor Lactobacillus kandleri, Lactobacillus kefiranofaciens, Lac tum, Eubacterium coprostanoligenes, Eubacterium cylin tobacillus kefiranofaciens subsp. kefiranofaciens, Lactoba droids, Eubacterium desmolans, Eubacterium dolichum, cillus kefiranofaciens subsp. kefirgranum, Lactobacillus Eubacterium eligens, Eubacterium exiguum, Eubacterium kefirgranum, Lactobacillus kefir, Lactobacillus kimchi, Lac fissicatena, Eubacterium formicigenerans, Eubacterium fos tobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus Sor, Eubacterium hadrum, Eubacterium hallii, Eubacterium lactis, Lactobacillus leichmannii, Lactobacillus lindneri, infinnum, Eubacterium lentum, Eubacterium limosum, Lactobacillus malefermentans, Lactobacillus mali, Lactoba Eubacterium minutum, Eubacterium moniliforme, Eubacte cillus maltaronicus, Lactobacillus manihotivorans, Lacto rium multiforme, Eubacterium nitritogenes, Eubacterium bacillus mindensis, Lactobacillus minor; Lactobacillus modatum, Eubacterium oxidoreducens, Eubacterium plautii, mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lac Eubacterium plexicaudatum, Eubacterium pyruvativorans, tobacillus Oris, Lactobacillus panis, Lactobacillus pantheris, Eubacterium ramulus, Eubacterium rectale, Eubacterium Lactobacillus parabuchneri, Lactobacillus paracasei, Lacto ruminantium, Eubacterium saburreum, Eubacterium saphe bacillus paracasei Subsp. paracasei, Lactobacillus paracasei num, Eubacterium Siraeum, Eubacterium suis, Eubacterium Subsp. tolerans, Lactobacillus paracollinoides, Lactobacil sulci, Eubacterium tarantellae, Eubacterium tardum, Eubac lus parakefiri, Lactobacillus parallimentarius, Lactobacillus terium tenue, Eubacterium timidum, Eubacterium tortuosum, paraplantarum, Lactobacillus pentosus, Lactobacillus pero Eubacterium uniforme, Eubacterium ventriosum, Eubacte lens, Lactobacillus piscicola, Lactobacillus plantarum, Lac rium xylamophilum, Eubacterium yuri, Eubacterium yuri tobacillus pontis, Lactobacillus psittaci, Lactobacillus reu Subsp. margaretiae, Eubacterium yuri Subsp. schtitka, teri, Lactobacillus rhamnosus, Lactobacillus rimae, Eubacterium yuri Subsp. Yurii, Abiotrophia adiacens, Lactobacillus rogosae, Lactobacillus rossii, Lactobacillus Abiotrophia balaetiopterae, Abiotrophia defectiva, Abiotro ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lac phia elegans, Atopobium fossor, Atopobium minutum, Atopo tobacillus sakei Subsp. carnosus, Lactobacillus sakei Subsp. bium parvulum, Atopobium rimae, Atopobium vaginae, sakei, Lactobacillus salivarius, Lactobacillus salivarius Gemella bergeri, Gemella cuniculi, Gemella haemolysans, Subsp. salicinius, Lactobacillus salivarius Subsp. salivarius, Gemella morbillorum, Gemella palaticanis, Gemella San Lactobacillus Sanfranciscensis, Lactobacillus satsumensis, guinis, Granulicatella adiacens, Granulicatella bal Lactobacillus sharpeae, Lactobacillus spicheri, Lactobacil aenopterae, Granulicatella elegans, Finegoldia magna, Lac lus Suebicus, Lactobacillus suntoryeus, Lactobacillus ther tobacillus acetotolerans, Lactobacillus acidifarinae, motolerans, Lactobacillus trichodes, Lactobacillus uli, Lac Lactobacillus acidipiscis, Lactobacillus acidophilus, Lacto tobacillus ultunensis, Lactobacillus vaccinostercus, bacillus acidophilus, Lactobacillus agilis, Lactobacillus Lactobacillus vaginalis, Lactobacillus versmoldensis, Lacto algidus, Lactobacillus alimentarius, Lactobacillus amy bacillus viridescens, Lactobacillus vitulinu, Lactobacillus lolyticus, Lactobacillus amylophilus, Lactobacillus amylo xylosus, Lactobacillus vananashiensis, Lactobacillus yama vorus, Lactobacillus animalis, Lactobacillus antri, Lactoba nashiensis Subsp. mali, Lactobacillus vananashiensis Subsp. cillus arizonensis, Lactobacillus aviarius, Lactobacillus yamanashiensis, Lactobacillus zeae, Lactobacillus zymae, aviarius Subsp. Ardfinosus, Lactobacillus aviarius Subsp. Actinomyces bernardiae, Actinomyces bovis, Actinomyces Aviarius, Lactobacillus bavaricus, Lactobacillus bifermen bowdenii, Actinomyces canis, Actinomyces cardiffensis, Acti tans, Lactobacillus brevis, Lactobacillus buchneri, Lactoba nomyces catuli, Actinomyces Coleocanis, Actinomyces denta cillus bulgaricus, Lactobacillus carnis, Lactobacillus casei, lis, Actinomyces denticolens, Actinomyces europaeus, Acti Lactobacillus casei Subsp. alactosus, Lactobacillus casei nomyces funkei, Actinomyces Georgia, Actinomyces Subsp. casei, Lactobacillus casei Subsp. pseudoplantarum, gerencseriae, Actinomyces graevenitzii, Actinomyces Lactobacillus casei Subsp. rhamnosus, Lactobacillus casei hongkongensis, Actinomyces hordeovulneris, Actinomyces Subsp. tolerans, Lactobacillus catenaformis, Lactobacillus howellii, Actinomyces humiferus, Actinomyces hyovaginalis, US 2008/0311640 A1 Dec. 18, 2008

Actinomyces israelii, Actinomyces marinammalium, Actino production and processing facilities, animal waste from Zoos, myces meyeri, Actinomyces naeslundii, Actinomyces nasi waste from fruit processing industries, waste from pulp and cola, Actinomyces neuii, Actinomyces neuii Subsp. anitratus, paper mills, Silvaculture residues, waste from wood process Actinomyces neuii Subsp. neuii, Actinomyces Odontolyticus, ing, waste from agricultural product processing, food waste, Actinomyces Oricola, Actinomyces pyogenes, Actinomyces Solids isolated from fermentation cultures, municipal sewer pyogenes, Actinomyces radicidentis, Actinomyces radingae, waste, animal manure, animal urine, animal parts, fish parts, Actinomyces slackii, Actinomyces suinastitidis, Actinomyces and combinations thereof. In further or alternative embodi suis, Actinomyces turicensis, Actinomyces urogenitalis, Acti ments of all aspects utilizing a biomass, the biomass may be nomyces vaccinaxillae, Actinomyces viscosus, Arcanobacte material Such as, but not limited to, glucose, beet Sugar, Sugar rium bernardiae, Arcanobacterium haemolyticum, Arcano bacterium hippocoleae, Arcanobacterium phocae, beet molasses, Sugar beet syrup, Sugar beetjuice, Sugar cane Arcanobacterium pluranimalium, Arcanobacterium pyo molasses, cane Sugar, Sugarcane Syrup, Sugarcane juice, corn genes, Actinobaculum Schaali, Actinobaculum suis, Actino syrup, cereal grains, oat flour, rice flour, corn flour, wheat baculum urinale, Bulleidia extructa, Collinsella aerofaciens, flour, potatoes, tomatoes, potato juice, tomato pulp, tomato Collinsella intestinalis, Collinsella stercoris, Cryptobacte juice, potato pulp, cheese whey, Sorghum, corn mash, wheat rium curtum, Holdemania filiformis, Rothia aeria, Rothia mash, oat mash, blackstrap molasses, citrus molasses, invert amarae, Rothia dentocariosa, Rothia mucilaginosa, Rothia Sugar, Sucrose, fructose, glucose, wood Sugar, cellulose, nasimurium, Pseudoramibacter alactolyticus, Mogibacte Xylose, plant parts, fruit, vegetable, bovine manure, poultry rium diversum, Mogibacterium neglectum, Mogibacterium manure, equine manure, porcine manure, bovine urine, poul pumilum, Mogibacterium timidum, Mogibacterium vescum, try urine, equine urine, porcine urine, wood shavings, wood Slackia exigua, Slackia heliotrinireducens, and Eggerthella chips, shredded paper, cotton burrs, grain, chaff, seed shells, lenta (hereinafter referred to as “anaerobic bacteria' or a hay, alfalfa, grass, leaves, seed pods, corn shucks, weeds, similar term). aquatic plants, algae, fungus, and combinations thereof. 0010. In further or alternative embodiments of all aspects 0014. In further or alternative embodiments of all aspects, using Substantially purified anaerobic bacterial strains, the the processes of anaerobically fermenting biomass are con bacteria may be collected from bovine rumen, Soil samples, tinuous processes. In still further or alternative embodiments sludge, anaerobic bacteria cultures, aerobic bacteria cultures, of all aspects, the processes of anaerobically fermenting bio anaerobic sediments from fresh or brackish waters, sewage, animals, animal feces, insect digestive tract, dental samples, mass are batch processes. hydrothermal soils, hydrothermal pools and deep water hydrothermal vents. Chemical Products and Uses of Chemical Products 0011. In further or alternative embodiments of all aspects 0015. In further or alternative embodiments of all aspects using Substantially purified anaerobic bacterial strains, the in which chemical products are produced by anaerobically bacteria may be obtained by genetic modification of known bactial strains. In further or alternative embodiments of all fermenting biomass, the chemical products may be gaseous, aspects using Substantially purified anaerobic bacterial non-gaseous, or combinations thereof. In further or alterna strains, the genetic modification results from transformation tive embodiments of such aspects, the non gaseous chemical procedures, bacterial conjugation, transduction, interaction products may be solids, solvents, Volatile organic acids, salts of bacterial strains with mutagens, and combinations thereof. of volatile organic acids, or combinations thereof. In further In further or alternative embodiments, the mutagens include, or alternative embodiments of Such aspects, the gaseous but not limited to, chemicals, ultraviolet light, and radioactive chemical products may be hydrogen, carbon dioxide, carbon elements. monoxide, methane, hydrogen Sulfide, ammonia, nitrogen, and combinations thereof. In further or alternative embodi 0012. In further or alternative embodiments of all aspects ments of such aspects, the gaseous chemical products may be using Substantially purified anaerobic bacteria cultures, the hydrogen, carbon dioxide, carbon monoxide, hydrogen Sul Substantially purified anaerobic bacteria cultures incorporate, fide, ammonia, nitrogen, and combinations thereof. In further along with appropriate growth media, Substantially purified or alternative embodiments of all aspects, the solid chemical single bacterial strains. In further or alternative embodiments products may be solids which comprises Sulfur, and in further of all aspects utilizing Substantially purified anaerobic bacte or alternative embodiments of such aspects, the Solid chemi rial strains, the substantially purified anaerobic bacterial cal products may be elemental sulfur. In still further or alter strains are part of inoculants. In further or alternative embodi native embodiments of Such aspects, the chemical products ments of all aspects using Substantially purified anaerobic may be solvents, such as, but not limited to, acetone, butanol, bacterial strains, the substantially purified single bacterial propanol, isopropanol, 1,2-propanediol, ethanol, methanol, strains are at least 95% purified. In still further or alternative and combinations thereof. In further or alternative embodi embodiments of all aspects using Substantially purified ments of Such aspects, the chemical products are volatile anaerobic bacterial strains, the Substantially purified single organic acids, such as, but not limited to formic acid, acetic bacterial strains are at least 99% purified. In even further or acid, propionic acid, butyric acid, Valeric acid, and combina alternative embodiments of all aspects using Substantially tions thereof. In further or alternative embodiments of such purified anaerobic bacterial strains, the substantially purified aspects, the chemical products are salts of Volatile organic single bacterial strains are at least 99.5% purified. acids in which the anion include, but are not limited to, Biomass formate, acetate, propionate, butyrate, Valerate, and combi nations thereof. In further or alternative embodiments of such 0013. In further or alternative embodiments of all aspects aspects, the chemical products are salts of the Volatile organic using biomass, the biomass comprises material obtained from acids the cations may alkali metal ions, alkaline earth ions, energy crops, Surplus agricultural products, waste from Sugar ammonium ion, or combinations thereof. In still further or US 2008/0311640 A1 Dec. 18, 2008

alternative embodiments of Such aspects, cations of the salts energy sources is methane. In further or alternative embodi of volatile organic acids may be Na", K", Ca", Mg", NH, ments, the power generation system may be fuel cells, inter and combinations thereof. nal combustion generators, turbine generators, and combina 0016. In one aspect are compositions which include at tions thereof. In further or alternative embodiments, the least one chemical product produced by anaerobically fer power generation systems are part of power stations. In fur menting biomass using Substantially purified anaerobic bac ther or alternative embodiments, the power stations are teria cultures. In an embodiment of this aspect, the Substan decentralized power stations. In further or alternative tially purified anaerobic bacteria cultures have not been embodiments, the fuel cells may molten carbonate fuel cells, Subjected to heatshocking processes, and in further or alter solid oxide fuel cells, and combinations thereof. In further or native embodiments, the substantially purified anaerobic bac alternative embodiments, the fuel cells are used to generate teria cultures are used as inoculants in anaerobic fermentation electricity for residential consumption, commercial con apparatuses. Sumption, motor-vehicle consumption, or combinations 0017. In another aspectare feedstocks for chemical indus thereof. In still further or alternative embodiments, the elec tries which are chemical products produced by anaerobically tricity generated by such fuel cells may be used locally, added fermenting biomass. In an embodiment of this aspect, the to the grid, or combinations thereof. In further or alternative chemical industries utilizing such feedstocks include poly embodiments, the turbine generators are used to generate mer industries, industrial synthesis industries, photographic electricity from steam created by heating water, wherein the industries, coatings industries, fertilizer industries, printing heat is generated by combustion of Such compositions com industries, and combinations thereof. prising chemical products produced by anaerobically fer 0018. In another aspect are energy sources for a power menting biomass. In further or alternative embodiments, the generation systems which are gaseous chemical products and electricity generated by such turbines may be used for resi Solvents produced by anaerobically fermenting biomass. In dential consumption or commercial consumption. In still fur an embodiment of this aspect, the power generation systems ther or alternative embodiments, the electricity generated by may be fuel cells, internal combustion generators, turbine Such turbines may be used locally, added to the grid, or generators, Stirling engines, and combinations thereof. In combinations thereof. In further or alternative embodiments, further oralternative embodiments, the power generation sys the chemical product may be used as an energy source for heat tems are part of power stations. In further or alternative generation systems. In still further or alternative embodi embodiments, the power stations are decentralized power ments, the heat generation systems may be furnaces, stove stations. In further or alternative embodiments, the fuel cells tops, ovens, barbeques, and driers. In still further or alterna may be alkaline fuel cells, phosphoric acid fuel cells, molten tive embodiments, such an energy source may be a carbonate fuel cells, Solid oxide fuel cells, proton exchange replacement for, or a compliment to, natural gas and methane. membrane fuel cells, and combinations thereof. In further or 0020. In another aspect are compositions which contain alternative embodiments, the fuel cells are used to generate chemical products formed by hydrogenation of starting mate electricity for residential consumption, commercial con rials using hydrogen and catalysts, wherein the hydrogen is Sumption, motor vehicle consumption, or combinations produced by anaerobically fermenting a biomass using Sub thereof. In still further or alternative embodiments, the elec stantially purified anaerobic bacteria cultures. Hydrogenation tricity generated by such fuel cells may be used locally, added occurs by admixing starting materials and catalysts, with to the grid, or combinations thereof. In further or alternative hydrogen. In an embodiment of this aspect, the Substantially embodiments, the turbine generators are used to generate purified anaerobic bacteria cultures have not been subjected electricity from steam created by heating water, wherein the to heatshocking processes. In further or alternative embodi heat is generated by combustion of compositions comprising ments, the chemical product is aniline and the starting mate chemical products produced by anaerobically fermenting rial is nitrobenzene. In further or alternative embodiments, biomass. In further or alternative embodiments, the electricity the catalysts may be NiS/CuS catalysts or Cu catalysts. In generated by Such turbines may be used for residential con further or alternative embodiments, the aniline may be used in Sumption or commercial consumption. In still further or alter the synthesis of pharmaceuticals, polymers, dyes, or solvents. native embodiments, the electricity generated by Such tur 0021. In another aspect are compositions which contain bines may be used locally, added to the grid, or combinations chemical products formed by admixing minerals and Volatile thereof. organic acids, wherein the Volatile organic acids are produced 0019. In another aspect are compositions which contain by anaerobically fermenting a biomass using Substantially chemical products formed by admixing carbon oxides, cata purified anaerobic bacteria cultures. In an embodiment of this lysts, and hydrogen, in which the hydrogen has been produce aspect, the Substantially purified anaerobic bacteria cultures by anaerobically fermenting biomass using Substantially have not been Subjected to heatshocking processes. In further purified anaerobic bacteria cultures. In an embodiment of this or alternative embodiments, the Volatile organic acid is acetic aspect, the Substantially purified anaerobic bacteria cultures acid. In further or alternative embodiments, the mineral is have not been Subjected to heatshocking processes. In further dolomite and the chemical product is calcium magnesium or alternative embodiments, the chemical product is methane. acetate. In further or alternative embodiments, the calcium In further or alternative embodiments, the carbon oxide may magnesium acetate may be used as?in anti-freeze agents, be carbon monoxide, carbon dioxide, or combination thereof. deicing agents, or anti-icing agents. In further or alternative embodiments, the catalysts contain 0022. In another aspect are compositions which contain activated nickel, and in still further or alternative embodi chemical products formed by admixing oxides, liquid ammo ments the catalyst is NiO/MgO. In further or alternative nia and Volatile organic acids, wherein the Volatile organic embodiments, are energy sources for a power generation acids are produced by anaerobically fermenting biomass systems which contain Such chemical products. In still further using Substantially purified anaerobic bacteria cultures. In an or alternative embodiments, the chemical product of such embodiment of this aspect, the Substantially purified anaero US 2008/0311640 A1 Dec. 18, 2008

bic bacteria cultures have not been subjected to heatshocking In further or alternative embodiments, the sterilization and processes. In further or alternative embodiments, the volatile deoxygenation systems are steam heat exchanger systems. In organic acid is acetic acid. In further or alternative embodi still further or alternative embodiments, the sterilization and ments, the oxide is Zinc oxide and the chemical product is Zinc deoxygenation systems are autoclaves. ammonium acetate. In further or alternative embodiments, 0028. In further or alternative embodiments, the steriliza the calcium magnesium acetate may be used as/infertilizer or tion and deoxygenation systems of the anaerobic fermenta seed germination enhancers. tion apparatuses are assemblages which incorporate at least Anaerobic Fermentation Apparatuses one vat, at least one source of steam, and at least one turbine. The sources of steam may be sources of high pressure steam, 0023. In one aspect are anaerobic fermentation appara which include, but are not limited to, boilers. In further or tuses which use populations of Substantially purified anaero alternative embodiments, the turbines of the sterilization and bic bacterial strains for anaerobically fermenting biomass deoxygenation systems may be used to produce low pressure into chemical products. The aforementioned anaerobic fer mentation apparatuses include (a) sterilization and deoxygen steam and to generate electricity, and in still further or alter ation systems, (b) anaerobic digesters, each of which contains native embodiments, such turbines include, but are not lim a population of Substantially purified anaerobic bacteria and ited to, backpressure turbine generators which generate low are equipped with anaerobic digester control systems, (c) pressure steam to sterilize and deoxygenate biomass and pro plurality of pipelines and pumps for introducing and re-cir duce electricity as power for local energy consumer, or for culating biomass, and (d) removal pipelines connected to the addition to grid systems of centralized power stations. Such anaerobic digesters for removing chemical products from the local energy consumer may include, but are not limited to, anaerobic digesters. In addition to this aspect, the population facilities for anaerobic fermentation, factories, buildings, of substantially purified anaerobic bacterial strain has not facilities for processing farm produce, and combinations been subjected to a heatshocking process. therein. 0024. In an embodiment of the aforementioned aspect, the 0029. In further or alternative embodiments, the concen anaerobic fermentation apparatuses further include pipelines trating systems of the anaerobic fermentation apparatuses are in communication with the sterilization and deoxygenation centrifugation systems used to concentrate biomass by means systems having pumps which are used for transferring steril of centrifugation processes. In further or alternative embodi ized and deoxygenated biomass to concentrating systems. In ments, the concentrating systems are reverse osmosis systems further or alternative embodiments, the anaerobic fermenta used to concentrate biomass by means of reverse osmosis tion apparatuses also incorporate pipelines in communication processes. with concentrating systems and have pumps for transferring 0030. In further or alternative embodiments, the removal deoxygenated, concentrated, sterilized, detoxified, and/or pipelines connected to anaerobic digesters have at least one pre-digested biomass to at least one anaerobic digester. output pipeline for gaseous chemical products and at least one 0.025 Infurther or alternative embodiments, the anaerobic output pipeline for non-gaseous chemical products. In further fermentation apparatuses may incorporate anaerobic digest or alternative embodiments, gaseous chemical products may ers, each of which may contain a population of Substantially be removed from anaerobic digesters using at least one output purified anaerobic photo-fermentation bacteria and are pipeline, and in still further or alternative embodiments, gas equipped with anaerobic digester control systems. In further eous chemical products are removed from anaerobic digest or alternative embodiments, the anaerobic fermentation appa ers by means of a vacuum applied to at least one output ratuses may incorporate anaerobic digesters, each of which pipeline. In further or alternative embodiments, gas scrubbers may contain a population of Substantially purified anaerobic are connected to at least one output pipeline and gas compres acetogenic bacteria and are equipped with anaerobic digester sors are connected to the scrubbers, and at least one gas control systems. In further or alternative embodiments, the storage tank. In further or alternative embodiments, non anaerobic fermentation apparatuses may incorporate anaero gaseous chemical products may be removed from anaerobic bic digesters, each of which contain may a population of digesters using at least one output pipeline, and in further or Substantially purified anaerobic solventogenic bacteria and alternative embodiments, the non-gaseous chemical products are equipped with anaerobic digester control systems. In still are volatile organic acids which are removed from output further or alternative embodiments, the anaerobic fermenta pipelines by reverse osmosis systems which are coupled to tion apparatuses may incorporate anaerobic digesters, each of the output pipelines and to at least one storage tank. Similarly, which may contain a population of Substantially purified in further or alternative embodiments, the non-gaseous anaerobic proteolytic bacteria and are equipped with anaero chemical product may be salts of volatile organic acids which bic digester control systems. may be removed from output pipelines by reverse osmosis 0026. In further or alternative embodiments, the anaerobic systems which are coupled to output pipelines and at least one fermentation apparatuses may be stand alone systems for storage tank. In still further or alternative embodiments, the anaerobically fermenting biomass into chemical products, or Solid non-gaseous chemical products may be removed from in still further or alternative embodiments, the anaerobic fer output pipelines by filtration systems which are coupled to the mentation apparatuses may be components of assemblages output pipeline and to at least one storage tank. In addition, in for generating power. further or alternative embodiments the solid chemical product 0027. In further or alternative embodiments, the steriliza is elemental sulfur which may be removed from output pipe tion and deoxygenation systems of the anaerobic fermenta lines by filtration systems which are coupled to the output tion apparatuses are used to sterilize and deoxygenate biom pipelines and at least one storage tank. In further or alternative ass by means of steam treatment processes, wherein the steam embodiments, such filtration systems are cross-flow filtration treatment processes may be pressurized steam treatment pro systems. In still further or alternative embodiments, the non cesses which use low pressure steam or high pressure steam. gaseous chemical products may be solvents which are US 2008/0311640 A1 Dec. 18, 2008

removed from the output pipelines by osmosis systems which alternative embodiments, the temperature sensors selected are coupled to the output pipelines and to at least one storage from thermometers, thermopiles, thermocouples, and combi tank nations thereof. In further or alternative embodiments, the 0031. In further or alternative embodiments, the gaseous metrology tools may be located on-line, or in further or alter chemical products which have been removed from the native embodiments, the metrology tools may be located anaerobic digester via output pipelines may be isolated and in-line. purified by means of a differential compression process. In 0036. In further or alternative embodiments, the anaerobic further or alternative embodiments, the volatile organic acids fermentation apparatuses may be incorporated into central which have been removed from the anaerobic digester via ized power generation facilities, and in still further or alter output pipelines may be isolated and purified using distilla native embodiments, the anaerobic fermentation apparatuses tion. In still further or alternative embodiments, the solvents may be incorporated into decentralized power generation which have been removed from the anaerobic digester via facilities. output pipelines may be isolated and purified using distilla 0037. In another aspect are assemblages combining tion. In further or alternative embodiments, the salts of vola Sources of steam, turbines, and digesters. In an embodiment tile organic acids which have been removed from the anaero of this aspect, the Sources of steam may be sources of high bic digester via output pipelines may be isolated and purified pressure steam. In further or alternative embodiments, the by reverse osmosis. sources of high pressure steam may be boilers. In still further 0032. In further or alternative embodiments, anaerobic or alternative embodiments, the sources of high pressure digester control systems combine at process control tools, steam are boilers heated by combustion of hydrogen, wherein metrology tools to acquire metrology data relating to anaero the hydrogen has been produced by anaerobically fermenting bic fermentation parameters; and process controllers opera biomass. In further or alternative embodiments, the digester tively coupled to the process control tools and the metrology may beat least one anaerobic digester. In further or alternative data, wherein the process controllers consists of decision embodiments, the turbines of the assemblages may be used to making units, input/output boards, and database units to store produce low pressure steam and to generate electricity. In the metrology data. Such anaerobic digester control systems further or alternative embodiments, the low pressure steam may be used to optimally operate anaerobic digesters. produced by such turbines may be used for biomass steriliza 0033. In further or alternative embodiments, process con tion and deoxygenation. In still further or alternative embodi trol tools are used to adjust operating parameters of the ments, the electricity generated by Such turbines may be used anaerobic digester, wherein the operating parameters are as power for local energy consumers or for addition to grid related to anaerobic fermentation parameters. In further or systems of centralized power stations. In even further or alter alternative embodiments, such process control tools are native embodiments, the local energy consumers may be valves or any means for adjusting the temperature of the facilities for anaerobic fermentation, factories, buildings, digester. homes, facilities for processing farm produce, and combina 0034. In further or alternative embodiments, the decision tions thereof. making units are used in feedback control processes to acquire metrology data from the input/output boards, then Knowledge Management System determine control adjustments to maintain anaerobic fermen 0038. In another aspectare methods for identifying bacte tation parameters within defined operational ranges, then rial species for use in the anaerobic production of hydrogen modify the magnitude of the control adjustments, and return from a particular biomass. The methods involve collecting the modified control adjustments to the input/output boards, appropriate information from various bacterial species and whereby by the modified control adjustments are sent to deriving probabilistic models from the information, the process control tools to adjust operating parameters, which probabilistic models being indicative of identification of bac are related to anaerobic fermentation parameters of the terial species for use in the anaerobic production of hydrogen anaerobic fermentation digester. In further or alternative and other chemical products from particular biomass. In an embodiments, the feedback control processes are continuous, embodiment of this aspect, the identification of bacterial spe automatic feedback control loops, while in still further or cies for use in the anaerobic production of hydrogen and other alternative embodiments, the feedback control processes are chemical products from particular biomass includes use of intermittent, manual feedback control loops. In still further or cultivation systems to identify various bacterial species and alternative embodiments, the process controller decision collecting appropriate information from the cultivation sys making units may be computers, PROMs, EPROMs, tem. In further or alternative embodiments, the cultivation EEPROMs, and combinations thereof. systems includes growing various bacterial species on par 0035. In further or alternative embodiments, the anaerobic ticular substrates, under various growth conditions to opti fermentation parameters may be temperature, pH, pressure, mize the bacterial hydrogen production. In further or alterna gas flow, biomass concentration, and chemical products con tive embodiments, the various growth conditions are selected centration, wherein the metrology tool used to monitor Such from the group consisting of variation in temperature, pH, parameters may be pH sensors, stirrers, oxidation-reduction fermentation products, exposure to various natural gases or monitors, nutrient concentration, pressure sensors, gas flow compounds, antibiotic efficacy, antibiotic resistance, com sensors, gas sensors, temperature sensors, gas chromato pound stimulation-digestion, compound toxicity or Surviv graphic systems, flow injection analysis systems, High Per ability. In further or alternative embodiments, the collection formance Liquid Chromatographic systems, Mass specro of appropriate information comprises selecting response data photometry, and combinations thereof. In further or from the scientific literature. In further or alternative embodi alternative embodiments, the pH sensors are selected from ments, the appropriate information comprises characteristics, glass membrane pH electrodes, Solid state pH electrodes, wherein Such characteristics are bacteria growth on various optode pH sensors, and combinations thereof. In further or Substrates, bacteria sensitivity to various conditions and/or US 2008/0311640 A1 Dec. 18, 2008

bacteria production of various metabolites. In still further or products comprising recordable media and a plurality of com alternative embodiments, such characteristics are categorized puter-readable program instructions on the recordable media into traits which include, but are not limited to, acetic acid that are executable by the computer to perform a method major metabolic product, acetic acid minor metabolic prod comprising receiving a plurality of bacterial species, where uct, acetone, ADH, ALP alpha-fucosidase, alpha-galactosi relationships between species are defined, receiving a plural dase, ammonia production, alpha-glucosidase, arabinose, ity of appropriate information for the plurality of bacteria ArgA, bacillus, beta-galactosidase, beta-glucosidase, beta species, and generating the candidate bacterial species foruse glucuronidase, beta-NAG, beta-xylosidase, box car shape, in the anaerobic production of hydrogen from a particular butyric acid major metabolic product, butyric acid minor biomass by traversing the plurality of information received hi metabolic product, Butanol, CAMP. caproic acid major meta order to find the optimal match. bolic product, carbon dioxide production, catalase, cello 0041. In further or alternative embodiments, the program biose, cellulose, chartreuse fluorescence, chymotrypsin, CO products may collect appropriate information by conducting growth, coccus, desulfoviridin, double Zone beta-hemolysis, cultivation systems to identify various bacterial species or by esculin hydrolysis, ethanol production, F/F required, fruc selecting response data from the scientific literature. In fur tose, gelatin hydrolysis, glucose, glycogen, gram reaction, ther or alternative embodiments, the program product may growth in bile, His A, hydrogen production, hydrogen Sulfide have cultivation systems comprises growing various bacterial production, 1-arabinose, indole, isobutyric acid major meta species on particular Substrates, under various growth condi bolic product, isobutyric acid minor metabolic product, iso tions to optimize the bacterial hydrogen production. In further capronic acid major metabolic product, isocapronic acid or alternative embodiments, the program product may have minor metabolic product, isovaleric acid major metabolic various growth conditions selected from the group consisting product, isovaleric acid minor metabolic product, lactate con of variation in temperature, pH, fermentation products, expo Verted to propionate, lactic acid major metabolic product, Sure to various natural gases or compounds, drug efficacy, lactic acid minor metabolic product, lactose, leithinase, compound toxicity or Survivability. LeuA, lipase, maltose, mannitol, mannose, melezitose, meli 0042. In further or alternative embodiments, the program biose, methane production, milk clot formed, milk digested, product may collect appropriate information involving bac motile, N-Acetyl-beta-glucosaminidase, nitrate, ONPG teria growth on a Substrate, bacteria sensitivity to a condition (Beta-galactosidase), oxygen tolerance, Phe A, phenylacetic and/or bacteria production of metabolites, wherein the appro acid minor metabolic product, pigment, pitting of agar, ProA, priate information is categorized into traits which include, but propionic acid major metabolic product, Pyra, raffinose, red are not limited to, acetic acid major metabolic product, acetic fluorescence, reverse CAMP test, rhamnose, ribose, salicin, acid minor metabolic product, ADH, ALP alpha-fucosidase, sensitive to colistin, sensitive to kanamycin, sensitive to SPS, alpha-galactosidase, alpha-glucosidase, arabinose, ArgA, sensitive to Vancomycin, Sorbitol, spore former, starch bacillus, beta-galactosidase, beta-glucosidase, beta-glucu hydrolysis, strictly anaerobic, Subterminal spore location, ronidase, beta-NAG, beta-xylosidase, box car shape, butyric Succinic acid major metabolic product, Succinic acid minor acid major metabolic product, butyric acid minor metabolic metabolic product, Sucrose, terminal spore location, threo product, CAMP. caproic acid major metabolic product, cata nine converted to propionate, trehalose, trypsin, Tyra, urease, lase, cellobiose, chartreuse fluorescence, chymotrypsin, CO Valeric acid major metabolic product, Valeric acid minor growth, coccus, desulfoviridin, double Zone beta-hemolysis, metabolic product, Xylan, and Xylose. Such categorization in esculin hydrolysis, F/F required, fructose, gelatin hydrolysis, the case of information regarding bacteria growth on various glucose, glycogen, gram reaction, growth in bile, His A, substrates may allow identification of bacteria strains which 1-arabinose, indole, isobutyric acid major metabolic product, utilize specific Substances as food sources, or it may allow the isobutyric acid minor metabolic product, isocapronic acid identification of optimal food sources for specific bacterial major metabolic product, isocapronic acid minor metabolic strains. In addition, categorization of the information regard product, isovaleric acid major metabolic product, isovaleric ing bacteria sensitivity to various conditions may allow for acid minor metabolic product, lactate converted to propi identification of optimal conditions for the anaerobic fermen onate, lactic acid major metabolic product, lactic acid minor tation of various Substances, while categorization of the infor metabolic product, lactose, leithinase, LeuA, lipase, maltose, mation regarding bacteria production of various metabolites mannitol, mannose, melezitose, melibiose, milk clot formed, may allow for identification the metabolites produced by milk digested, motile, N-Acetyl-beta-glucosaminidase, various bacterial strains using various food sources. nitrate, ONPG(Beta-galactosidase), oxygen tolerance, Phe A. 0039. In further or alternative embodiments, the appropri phenylacetic acid minor metabolic product, pigment, pitting ate information may be bacterial response which may be ofagar, ProA, propionic acid major metabolic product, Pyra, positive, negative, a variable response, or non responsive. In raffinose, red fluorescence, reverse CAMP test, rhamnose, still further or alternative embodiments, the bacterial ribose, salicin, sensitive to colistin, sensitive to kanamycin, response is measured by Smell, color, growth, non-growth, sensitive to SPS, sensitive to Vancomycin, sorbitol, spore death, symbiosis, inhibition, and non-Symbiosis. In further or former, starch hydrolysis, strictly anaerobic, Subterminal alternative embodiments, the appropriate information is col spore location, Succinic acid major metabolic product, Suc lected in computer readable media. In further or alternative cinic acid minor metabolic product, Sucrose, terminal spore embodiments, the probabilistic models are derived using location, threonine converted to propionate, trehalose, minimizing parameters based on the appropriate information. trypsin, Tyr A, urease, Valeric acid major metabolic product, 0040. In another aspect are program products for use in Valeric acid minor metabolic product, Xylan, and Xylose. computers that executes program instructions recorded in a 0043. In further or alternative embodiments, the appropri computer-readable media to produce candidate bacterial spe ate information may be bacterial response which may be cies for use in the anaerobic production of hydrogen and other positive, negative, or non responsive. In further or alternative chemical products from particular biomass, the program embodiments, the program product may measures bacterial US 2008/0311640 A1 Dec. 18, 2008

response by Smell, color, growth, non-growth, death, Symbio the various growth conditions are selected from the group sis, and non-Symbiosis. In further or alternative embodi consisting of variation in temperature, pH, fermentation ments, the program product may collect the appropriate infor products, exposure to various natural gases or compounds, mation in computer readable media. drag efficacy, compound toxicity or Survivability. In further or 0044. In another aspect are program products for use in alternative embodiments, the appropriate information may be computers that executes program instructions recorded on bacterial response that may be positive, negative, or non computer-readable media to obtain candidate bacterial spe responsive, and which may be measured by Smell, color, cies for use in the anaerobic production of hydrogen from a growth, non-growth, death, symbiosis, and non-symbiosis. In particular biomass, the program products comprise record further or alternative embodiments, the appropriate informa able media; and a plurality of computer-readable program tion is collected in computer readable media. instructions on the recordable media that are executable by the computer to perform a method comprising: a) determin Nutrient/Biomass Preparation ing a bacterial species of interest; b) determining the appro 0047. In another aspectare methods of anaerobically pro priate information for the bacterial species of interest; and c) ducing hydrogen and other chemical products comprising repeating steps a-b for other bacterial species; and d) com obtaining biomass material Suitable as nutrients for isolated paring appropriate information collected from step c to assess non-heatshocked anaerobic bacteria; concentrating or dilut optimal candidate bacterial species for use in the anaerobic ing the biomass material by at least a factor of 2; Sterilizing the production of hydrogen from a particular biomass. biomass material, deoxygenating the biomass material; and 0045. In an embodiment of the aforementioned aspect, the fermenting the concentrated (or diluted), sterilized, deoxy program product may collect appropriate information by con genated, detoxified, and/or pre-digested biomass material ducting cultivation systems to identify various bacterial spe with the isolated non-heatshocked anaerobic bacteria under cies or selecting response data from the scientific literature, anaerobic conditions so as to produce hydrogen and other wherein the appropriate information comprises information chemical products. In one embodiment, the biomass material regarding bacteria growth on a Substrate, bacteria sensitivity is concentrated by at least a factor of 2; in an alternative to a condition and/or bacteria production of metabolites, embodiment, the biomass material is diluted by at least a which may be further categorized into traits which include, factor of 2. In an embodiment of this aspect, the hydrogen is but are not limited to, acetic acid major metabolic product, produced continuously. In further or alternative embodi acetic acid minor metabolic product, ADH, ALP alpha-fu ments, the fermentation occurs in anaerobic digesters, land cosidase, alpha-galactosidase, alpha-glucosidase, arabinose, fills, trenches, or combinations thereof. In further or alterna ArgA, bacillus, beta-galactosidase, beta-glucosidase, beta tive embodiments, the concentration step is completed before glucuronidase, beta-NAG, beta-xylosidase, box car shape, initiating the sterilization step and/or the deoxygenation step. butyric acid major metabolic product, butyric acid minor In further or alternative embodiments, the dilution step is metabolic product, CAMP. caproic acid major metabolic completed before initiating the sterilization step and/or the product, catalase, cellobiose, chartreuse fluorescence, chy deoxygenation step. In further or alternative embodiments, motrypsin, CO. growth, coccus, desulfoviridin, double Zone the concentration step is initiated after completion of the beta-hemolysis, esculin hydrolysis, F/F required, fructose, sterilization step and/or the deoxygenation step. In further or gelatin hydrolysis, glucose, glycogen, gram reaction, growth alternative embodiments, the concentration, sterilization and in bile, His A, I-arabinose, indole, isobutyric acid major meta deoxygenation steps overlap. In further or alternative bolic product, isobutyric acid minor metabolic product, iso embodiments, the sterilization step is completed before initi capronic acid major metabolic product, isocapronic acid ating the concentration step and/or the deoxygenation step. In minor metabolic product, isovaleric acid major metabolic further or alternative embodiments, the sterilization step is product, isovaleric acid minor metabolic product, lactate con initiated after completion of the concentration step and/or the Verted to propionate, lactic acid major metabolic product, deoxygenation step. In further or alternative embodiments, lactic acid minor metabolic product, lactose, leithinase, the biomass material is concentrated by at least a factor of 4. LeuA, lipase, maltose, mannitol, mannose, melezitose, meli In further or alternative embodiments, the dilution step is biose, milk clot formed, milk digested, motile, N-Acetyl initiated after completion of the sterilization step and/or the beta-glucosaminidase, nitrate, ONPG(Beta-galactosidase), deoxygenation step. In further or alternative embodiments, oxygen tolerance, Phea, phenylacetic acid minor metabolic the dilution, sterilization and deoxygenation steps overlap. In product, pigment, pitting of agar, ProA, propionic acid major further or alternative embodiments, the sterilization step is metabolic product, Pyra, raffinose, red fluorescence, reverse completed before initiating the dilution step and/or the CAMP test, rhamnose, ribose, salicin, sensitive to colistin, deoxygenation step. In further or alternative embodiments, sensitive to kanamycin, sensitive to SPS, sensitive to Vanco the sterilization step is initiated after completion of the dilu mycin, Sorbitol, spore former, starch hydrolysis, strictly tion step and/or the deoxygenation step. In further or alterna anaerobic, Subterminal spore location, Succinic acid major tive embodiments, the biomass material is diluted by at least metabolic product, Succinic acid minor metabolic product, a factor of 4. Sucrose, terminal spore location, threonine converted to pro 0048. In further or alternative embodiments, the biomass pionate, trehalose, trypsin, Tyra, urease, Valeric acid major material is concentrated by centrifugation. In further or alter metabolic product, Valeric acid minor metabolic product, native embodiments, the biomass material is concentrated by Xylan, and Xylose. reverse osmosis. 0046. In further or alternative embodiments, the program 0049. In further or alternative embodiments, the biomass product may involve a cultivation system which comprises material is sterilized with pressurized steam. In further or growing various bacterial species on particular substrates, alternative embodiments, the biomass material is sterilized by under various growth conditions to optimize the bacterial autoclaving. In further or alternative embodiments, the bio hydrogen production. In further or alternative embodiments, mass material is deoxygenated with pressurized steam. In US 2008/0311640 A1 Dec. 18, 2008

further or alternative embodiments, the biomass material is Rhodopseudomonas, Rhodospirillum, Erythrobacter, Metha deoxygenated by autoclaving. In further or alternative nobacterium, Methanobrevibacter, Methanococcu, Metha embodiments, the deoxygenated biomass material is further nococcoides, Methanolobus, Methanolacinia, Methanomi deoxygenated by adding a reducing agent. In further or alter crobium, Methanogenium, Methanospirillum, native embodiments, the reducing agent is dithiothreitol, cys Methanoplanus, Methanothrix, Methanothermus, Methano teine, thioglycolate, or sodium sulfide. In further or alterna corpusculum, Methanoculleus, Methanohalobium, Methano tive embodiments, the biomass material is sterilized and halophilus, Methanosarcina, Methanosphaera, Eubacte deoxygenated with pressurized steam. In further or alterna rium, Abiotrophia, Atopobium, Gemella, Granulicatella, tive embodiments, the biomass material is sterilized and Finegoldia, Lactobacillus, Actinomyces, Arcanobacterium, deoxygenated by autoclaving. In further or alternative Bulleidia, Collinsella, Cryptobacterium, Holdemania, embodiments, the sterilized and deoxygenated biomass mate Rothia, Pseudoramibacter, Mogibacterium, Slackia, and rial is further deoxygenated by adding a reducing agent. Eggerthella; (b) concentrating the biomass material by at 0050. In further or alternative embodiments, the deoxy least a factor of 2; (c) Sterilizing the biomass, (d) deoxygen genated biomass material is suitable for producing hydrogen ating the biomass material; and (e) fermenting the concen and other chemical products using isolated non-heatshocked trated, sterilized, detoxified, deoxygenated and/or pre-di anaerobic bacteria. In further or alternative embodiments, the gested biomass material with Such isolated non-heatshocked biomass material is deoxygenated with oxygen scavengers. In anaerobic bacteria under anaerobic conditions so as to pro further or alternative embodiments, the biomass material is duce hydrogen. In an embodiment of this aspect, the hydro deoxygenated with oxygen scavenger microorganisms. In gen is produced continuously. In further or alternative further or alternative embodiments, the biomass material is embodiments, the fermentation occurs in anaerobic digesters, deoxygenated with a cellular membrane preparation of oxy landfills, trenches, or combinations thereof. In further or gen Scavenger microorganisms. In further or alternative alternative embodiments, the concentration step is completed embodiments, the deoxygenated biomass material contains before initiating the sterilization step and/or the deoxygen less than about 100 parts per million of oxygen. In further or ation step. In further or alternative embodiments, the concen alternative embodiments, the deoxygenated biomass material tration step is initiated after completion of the sterilization contains less than about 50 parts per million of oxygen. In step and/or the deoxygenation step. In further or alternative further or alternative embodiments, the deoxygenated biom embodiments, the concentration, Sterilization and deoxygen ass material contains less than about 20 parts per million of ation steps overlap. In further or alternative embodiments, the oxygen. In further or alternative embodiments, deoxygenated sterilization step is completed before initiating the concentra water is added to the concentrated, sterilized, deoxygenated, tion step and/or the deoxygenation step. In further or alterna detoxified, and/or pre-digested biomass material to facilitate tive embodiments, the sterilization step is initiated after growth of the isolated non-heatshocked anaerobic bacteria. In completion of the concentration step and/or the deoxygen further or alternative embodiments, the isolated non-heat ation step. In further or alternative embodiments, the biomass shocked anaerobic bacteria are mixed with deoxygenated material is concentrated by at least a factor of 4. water prior to the fermentation step. 0052. In further or alternative embodiments, the biomass 0051. In another aspectare methods of anaerobically pro material is concentrated by a method selected from centrifu ducing hydrogen which comprise (a) obtaining biomass gation, reverse osmosis, drying or a combination thereof. In material Suitable as nutrients for isolated non-heatshocked further or alternative embodiments, the biomass material is bacteria selected from the group consisting of the following sterilized by a method selected from pressurized steam or bacteria genera Acetivibrio, Acetoanaerobium, Acetofilamen autoclaving. In further or alternative embodiments, the bio tum, Acetogenium, Acetothermus, Acidaminobacter, Anaero mass material is deoxygenated by a method selected from biospirillum, Anaerorhabdus, Anaerovibrio, Atopobium, pressurized steam, autoclaving, addition of a reducing agent, Bacteroides, Bifidobacterium, Bilophila, Butyrivibrio, or a combination thereof. In further or alternative embodi Campylobacter, Catonella, Centipeda, Dialister; Dichelo ments, the reducing agent is dithiothreitol, cysteine, thiogly bacter, Fervidobacterium, Fibrobacter, Fusobacterium, collate, or Sodium Sulfide. In the resulting deoxygenated bio Halanaerobacter; Halanaerobium, Ilvobacter; Johnsonella, mass material is suitable for producing hydrogen using the Lachnobacterium, Leptotrichia, Malonomonas, Megamonas, listed isolated non-heatshocked bacteria species of this Mitsuokella, Oxalobacter, Pectinatus, Pelobacter, Porphy aspect. romonas, PrevOtella, Propionibacterium, Propionigenium, 0053. In further or alternative embodiments, the biomass Propionispira, Rikenella, Roseburia, Ruminobacter, material is deoxygenated using an agent selected from the Sebaldella, Selenomonas, Sporomusa, Succinimonas, Suc group consisting of oxygen Scavengers, oxygen Scavenging cinivibrio, Syntrophobacter, Syntrophomonas, Sutterella, microorganisms, cellular membrane preparation of oxygen Saponavida, Thermobacteroides, Thermosipho, Thermo Scavenging microorganisms, and combinations thereof. In toga, Tissierella, Wolinella, Zymophilus, Desulfobacter, Des further or alternative embodiments, the deoxygenated biom ulfobacterium, Desulfobulbus, Desulfococcus, Desulfomi ass material contains less than about 20 parts per million of crobium, Desulfomonas, Desulfomonile, Desulfonema, oxygen. In further or alternative embodiments, the deoxygen Desulfosarcina, Desulfotomaculum, Desulfovibrio, Des ated water is added to the concentrated, sterilized, detoxified, ulfurella, Desulfuromonas, Thermodesulfobacterium, deoxygenated, and/or pre-digested biomass material to facili Acidaminococcus, Megasphaera, Syntrophococcus, Veil tate growth of the listed isolated non-heatshocked bacteria of lonella, Coprococcus, Peptococcus, Peptostreptococcus, this aspect. Ruminococcus, Sarcina, Clostridium, Amoebobacter; Chro 0054. In another aspectare methods of anaerobically pro matium, Lamprobacter; Thiocapsa, Thiocystis, Thiodictyo, ducing hydrogen comprising (a) obtaining a biomass material Thiopedia, Thiospirillum, Ectothiorhodospira, Rhodobacter, suitable as a nutrient for isolated non-heatshocked bacteria Rhodocyclus, Rhodomicrobium, Rhodopilla, selected from the group consisting of anaerobic bacteria (see US 2008/0311640 A1 Dec. 18, 2008

above); (b) concentrating the biomass material by at least a genation steps. In further or alternative embodiments, the factor of 2 using centrifugation, reverse osmosis or a combi biomass material is concentrated by at least a factor of 4. In nation thereof; (c) Sterilizing the biomass, (d) deoxygenating further or alternative embodiments, the reducing agent is the biomass material; and (e) fermenting the concentrated, dithiothreitol, cysteine, thioglycollate, or sodium sulfide. In sterilized, detoxified, deoxygenated and/or pre-digested bio the resulting deoxygenated biomass material is suitable for mass material with the listed isolated non-heatshocked producing hydrogen using isolated non-heatshocked anaerobic bacteria under anaerobic conditions so as to pro Clostridia. duce hydrogen. 0059. In further or alternative embodiments, the biomass 0055. In an embodiment of this aspect, the listed bacteria material is further deoxygenated using an agent selected from are collected from bovine rumen, Soil samples, sludge, the group consisting of oxygen Scavengers, oxygen scaveng anaerobic bacteria cultures, aerobic bacteria cultures, anaero ing microorganisms, cellular membrane preparation of oxy bic sediments from fresh or brackish waters, sewage, animals, gen Scavenging microorganisms, and combinations thereof. animal feces, insect digestive tract; dental samples, hydro In further or alternative embodiments, the deoxygenated thermal soils, hydrothermal pools or deep water hydrother water is added to the concentrated, sterilized, deoxygenated, mal vents. In further or alternative embodiments, the hydro detoxified, and/or pre-digested biomass material to facilitate gen is produced continuously. In further or alternative growth of the isolated non-heatshocked Clostridia. embodiments, the fermentation occurs in anaerobic digesters, 0060. In another aspectare methods of anaerobically pro landfills, trenches, or combinations thereof. In further or ducing hydrogen comprising obtaining a biomass material alternative embodiments, the concentration step occurs prior suitable as a nutrient for isolated non-heatshocked Clostridia, to, after, or simultaneously with the sterilization and/or wherein the biomass material comprises molasses, raw paper, deoxygenation steps. In further or alternative embodiments, agricultural waste, or mulch, concentrating the biomass mate the biomass material is concentrated by at least a factor of 4. rial by at least a factor of 2 via centrifugation, reverse osmosis Infurther or alternative embodiments, the biomass material is or a combination thereof. Sterilizing the biomass material by deoxygenated by a method selected from pressurized steam, a method selected from pressurized steam or autoclaving, autoclaving, addition of reducing agents, or combinations deoxygenating the biomass material by a method selected thereof. In further or alternative embodiments, the reducing from pressurized steam, autoclaving, addition of reducing agent is dithiothreitol, cysteine, thioglycollate, or sodium agents, or combinations thereof, wherein the deoxygenated sulfide. In further or alternative embodiments, the resulting biomass material contains less than about 20 parts per million deoxygenated biomass material is suitable for producing of oxygen; and fermenting the concentrated, sterilized, hydrogen using the listed isolated non-heatshocked bacteria deoxygenated, detoxified, and/or pre-digested biomass mate of this aspect. rial with the isolated non-heatshocked anaerobic bacteria 0056. In further or alternative embodiments, the biomass under anaerobic conditions in an anaerobic digester so as to material is deoxygenated using an agent selected from the produce hydrogen. In an embodiment of this aspect, the group consisting of oxygen Scavengers, oxygen Scavenging hydrogen is produced continuously. In further or alternative microorganisms, cellular membrane preparation of oxygen embodiments, the fermentation occurs in anaerobic digesters, Scavenging microorganisms, and combinations thereof. In landfills, trenches, or combinations thereof. In further or further or alternative embodiments, the deoxygenated biom alternative embodiments, the concentration step occurs prior ass material contains less than about 20 parts per million of to, after, or simultaneously with the sterilization and/or oxygen. In further or alternative embodiments, the deoxygen deoxygenation steps. In further or alternative embodiments, ated water is added to the concentrated, sterilized, detoxified, the biomass material is concentrated by at least a factor of 4. deoxygenated, and/or pre-digested biomass material to facili 0061. In further or alternative embodiments, the reducing tate growth of the listed isolated non-heatshocked bacteria of agent is dithiothreitol, cysteine, thioglycollate, or sodium this aspect. sulfide. In further or alternative embodiments, the resulting 0057. In another aspectare methods of anaerobically pro deoxygenated biomass material is suitable for producing ducing hydrogen comprising obtaining a biomass material hydrogen using isolated non-heatshocked Clostridia. In fur suitable as a nutrient for isolated non-heatshocked Clostridia, ther or alternative embodiments, the biomass material is fur concentrating the biomass material by at least a factor of 2 via ther deoxygenated using an agent selected from the group centrifugation, reverse osmosis or a combination thereof. consisting of oxygen scavengers, oxygen scavenging micro sterilizing the biomass material by a method selected from organisms, cellular membrane preparation of oxygen Scav pressurized steam or autoclaving, deoxygenating the biomass enging microorganisms, and combinations thereof. In further material by a method selected from pressurized steam, auto or alternative embodiments, the deoxygenated water is added claving, addition of reducing agents, or combinations thereof, to the concentrated, sterilized, deoxygenated, detoxified, and/ wherein the deoxygenated biomass material contains less or pre-digested biomass material to facilitate growth of the than about 20 parts per million of oxygen; and fermenting the isolated non-heatshocked Clostridia. concentrated, deoxygenated, detoxified, and/or pre-digested 0062. In another aspectare methods for producing a nutri biomass material with the isolated non-heatshocked anaero ent suitable for non-heatshocked anaerobic bacteria to pro bic bacteria under anaerobic conditions in an anaerobic duce hydrogen comprising removing Substantially all of the digestor so as to produce hydrogen. oxygen from a biomass by a method selected from pressur 0058. In an embodiment of this aspect, the hydrogen is ized steam, autoclaving, addition of a reducing agent, or produced continuously. In further or alternative embodi combinations thereof. ments, the fermentation occurs in anaerobic digester, land 0063. In another aspectare nutrients suitable for non-heat fills, trenches, or combinations thereof. In further or alterna shocked anaerobic bacteria to produce hydrogen comprising tive embodiments, the concentration step occurs prior to, prepared by removing Substantially all of the oxygen from a after, or simultaneously with the sterilization and/or deoxy biomass by a method selected from pressurized steam, auto US 2008/0311640 A1 Dec. 18, 2008

claving, addition of a reducing agent, or combinations tures, aerobic bacteria cultures, anaerobic sediments from thereof. In an embodiment of this aspect, the nutrients are fresh or brackish waters, sewage, animals, animal feces, further prepared by concentrating the biomass using centrifu insect digestive tract, dental samples, hydrothermal soils, gation, reverse osmosis or a combination thereof, and steril hydrothermal pools or deep water hydrothermal vents. In izing the biomass by means of pressurized steam or autoclav further or alternative embodiments, the apparatuses further 1ng. contain Substantially deoxygenated water. 0068. In further or alternative embodiments, the multi Isolation/Enrichment System plicities of growth conditions are selected from the group 0064. In another aspectare methods for selecting bacteria consisting of variation in temperature, pressure, pH, fermen for use in anaerobic hydrogen production comprising isolat tation products, exposure to various natural gases or com ing anaerobic bacterial Strains for their ability to use biomass pounds, drug efficacy, compound toxicity or Survivability. In as nutrients and measuring collected hydrogen gas produced, further or alternative embodiments, the sealed chambers have wherein the anaerobic bacteria do not require heatshocking in shapes selected from the group consisting of circular, cylin order to anaerobically produce hydrogen and other chemical drical, spherical, square, or rectangular forms. In further or products from the biomass. In an embodiment of their aspect, alternative embodiments, the sealed chambers further have the bacteria are placed into a sealed chamber with substan shapes having volumes of 100 cm to 50,000 cm. In further tially deoxygenated water and various types of biomass. In or alternative embodiments, the apparatuses further comprise further or alternative embodiments, the sealed chamber ulti at least one bacterial strain that produces hydrogen and other mately hosts an anaerobic environment. In further or alterna chemical products anaerobically. In further or alternative tive embodiments, the sealed chamber can provide a multi embodiments, the apparatuses further contain particular bio plicity of growth conditions. In further or alternative mass samples the bacteria use as nutrients for producing embodiments, the multiplicity of growth conditions are hydrogen gas and other chemical products. selected from the group consisting of variation in tempera 0069. In further or alternative embodiments, the bacteria ture, pressure, pH, fermentation products, exposure to various selected from the sealed chambers are removed without sub natural gases or compounds, drug efficacy, drug resistance, stantially disturbing the anaerobic environment. In further or compound toxicity or survivability. In further or alternative alternative embodiments, the bacteria are selected from the embodiments, the sealed chamber has a shape selected from sealed chambers by use of needles or by means of a vacuum. the group consisting of a circular, cylindrical, spherical, In further or alternative embodiments, the apparatus com square, or rectangular form. In further or alternative embodi prises at least two substantially parallel glass plates. In further ments, the sealed chamber has a volume of 100 cm to 50,000 or alternative embodiments, the apparatus further comprises a cm. seal on at least one side that is penetrable by needles. 0065. In further or alternative embodiments, isolated non heatshock anaerobic bacteria are taken from the sealed cham Use of Bacteria and Bacterial Cultures to Produce Hydrogen ber and further selected by cultivation systems. In further or and Other Chemical Products alternative embodiments, the cultivation systems comprise 0070. In another aspect are bacterial cultures which com growing various bacterial species on particular substrates, prise purified bacterial strains, in which the bacterial strains under various growth conditions to optimize the bacterial have not undergone aheatshocking process and they produce hydrogen production. In further or alternative embodiments, hydrogen by anaerobically fermenting biomass. In further or the isolated non-heatshock anaerobic bacteria are selected by alternative embodiments, the biomass has been deoxygen its production of hydrogen, including by way of example ated. In further or alternative embodiments, are the biomass only, at least 5%, at least 10%, at least 15%, at least 20%, at has been sterilized. Further or alternative embodiments are least 25%, or at least 30% of the total theoretical amount of the biomass has been concentrated. In further or alternative hydrogen that could be produced from a particular biomass. embodiments, the biomass has been deoxygenated and con In further or alternative embodiments, the isolated non-heat centrated. In further or alternative embodiments, the biomass shock anaerobic bacteria are selected by its production of 2 has been sterilized, deoxygenated, concentrated, detoxified, moles of hydrogen per mole of substrate produced. In further and/or pre-digested. In further or alternative embodiments, or alternative embodiments, the various growth conditions the bacterial strains have been selected by means of a Knowl are selected from the group consisting of variation in tem edge Management System. In further or alternative embodi perature, pressure, pH, fermentation products, exposure to ments, the bacterial strains have been selected by means of various natural gases or compounds, drug efficacy, compound isolation/enrichment systems. In further or alternative toxicity or survivability. embodiments, the bacterial strains have been isolated by 0.066. In further or alternative embodiments, the isolated means of isolation/enrichment systems. In further or alterna non-heatshock anaerobic bacteria are taken from the sealed tive embodiments, the bacterial strains have been isolated by chamber and further selected in cultivation systems after 1-7 means of isolation/enrichment systems and have been days. In further or alternative embodiments, the isolated non selected by means of a Knowledge Management System. In heatshock anaerobic bacteria are taken from the sealed cham further or alternative embodiments, the biomass comprises ber and further selected in cultivation systems after 1-20 material from at least one member of the genus Capsicum. In weeks. still further or alternative embodiments, the member of the 0067. In another aspect are apparatuses for selecting bac genus Capsicum is selected from the group consisting of teria for use in anaerobic hydrogen production which com Capsicum anuum, Capsicum baccatum, Capsicum chinense, prise sealed chambers capable for providing anaerobic envi Capsicum gemnifolium, Capsicum frutescens, Capsicum ronments and multiplicities of growth conditions. In an pubescens, and combinations thereof. In further or alternative embodiment of this aspect, the bacteria are collected from embodiments, the biomass comprises material from at least bovine rumen, Soil samples, sludge, anaerobic bacteria cul one member of the genus Allium. In still further or alternative US 2008/0311640 A1 Dec. 18, 2008 15 embodiments, the member of the genus Allium is selected species of interest; b) determining the appropriate informa from the group consisting of Allium sativum, Allium cepa, tion for the bacterial species of interest; c) repeating steps a-b Allium Schoenoprasum, Allium tuberosum, Allium ampelo for other bacterial species; and d) comparing appropriate prasum, and combinations thereof. In further or alternative information collected from step c to assess optimal candidate embodiments, the biomass comprises Sugar products. In fur bacterial species for use in the anaerobic production of hydro ther or alternative embodiments, the Sugar products include gen from a particular biomass. In further or alternative material from at least one member of the genus Saccharum. In embodiments, the appropriate information is collected by still further or alternative embodiments, the member of the conducting cultivation systems to identify various bacterial genus Saccharum is a species selected from the group con species. In further or alternative embodiments, the cultivation sisting of Saccharum spontaneum, Saccharum robustum, systems comprise growing various bacterial species on par Saccharum officinarum, Saccharum barberi, Saccharum sin ticular substrates, under various growth conditions to opti ense, Saccharum edule, and combinations thereof. In further mize the bacterial hydrogen production. or alternative embodiments, the biomass comprises cellulose 0072. In further or alternative embodiments, the isolation/ products. In still further or alternative embodiments, the cel enrichment system is a sealed chamber containing Substan lulose products includes wood pulp. In further or alternative tially deoxygenated water and at least one biomass sample embodiments, the biomass comprises material from at least used to anaerobically ferment the at least one biomass. In one species selected from the group consisting of Solanum further or alternative embodiments, the isolation/enrichment Esculentum, Solarium melongena, Solarium tuberosum, system ultimately hosts an anaerobic environment. The iso Lycopersicon esculentum, Beta vulgaris, and combinations lation/enrichment system can provide a multiplicity of thereof. In further or alternative embodiments, the bacteria growth conditions. In further or alternative embodiments, the cultures are used to produce hydrogen and other chemical isolation/enrichment system has a shape selected from the products. In further or alternative embodiments, the chemical group consisting of a circular, cylindrical, spherical, square, products are selected from the group consisting of gases, or rectangular form. Solids, solvents, Volatile organic acids, salts of volatile 0073. In further or alternative embodiments, the bacterial organic acids, and combinations thereof. In further or alter strains are used to produce hydrogen by anaerobically fer native embodiments, the chemical product is a gas selected menting biomass in anaerobic fermentation apparatuses. In from the group consisting of carbon dioxide, carbon monox further or alternative embodiments, the anaerobic fermenta ide, hydrogen Sulfide, ammonia, nitrogen, and combinations tion apparatuses comprises: Sterilization and deoxygenation thereof. In further or alternative embodiments, the chemical systems; anaerobic digesters containing a population of sub product is a solid that comprises sulfur. In further or alterna stantially purified anaerobic bacteria and equipped with an tive embodiments, the solid is elemental sulfur. In further or anaerobic digester control systems; plurality of pipelines and alternative embodiments, the solvents are selected from the pumps for introducing and re-circulating biomass, and group consisting of acetone, butanol, ethanol, propanol, iso removal pipelines connected to the anaerobic digesters for propanol. 1.2-propanediol, and combinations thereof. In fur removing chemical products from the anaerobic digesters. In ther or alternative embodiments, the Volatile organic acids are further or alternative embodiments, the anaerobic digester selected from the group consisting of formic acid, acetic acid, control systems are used to optimally operate the anaerobic propionic acid, butyric acid, Valeric acid and combinations digesters and which comprise: process control tools; metrol thereof. In further or alternative embodiments, the salts of ogy tools to acquire metrology data relating to anaerobic Volatile organic acids comprise anions selected from the fermentation parameters; process controllers operatively group consisting of formate, acetate, propionate, butyrate, coupled to the process control tools and the metrology data, Valerate, and combinations thereof. In further or alternative wherein the process controllers comprise decision making embodiments, the salts of volatile organic acid comprise cat units, input/output boards, and database units to store the ions selected from the group consisting of alkali metal ions, metrology data. alkaline earth ions, ammonium ions, and combinations 0074. Other objects, features and advantages of the meth thereof. In further or alternative embodiments, the cations are ods and compositions described herein will become apparent Na", K", Ca, Mg", NH, and combinations thereof. In from the following detailed description. It should be under further or alternative embodiments, the bacteria cultures stood, however, that the detailed description and the specific comprise Substantially purified single bacterial strains. In examples, while indicating specific embodiments, are given further or alternative embodiments, the substantially purified by way of illustration only, since various changes and modi single bacterial strains are at least 95% purified. In further or fications within the spirit and scope of the invention will alternative embodiments, the Substantially purified single become apparent to those skilled in the art from this detailed bacterial strains are at least 99% purified. In further or alter description. native embodiments, the Substantially purified single bacte rial strains are at least 99.5% purified. 0071. In further or alternative embodiments, the Knowl BRIEF DESCRIPTION OF THE DRAWINGS edge Management System comprises a program product for use in a computer that executes program instructions recorded 0075 FIG. 1 presents one potential schematic showing the in a computer-readable media to obtain candidate bacterial various aspects of the present invention. species for use in the anaerobic production of hydrogen from 0076 FIG. 2 presents one potential schematic illustrating a particular biomass. In further or alternative embodiments, the use of the Knowledge Management System for utilization the program product comprises a recordable medium and a of a particular biomass. plurality of computer-readable program instructions on the 0077 FIG.3 presents one potential screen shot of a display recordable media that are executable by the computer to page from the Knowledge Management System that lists perform a method comprising: a) determining a bacterial bacterial species and traits. US 2008/0311640 A1 Dec. 18, 2008

0078 FIG. 4 presents one potential screenshot of a display electron acceptor. Some bacteria respire by using nitrate or page from the Knowledge Management System showing a Sulfate ions as the terminal electron acceptor during respira preferred test that displays specific traits the bacteria are tion. tested for a response. 0096. The term “assemblage' as used herein, refers to 007.9 FIG.5 presents one potential screenshot of a display combination of components interconnected to achieve a page from the Knowledge Management System detailing desired function. The assemblage may be a stand alone sys appropriate information collected on the bacteria Bilophila tem or a system incorporated into another assemblage. The Wadsworthia. function of the assemblage includes, but is not limited to, the 0080 FIG. 6 presents one potential screenshot of a display production of chemical products by anaerobically fermenting page from the Knowledge Management System detailing a biomass, the monitoring and control of anaerobic fermenta description of a catalase indicator test. tion systems, and generation power and low pressure steam. 0081 FIG. 7 presents one potential bock diagram of a 0097. The terms “biomass, as used herein, refers to any system in the Knowledge Management System that performs animal or plant derived material that contains one or more sample processing and analysis in a biological workstation. components that can be converted, bioconverted or biode 0082 FIG. 8 presents one potential block diagram of a graded into a useful material by anaerobic fermentation. computer in the Knowledge Management System, illustrat 0098. The term “bacteria culture', as used herein, refers to ing the hardware components included in a computer that can growing of bacteria in a specially prepared nutrient medium. provide the functionality of workstations and computers. 0099. The term “energy crops, as used herein, refers to 0083 FIG. 9 presents one potential diagram of one plants which are grown for use as a source of biomass for fuel. embodiment of the isolation/enrichment system used to iso 0100. The term “feedstock', as used herein, refers to a late anaerobic bacteria. Source of chemical products used industrially to generate new 0084 FIG. 10 presents one potential process flow diagram chemical products and compositions, and it also refers to of a preferred embodiment of the anaerobic digester system. biomass sources. 0101 The term “fermentation', as used herein, refers to a 0085 FIG. 11 presents one potential procedure to prepare biological process in which organic compounds are partially biomass material. degraded; for example, yeasts ferment Sugars to alcohol. I0086 FIG. 12 presents one potential procedure to deoxy 0102 The terms “heatshocking and “heatshocking pro genate biomass. cess', as used herein, refers to the process of heating a micro 0087 FIG. 13 presents one potential procedure to concen bial community, Such as an inoculum or a sample containing trate biomass. multiple bacterial strains, to eliminate undesired strains, such 0088 FIG. 14 presents one potential schematic showing as non-spore forming (vegetative) bacteria, e.g. methano the multi fermentation scheme used for optimal hydrogen gens, with desired strains, such as spore forming bacteria, production. surviving the procedure for future growth in better conditions. I0089 FIG. 15 presents one potential schematic of the A non-limiting example of a heatshocking process is to heat showing the various uses for the anaerobic fermentation prod up the mixture of bacteria to 105° C. for up to 2 hours. uctS. 0103) The terms “heatshocking and “heatshocking pro 0090 FIG. 16 presents one potential schematic of the cess', as used herein, may also refer to the induction of machinery components used for hydrogen production. specific chemical pathways and/or a redirection of protein 0091 FIG. 17 presents one potential schematic demon synthesis that involves diversion from induction and synthe strating the use of a turbine system to generate low pressure sis of other proteins. steam for sterilization and deoxygenation of a biomass 0104. The term “heatshocked’, as used herein, refers to 0092 FIG. 18 presents one potential schematic of one having undergone a heatshocking process. embodiment of the anaerobic digester control system 0105. The term “metrology tool', as used herein, refers to as measurement tool, with the information obtained referred DETAILED DESCRIPTION OF THE INVENTION to herein as “metrology data”. 0106 The term “substantially deoxygenated, as used Glossary of Certain Terms herein, refers to a biomass or bacteria culture media in which there has been a reduction of the oxygen concentration. By 0093. The term “anaerobic' or “anoxic', as used herein, way of example only, the oxygen concentration may be less refers to an environment in which the oxygen has been Sub than 10000 ppm, or the oxygen concentration may be less stantially removed. By way of example only, the oxygen than 1000 ppm, or the oxygen concentration may be less than concentration may be less than 10000 ppm, or the oxygen 100 ppm, or the oxygen concentration may be less than 50 concentration may be less than 1000 ppm, or the oxygen ppm, or the oxygen concentration may be less than 20 ppm. In concentration may be less than 100 ppm, or the oxygen con embodiments when “substantially deoxygenated’ refers spe centration may be less than 50 ppm, or the oxygen concen cifically to a reduction of the concentration of oxygen in an tration may be less than 20 ppm aqueous solution, then the term 'substantially deoxygenated 0094. The term “anaerobic fermentation', as used herein, means less than 5 ppm of oxygen in the aqueous solution, refers to an anaerobic process in which biomass is degraded to including less than 4 ppm of oxygen, less than 3 ppm of chemical products such as, by way of example only, hydro oxygen, less than 2 ppm of oxygen, and less than 1 ppm of gen, carbon dioxide, Volatile organic acids, hydrogen Sulfide, OXygen. methane, Sulfur, and carbon monoxide. 0107 The term “substantially parallel.” as used herein, 0095. The term “anaerobic respiration', as used herein, refers to the distance between walls of the isolation/enrich refers to a process in which organic Substrates are degraded to ment system which may differ by a certain amount along the CO, but using a substance other than oxygen as the terminal length of the isolation/enrichment system. By way of US 2008/0311640 A1 Dec. 18, 2008

example only, the distance between walls of the isolation/ light and wind. Process technologies include thermochemi enrichment system may differ up to 5% along the length of the cal, biological, electrolytic and photolytic. Associated with isolation/enrichment system, or the distance between walls of each potential feedstock or energy source are unique chal the isolation/enrichment system may differ up to 1% along lenges and benefit trade-offs. Fossil feedstocks, for example, the length of the isolation/enrichment system, or the distance are widely available and relatively well understood and between walls of the isolation/enrichment system may differ accepted, but until carbon sequestration becomes feasible, up to 0.5% along the length of the isolation/enrichment sys greenhouse gas emissions make fossil feedstocks environ tem. Perfectly parallel walls would have no difference in mentally difficult. Biomass, on the other hand, has very small width along the length of the isolation/enrichment system. or even negative net carbon emissions, but has presented 0108. The term “substantially purified, as used herein, challenges with biomass collection, transportation, availabil refers to a bacterial culture in which there has been a reduc ity, and storage. Hydrogen produced from biomass fermen tion of some biological component in the culture media Such tation has not been cost competitive with gasoline because as, by way of example only, viruses, pathogens, toxins and biomass and capital costs are high. Improved biomass/agri bacteria strains, other than the bacterial strain of interest. culture technology (higher yields per acre, etc.); lower cost 0109) Hydrogen Production biomass collection, transportation, and storage options; and 0110. One approach to hydrogen production that may improved biomass preparation are needed. In addition, bio prove economically viable uses biological conversion of mass sources are often seasonal in nature, therefore biomass energy crops and industrial and agricultural wastes and resi flexible processes and/or cost effective biomass storage are dues into hydrogen and other chemical products. Hydrogen needed for year round operation. gas is an environmentally friendly energy source, as it may be 0113 All renewable energy sources are ultimately based used to generate electricity without producing greenhouse on Solar energy which is made available through photovoltaic gas emissions. Currently, most hydrogen is produced by cells, wind energy or stored as chemical energy in biomass. “steam reforming of methane, which predominantly relies Thus, biomass essentially stores Solar energy in chemical on natural gas as a source of methane and energy. In this form, wherein the potential energy is releases in the form of process, natural gas and a chemical catalyst are mixed with hydrogen during anaerobic fermentation. In addition, hydro steam at high temperatures and pressure to form hydrogen gen production may also result from direct photobiological and carbon dioxide. In this process a non-renewable energy processes. Biomass sources available for conversion into Source is used to generate hydrogen, thus; it would be ideal to hydrogen include, but are not limited to, dedicated bioenergy replace the fossil fuels with renewable energy sources, such crops and/or less expensive residues, such as organic waste as hydrogen production via biomass fermentation. Potential from regular agricultural farming and wood processing (bio hydrogen production may be derived from renewable energy mass residues). In contrast, direct photobiological hydrogen Sources Such as, but not limited to, energy crops, Surplus production, does not require biomass, as water is directly agricultural products, waste from Sugar production and pro cleaved by photosynthetic microorganisms. Regardless, cessing facilities, animal waste from Zoos, waste from fruit hydrogen production by these biological means is attractive processing industries, waste from pulp and paper mills, sil due to the renewable energy nature of Solar energy. Vaculture residues, waste from wood processing, waste from 0114. A distinction can be made between the use of dry agricultural product processing, food waste, Solids isolated biomass (such as wood) and the use of wet biomass sources from fermentation cultures, municipal sewer waste, animal Such as the organic fraction of domestic waste, agro-indus manure, animal urine, animal parts, fish parts, and combina trial wastes and slurries, and wastewater. Dry biomass is used tions thereof. for thermal conversion processes that require minimal water 0111. The future energy economy may have an important content such as green electricity generation (via combustion role for hydrogen as a clean, carbon dioxide neutral energy or gasification) or the production of renewable diesel fuel source for use in, by way of example only, fuel cell vehicles through gasification, followed by Fischer-Tropsch Synthesis. and for decentralized electricity generation in stationary fuel Wet biomass and residues are typically less suitable for ther cell systems. In fuel cells, hydrogen can be converted to mal conversion because transport and drying require a con electricity very efficiently, producing only water as a waste siderable amount of energy, which leads to a limited or even product, thus drastically reducing carbon dioxide, nitric negative overall carbon dioxide reduction. The available oxide, particulate and other emissions that accompany the use amount of wet biomass and residues is however considerable, of fossil fuels. Although the use of hydrogen produced from Such that their use as sources for renewable energy production fossil sources can lead to a substantial reduction of emissions, is desirable. Biological conversion processes are particularly the energy efficiency of the production-to-end-use chain useful for this application because they are catalyzed by (from natural gas to hydrogen to electricity) is limited. This is microorganisms in an aqueous environment at low tempera due to energy losses in the hydrogen production phase with ture and pressure. Furthermore these techniques are well concomitant carbon dioxide capture. In the long run, hydro Suited for decentralized energy production in Small-scale gen would ideally be produced from renewable sources such installations in locations where biomass or wastes are avail as the electrolysis of water with renewable electricity, or by able, thus avoiding energy expenditure and costs for trans means of biomass gasification, or biological fermentation or port. The general expectation is that biological processes may photobiological hydrogen production. With large-scale play a substantial role in the production of renewable gaseous implementation of renewable energy production, hydrogen and liquid biofuels including methane, hydrogen, bioethanol can be a clean carrier of energy for storage and transport. and ABE (Acetone-Butanol-Ethanol). 0112 Hydrogen can be produced from a variety of feed 0115 Hydrogen production from biomass has been stocks using a variety of process technologies. Feedstock thought to be an expensive process. This is reflected when options include fossil resources such as coal, natural gas, and comparing the higher costs for biomass, distribution, and petroleum, and renewable resources such as biomass, Sun fixed capital costs relative to other production methods such US 2008/0311640 A1 Dec. 18, 2008 as natural gas reforming. In addition, due to the heterogeneity 0119 Anaerobic microbiological decomposition is a pro of biomass, the localized production of biomass, and the cess in which micro-organisms derive energy and grow by relatively high costs of gathering and transporting biomass metabolizing organic material in an oxygen-free environment Sources, digester complexes dedicated for biomass gasifica resulting in the production of various end-products. The tion are limited to midsize-scale operations. Additionally, anaerobic digestion process can be subdivided into four factors not associated in the economic analysis are fertilizer phases, each requiring its own characteristic group of micro costs, the environmental impact associated with the produc organisms: hydrolysis (conversion of non-soluble biopoly tion, harvest, and transport of biomass, or any potential deg mers to soluble organic compounds), acidogenesis (conver radation in land quality associated with intensive bioenergy sion of soluble organic compounds to Volatile organic acids, crop farming. Increased efficiency of continuous hydrogen also referred to as volatile fatty acids, and carbon dioxide), production may be obtained by improvements in Systems, acetogenesis (conversion of carbohydrates or Volatile organic methods and compositions used for anaerobic bacterial fer acids to acetate and hydrogen), and methanogenesis (conver mentation. The ability to effectively utilize a variety of bio sion of organic acids or acetate and carbon dioxide plus mass types, and the development of localized energy produc hydrogen to methane gas). The acidogenic bacteria excrete tion from decentralized fuel cell power stations, may enzymes for hydrolysis and convert Soluble organics to Vola ultimately decrease biomass harvest and transportation tile organic acids and alcohols. Volatile organic acids and 1SSU.S. alcohols are then converted by acetogenic bacteria into acetic Production of Hydrogen and Other Chemical Products from acid or hydrogen and carbon dioxide. Methanogenic bacteria Biomass then use acetic acid, hydrogen and carbon dioxide to produce 0116. The production of hydrogen is a ubiquitous, natural methane. phenomenon under anaerobic conditions. A wide variety of I0120 Bio-methane production through anaerobic diges bacteria, from Swamps, sewage, hot springs, and the rumen of tion of wastes and wastewater using mixtures of bacteria cattle are able to convert organic matter to hydrogen, carbon species is an established technology. The final biogas product dioxide and other chemical products including, but not lim is a mixture of methane (55-75 Vol.%) and carbon dioxide ited to, acetic acid, butryric acid, lactate, and ethanol. In (25-45 Vol.%) which can be used for heating, upgrading to general, these bacteria live in the close proximity to other natural gas quality or co-generation of electricity and heat. bacteria which consume the chemical products, including The biogas yield varies with the type and concentration of the hydrogen, producing their own endproducts like methane and biomass and process conditions. However, there are many carbon dioxide. In this way, a stable ecosystem is formed disadvantages of conventional anaerobic treatment for meth wherein hydrogen consumers remove hydrogen, and allow ane production, including a high sensitivity of methanogenic continued growth of hydrogen producers, which would oth bacteria to a large number of chemical compounds, the first erwise be inhibited by high concentrations of hydrogen. start-up of an installation without the presence of proper seed 0117. In anaerobic environments, hydrogen is commonly sludge can be time-consuming due to the low growth yield of produced during microbial breakdown of organic com anaerobic bacteria, and the anaerobic treatment can be pounds. When organic compounds are the Sole carbon and accompanied by odor due to the formation of sulfide when energy source providing metabolic energy under anaerobic treating waste water containing Sulfurous compounds. Dur conditions, the process is termed "dark hydrogen fermenta ing this methane production through anaerobic digestion of tion', or “dark fermentation’. When light is required to pro wastewater and residues (including sewage sludge, manure vide additional energy, the process belongs to the category of and the organic fraction of municipal waste) hydrogen is an photobiological processes and is termed "photo-fermenta intermediary product, which is not available because it is tion. rapidly taken up and converted into methane by methane 0118 When bacteria grow on organic substrates (het producing microorganisms. Also present are organisms that erotrophic growth), these substrates are degraded by oxida compete for nutrients and create undesirable end products. tion to provide building blocks and metabolic energy for The final gas produced in this method is also contaminated growth. This oxidation generates electrons which need to be with hydrogen Sulfide, ammonia, and siloxanes which dam disposed of in order to maintain electrical neutrality. In aero age energy producing equipment. This method can also be bic environments, oxygen is reduced and wateris the product, time consuming and resource intensive due to extended however, in anaerobic environments no oxygen is present and hydraulic retention times. Thus, to achieve efficient hydrogen therefore other compounds act as electron acceptors. For production from anaerobic fermentation, as achieved using instance, protons accept electrons and are thereby reduced to the methods disclosed herein, methanogenic bacteria mixed molecular hydrogen (H). In “dark fermentation” a wide vari with hydrogen producing bacteria, thereby eliminating the ety of bacteria use the reduction of protons to dispose of possibility of hydrogen consumption. The methods described reducing equivalents which results from primary metabolism. herein decouple hydrogen formation and consumption Such Other examples of alternative electron acceptors in anaerobic that hydrogen is available as the final product. environments are nitrate with nitrogen gas (N) as the product Anaerobic Production of Hydrogen and Other Chemical or sulfate with dihydrogensulfide (H2S) as the reduced prod Products from Biomass uct. Even organic compounds can act as electron acceptors. 0121 The present approach takes advantage of emulating For instance, the microbial production of butanol occurs a natural environment for anaerobic bacterial growth in order through the reduction of butyric acid. The capacity to reduce to achieve optimal hydrogen production. This is contrary to other electron acceptors than oxygen requires the presence of previous less efficient attempts at hydrogen production using a specific enzyme system in the micro-organisms: hydrogen anaerobic fermentation by genetically modifying bacteria or producing bacteria possess hydrogenase enzymes; nitrate by forcing bacteria to exist in unnatural environments and reducing bacteria possess an elaborate set of enzymes cata coaxing them to produce hydrogen. FIG. 1 depicts one lyzing the stepwise reduction of nitrate to nitrogen etc. approach of the methods described herein, wherein hydrogen US 2008/0311640 A1 Dec. 18, 2008

is produced under anaerobic conditions, using anaerobic bac leric, Valeric, isocaproic, caproic, and heptanoic acids. By teria to anaerobically ferment different types of biomass. way of example alcohol products from fermentation include Central to the approach is the Anaerobic Digester, in which ethanol, ethanol, propanol, isobutanol, butanol, isopentanol, the natural environment for anaerobic bacterial growth is pentanol, acetone, propanol, isopropanol, and 1, 2-pro emulated to achieve optimal hydrogen production. The panediol. By way of example non-volatile products from digester operating parameters (such as, by way of example fermentation include pyruvic, lactic, oxalacetic, oxalic, only, pH, pressure and temperature) are monitored and methyl malonic, malonic, fumaric, Succinic. adjusted accordingly in order to control and maintain the appropriate environment. Similarly, the concentrations of the Knowledge Management System fermentation products are monitored and the products 0.124 One aspect of the methods described herein is the removed from the digester in order to maintain optimal creation of a database by compilation of information regard anaerobic fermentation conditions in the digester, with opti ing the hydrogen producing characteristics of various isolated mal hydrogen production. By way of example only, Such bacteria genera and species, along with optimal biomass used products removed may include organic acids/solvents/solids, by the respective bacteria and optimal conditions. This data bacterial biomass, carbon dioxide and hydrogen. base is incorporated into a Knowledge Management System, 0122 One aspect of the approach, shown in FIG. 1, is the which is an effective tool for exploitation of anaerobic hydro selective isolation of anaerobic bacteria, which use particular gen production as an alternative energy source. The type of types of biomass, and are abundant hydrogen producers. This biomass available for hydrogen production via anaerobic fer selection, modification and maximization is achieved by uti mentation may vary. As shown in FIG. 2, the Knowledge lizing an anaerobic bacteria isolation/enrichment system in Management System allows for the identification of bacteria, which a variety of food sources are arranged, and the system with known hydrogen producing characteristics for a particu is deoxygenated and inoculated with a mixture of bacteria lar biomass, and the optimal digesteroperating parameters for obtained from different sources. The bacteria are allowed to anaerobic fermentation of the specific biomass available. grow in the isolation/enrichment system, and those that thrive This system therefore can minimize the time required for the on a particular biomass are removed and evaluated for spe discovery, research, and development process, and thereby cific Substrate utilization and end products generated. These increase the efficiency of an energy plant based on anaerobic bacteria are placed in a separate cultivation system for con fermentation. tinued growth using the optimal biomass indicated from the 0.125. The Knowledge Management System software isolation/enrichment system. The cultivation systems allow includes user friendly options that aid the user in navigating for the isolation of bacteria on particular substrates under the system. Such options include creating new identifications, various growth conditions to optimize hydrogen production. opening identifications, saving an identification, opening a Various growth conditions may include variation in tempera Session, saving a session, importing data, finding an organ ture, pH, fermentation products, exposure to various natural ism, finding an indicator test, finding a reference, finding a gases or compounds, drug efficacy, compound toxicity or photo, comparing organisms, showing related organisms, survivability. The cultivation system aids in determining the finding common traits, adding organisms, adding traits, add primary food source for particular bacteria strains and the ing the source, user login, user logout, checkout of organisms, optimal growing conditions. The isolated bacteria are then checking of organisms, testing the server, reports on identifi harvested from the cultivation system and stored for later use ability, column sorting, preferred tests, Sorting organisms, as inoculum for hydrogen production in anaerobic digesters. starting over, and Switch to relatedness mod. The Knowledge Of importance to the successful operation of the anaerobic Management System houses data that can be quickly digester is the selection of nutrients for a particular bacteria accessed and manipulated into a user friendly form such as a species, and the preparation, detoxification, sterilization and/ graph or spreadsheet. In particular, the Knowledge Manage or pre-digestion of nutrients prior to introduction into the ment System can narrow down which bacteria uses particular digester. The selection of nutrients is known from the enrich nutrients for producing a specific amount of hydrogen. ment/incubation process, in which the bacteria species was 0.126 FIG. 3 is an example of a screen shot from the originally isolated. Finally, the information obtained for a Knowledge Management System that displays various traits particular species of bacteria, Such as, by way of example that were determined by indicator tests listed along the top only, optimal biomass, optimal growth pH, optimal growth row of columns and the various organisms listed in the first temperature, and fermentation products, are compiled into a left hand column. FIG. 4 is an example of a screen shot from database. A Knowledge Management System, which the Knowledge Management System that displays a Preferred includes the database of information, can be later utilized to Test which groups only a particular subset of traits. FIG. 5 is identify an optimal bacterial species for use in producing an example of a screen shot from the Knowledge Manage hydrogen from a particular biomass, rather than trying to ment System that displays appropriate information for the genetically engineer the bacteria to ferment the biomass. bacteria Bilophila wadsworthia. Such information may However, bacteria may be obtained by genetic modification include habitat, growth rate, identification, where it is nor of known bacterial strains and evaluated in the isolation/ mally associated or found, information from in vitro, in Vivo, enrichment system. The genetic modification of the bacterila and molecular studies, Subgroups or strains, growth condi strains may results from transformation procedures, bacterial tions, diameter, form, elevation, margin, color, density, gen conjugation, transduction, interaction of bactrial strains with eral information, colony morphology, cellular morphology, mutagens and combinations thereof. The mutagens used may references, photos of gram stain, photos of blood plates, and include, but are not limited to, chemicals, ultraviolet light, photos of BBE/LKV plates. and radioactive element. I0127. The Knowledge Management System houses 0123. By way of example volatile fatty acids produced appropriate information for each bacterial species. Appropri include formic, acetic, propionic, isobutyric, butyric, isova ate information may be collected from scientific literature and US 2008/0311640 A1 Dec. 18, 2008 20 may consist of information regarding bacteria growth on Vari automated testing system includes various identification ous Substrates, bacteria sensitivity to a condition and/or bac workstation that includes analytical instruments, such as a teria production of metabolites. Such traits may also be CCD camera or a microscope or other instruments for cap assayed for by indicator tests. By way of example, such traits turing the image of a bacterial sample or instruments that can may include growth, reactions or production from: acetic acid measure bacterial physiological changes, and a computer for major metabolic product, acetic acid minor metabolic prod data analysis which is capable of communicating with the uct, ADH, ALP, alpha-fucosidase, alpha-galactosidase, analytical instrument. alpha-glucosidase, arabinose, ArgA, bacillus, beta-galactosi 0.130. In an exemplary embodiment, the computer is a dase, beta-glucosidase, beta-glucuronidase, beta-NAG, beta desktop computer system, such as a computer that operates Xylosidase, box car shape, butyric acid major metabolic prod under control of the “Microsoft Windows: operation system uct, butyric acid minor metabolic product, CAMP. caproic of Microsoft Corporation or the “Macintosh' operating sys acid major metabolic product, catalase, cellobiose, chartreuse tem of Apple Computer, Inc., that communicates with the fluorescence, chymotrypsin, CO. growth, coccus, des instrument using a known communication standard Such as a ulfoviridin, double Zone beta-hemolysis, esculin hydrolysis, USB, parallel or serial interface. F/F required, fructose, gelatin hydrolysis, glucose, glycogen, I0131 For example, systems for analysis of bacterial gram reaction, growth in bile, His A, I-arabinose, indole, samples are provided. The system includes a processing sta isobutyric acid major metabolic product, isobutyric acid tion that performs the various indicator tests. For example, the minor metabolic product, isocapronic acid major metabolic system may include a processing station that performs the product, isocapronic acid minor metabolic product, isovaleric nitrate indicator test and a catalase indicator test. The nitrate acid major metabolic product, isovaleric acid minor meta indicator test determines an organism's ability to reduce bolic product, lactate converted to propionate, lactic acid nitrate to nitrite. A nitrate disk is placed on the plate at the time major metabolic product, lactic acid minor metabolic prod of inoculation. After 24-48 hours, reagents A (Sulfanilic acid uct, lactose, leithinase, LeuA, lipase, maltose, mannitol, man dissolved in glacial acetic acid) and B (1,6-Cleve's acid dis nose, melezitose, melibiose, milk clot formed, milk digested, Solved in glacial acetic acid) are added and a positive test is motile, N-Acetyl-beta-gulcosaminidase, nitrate, ONPG indicated by a bright magenta color. If no color is seen within (Beta-galactosidase), oxygen tolerance, Phea, phenylaceric a few minutes, Zinc dust is added to make Sure the nitrate has acid minor metabolic product, pigment, pitting of agar, ProA, not reduced beyond nitrite. Zinc dust, too, reduces nitrate to propionic acid major metabolic product, Pyra, raffinose, red nitrite. Magenta coloration at this point is interpreted as a fluorescence, reverse CAMP test, rhamnose, ribose, salicin, negative result because it indicates that the nitrate was not sensitive to colistin, sensitive to kanamycin, sensitive to SPS, previously reduced by the organism. No color change after sensitive to Vancomycin, Sorbitol, spore former, starch the addition of Zinc is a positive result, because the organism hydrolysis, strictly anaerobic, Subterminal spore location, reduced the nitrate beyond nitrite. The catalase indicator test, Succinic acid major metabolic product, Succinic acid minor an example of which is shown in FIG. 6, is when the presence metabolic product, Sucrose, terminal spore location, threo of catalase is determined by the addition of 15 percent hydro nine converted to propionate, trehalose, trypsin, Tyra, urease, gen peroxide to a loop of cells placed on a glass slide. The Valeric acid major metabolic product, Valeric acid minor formation of gas bubbles indicates a positive reaction. Thered metabolic product, Xylan, and Xylose. Bacterial response may blood cells in brucella and other blood agar plates contain include positive, negative, or no response. Bacterial response catalase and scraping the media may give a false-positive may also be measured by Smell, color, growth, non-growth, reaction. death, symbiosis and non-symbiosis. 0.132. The system may also include a spectral analysis 0128. The Knowledge Management System software also system that analyzes the bacterial color changes from a CCD includes statistical modeling programs which aid in identify camera which processes results from a nitrate indicator test or ing an optimal biomass for anaerobic bacterial hydrogen pro a gas formation analysis system that analyzes formation of duction. Such methods include Hidden Markov Models, phy gas by bacteria from a CCD camera which processes results logenetic inferences which include clustering methods such from a catalase indicator test; and a complete data analysis as (UPGMA) Unweighted Pair Group Methods using Arith system, Such as a computer programmed to identify which metic averages, distance and parsimony methods, maximum biomass is optimal for a particular bacteria to produce hydro likelihood estimation and Chomsky hierarchy. For example, gen anaerobically. The system can also include a control given a set of growth conditions by which indicator tests have system that determines when processing at each station is been performed to identify traits of bacteria, use of probabi complete and, in response, moves the sample to the next test listic modeling may infer whether using different growth station, and continuously processes samples one after another conditions deter or optimize production of hydrogen. Also until the control system receives a stop instruction. use of phylogenetic trees may aid to infer relationships 0.133 FIG. 7 is an example of a block diagram of a system between bacterial species and assist in determining for that performs sample processing and biological workstation example the likelihood of whether certain unperformed indi 702 and an analysis computer 740. At the biological worksta cator tests will result in either similar or dissimilar results tion, one or more samples 706 are received and prepared for compared to that of a related bacteria species. analysis at a sample processing station 708, where the above 0129. Also provided are systems that automate the optimal described indicator tests can take place. The samples are then biomass detection process for particular bacterial genera and moved to an analysis station 712, where analysis of the bac species using a computer programmed for identifying posi teria Such as spectral or gas formation analysis can be pro tive and negative results based upon the methods provided cessed and recorded. The samples are moved from the pro herein. The methods herein can be implemented, for example, cessing station 708 to the analysis station 712 by either a by use of the following computer systems and using the computer-controlled robotic device 710 or by manual pro following calculations, systems and methods. An exemplary cessing, not shown. US 2008/0311640 A1 Dec. 18, 2008

0134. The robotic device can include subsystems that the art will appreciate that the stations and computers illus ensure movement between all of the stations that preserve the trated in FIG. 7 can all have a similar computer construction, integrity of the samples 706 and ensure valid test results. The or can have alternative constructions consistent with the capa Subsystems can include, for example, a mechanical lifting bilities and respective functions described herein. The FIG. 8 device or arm that can pick up a sample from the sample construction is especially Suited for the data analysis com processing station 708 and then move to and deposit the puter 704 illustrated in FIG. 7. processed sample for analysis at the analysis station 712. 0140 FIG. 8 displays an exemplary computer 800 such as 0135 The analysis station 712 produces data that identi fies and quantifies the positive and negative results of the might comprise a computer that controls the operation of any indicator tests of the sample 706 being measured. Those of the stations and analysis computers 708, 712, and 704. skilled in the art will be familiar with the biological monitor Each computer 800 operates under control of a central pro ing systems, such as microscopes, that can be used to identify cessor unit (CPU) 802, such as a “Pentium microprocessor positive or negative bacterial reactions or a CCD camera, and associated integrated circuit chips, available from Intel which can be used to image the results. The data is provided Corporation of Santa Clara, Calif., USA. A computer user can from the analysis station 712 to the analysis computer 704, input commands and data from a keyboard and computer either by manual entry of measurements results into the mouse 804, and can view inputs and computer outputs at a analysis computer or by communication between the analysis display 806. The display is typically a video monitor or flat station and the analysis computer. For example, the analysis panel display. The computer 800 also includes a direct access station 712 and the analysis computer 704 can be intercon storage device (DASD) 808, such as a hard disk drive. The nected over a network 714 such that the data produced by the computer includes a memory 810 that typically comprises analysis station can be obtained by the analysis computer. The Volatile semiconductor random access memory (RAM). Each network 714 can comprise a local area network (LAN), or a computer includes a program product reader 812 that accepts wireless communication channel, or any other communica a program product storage device 814, from which the pro tions channel that is suitable for computer-to-computer data gram product reader can read data (and to which it can option exchange. ally write data). The program product reader can comprise, 0136. The processing function of the analysis computer for example, a disk drive, and the program product storage 704 and the control function of the biology workstation 702 device can comprise removable storage media Such as a mag can be incorporated into a single computer device, if desired. netic floppy disk, a CD-R disc, a CD-RW disc, or DVD disc. In that configuration, for example, a single general purpose 0141. Each computer 800 can communicate with the other computer can be used to control the robotic device, not FIG. 7 systems over a computer network 820 (such as, for shown, and to perform the data processing of the data analysis example, the local network 714 or the Internet or an intranet) computer 704. Similarly, the operations of the analysis station through a network interface 818 that enables communication 712, the sample processing operations of the sample process over a connection 822 between the network 820 and the ing station 708, or the other additional stations, not shown, computer. The network interface 818 typically comprises, for can be performed under the control of a single computer. example, a Network Interface Card (NIC) that permits com 0.137 Thus, the processing and analysis functions of the munication over a variety of networks, along with associated stations and computer 708, 712, and 704 can be performed by network access Subsystems. Such as a modem. a variety of computing devices, if the computing devices have 0142. The CPU 802 operates under control of program a Suitable interface to any appropriate Subsystems (such as a ming instructions that are temporarily stored in the memory mechanical arm of the robotic device, not shown) and have 810 of the computer 800. When the programming instructions Suitable processing power to control the systems and perform are executed, the computer performs its functions. Thus, the the data processing. programming instructions implement the functionality of the 0.138. The data analysis computer 704 can be part of the respective workstation or processor. The programming analytical instrument or another system component or it can instructions can be received from the DASD 808, through the be at a remote location. The computer system can communi program product storage device 814, or through the network cate with the instrument, for example, through a wide area connection 822. The program product storage drive 812 can network or local area communication network or other Suit receive a program product 814, read programming instruc able communication network. The system with the computer tions recorded thereon, and transfer the programming instruc is programmed to automatically carry out steps of the meth tions into the memory 810 for execution by the CPU 802. As ods herein and the requisite calculations. For embodiments notes above, the program product storage device can com that use color changing patterns (for a reference) based on the prise any one of multiple removable media having recorded indicator test used in the processing station, a user enters the computer-readable instructions, including magnetic floppy spectral identification for the bacteria sample. These data can disks and CD-ROM storage discs. Other suitable program be directly entered by the user from a keyboard of from other product storage devices can include magnetic tape and semi computers or computer systems linked by network connec conductor memory chips. In this way, the processing instruc tion, or on removable storage medium such as a data CD, tions necessary for operation in accordance with the methods minidisk (MD), DVD, floppy disk, jump disk or other suitable and disclosure herein can be embodied on a program product. storage medium. Next, the user initiates execution Software 0.143 Alternatively, the program instructions can be that operates the system in which the spectral identification is received into the operating memory 810 over the network constructed for the bacteria imaged. 820. In the network method, the computer 800 receives data 0139 FIG. 8 is a block diagram of a computer in the including program instructions into the memory 810 through system 800 of FIG. 7, illustrating the hardware components the network interface 818 after network communication has included in a computer that can provide the functionality of been established over the network connection 822 by well the stations and computers 708, 712, and 704. Those skilled in known methods that will be understood by those skilled in the US 2008/0311640 A1 Dec. 18, 2008 22 art without further explanation. The program instructions are sites according to their environmental tolerances and their then executed by the CPU 802 thereby comprising a computer carbon and energy requirements. process. 0.148. A typical Winogradsky column is usually a glass or 0144. It should be understood that all the stations and computers of the system 700 illustrated in FIG. 7 can have a plastic tube, about 30 cm tall and 5 cm diameter. Mud from construction similar to that shown in FIG. 8, so that details the bottom of a lake or river, supplemented with cellulose described with respect to the FIG. 8 computer 800 will be (e.g. newspaper), Sodium Sulphate and calcium carbonate, is understood to apply to all computers of the system 700. It then added to the lower one-third of the tube, and the remain should be appreciated that any of the communicating stations der of the tube is filled with water from the lake or river. The and computers can have an alternative construction, so long tube is then capped (not sealed), allowing airflow to the top of as they can communicate with the other communicating sta the water column, and placed near a window with Supplemen tions and computers illustrated in FIG. 7 and can support the tary strip lights. All the organisms are present initially in low functionality described herein. For example, if a workstation numbers, but after incubation for 2 to 3 weeks the different is unable to receive program instructions from a program types of microorganisms proliferate and occupy distinct product device, then it is not necessary for that workstation to Zones where the environmental conditions favor their specific include that capability, and that workstation will not have the activities. The large amount of cellulose added initially pro elements depicted in FIG. 8 that are associated with that motes rapid microbial growth which Soon depletes the oxy capability. gen in the sediment and in the water column. Only the very top of the column remains aerated because oxygen diffuses very slowly through water from the capped tube opening. The Bacteria Selection, Modification and Maximization only organisms that can grow in the anaerobic conditions are 0145 The various species of bacteria and various forms of those that ferment organic matter and those that perform biomass described herein may be used in all aspects of anaerobic respiration. For example, Clostridium species are anaerobic production of hydrogen and other chemical prod strictly anaerobic and start to grow when the oxygen is ucts presented herein. For example, the various species of depleted in the sediment. Cellulose-degrading Clostridium bacteria may be used in the anaerobic fermentation appara species degrade the cellulose to glucose and then ferment the tuses described herein, using any form of biomass presented glucose producing a range of simple organic compounds (e.g. as a food source. acetic acid), carbon dioxide, and hydrogen as the fermenta 0146 In contrast to known anaerobic methods, which gen tion end products. Sulphur-reducing bacteria, such as Des erate hydrogen from mixed bacteria cultures obtained from ulfo vibrio, utilize these fermentation products by anaerobic soil or other samples, the methods described herein produces respiration, using either Sulphate or other partly oxidised hydrogen using specific isolated anaerobic bacteria Strains, forms of Sulphur (e.g. thiosulphate) as the terminal electron chosen for the ability of particular strains to utilize specific acceptor, generating large amounts of HS by this process. biomass/nutrients as food sources. The approach is to isolate The HS can react with any iron in the sediment, producing and culture specific bacterial strains which are high hydrogen blackferrous sulphide, and some of the HS diffuses upwards producers for particular biomass. By way of example only, a into the water column, where it is utilized by other organisms. particular bacteria Strain may yield large quantities of hydro The diffusion of HS from the sediment into the water column gen from cellulosic material, whereas a different isolated enables anaerobic photosynthetic bacteria to grow, resulting strain may yield large quantities of hydrogen from sewage. in a Zone of green Sulphur bacteria immediately above the Thus, selection and isolation of specific bacterial strains with sediment, followed by a Zone of purple sulphur bacteria. The enhanced fermentation properties for specific biomass allows green and purple Sulphur bacteria gain energy from light for optimization of hydrogen production as the type of Sub reactions and produce their cellular materials from CO in strate available varies. much the same way as plants do. However, HS is used as the 0147 The Winogradsky column can be used to demon reductant instead of water and rather than generate oxygen strate the metabolic diversity of prokaryotes, and how the during photosynthesis elemental Sulphur is formed. The activities of different microorganisms enable other organisms purple Sulphur bacteria (e.g. Thiocapsa) typically have large to grow in an interdependent or symbiotic manner. In addi cells and they deposit Sulphur granules inside the cells, tion, the column demonstrates how elements, such as Sulphur, whereas green Sulphur bacteria have Smaller cells and typi nitrogen, carbon and other elements are cycled in natural cally deposit sulphur externally. Note that the sulphur (or environments. These columns are complete, self-contained sulphate formed from it) produced by these photosynthetic recycling systems, driven only by energy from light. All life bacteria returns to the sediment where it can be recycled by on earth can be categorized in terms of the organism's carbon Desulfovibrio as part of the sulphur cycle in natural waters. and energy source. Energy can be obtained from light reac Most of the water column above the photosynthetic bacteria is tions (phototrophs) or from chemical oxidations of organic or coloured bright red by a large population of purple non inorganic Substances (chemotrophs); the carbon for cellular Sulphur bacteria. These include species of Rhodopseudomo synthesis can be obtained from CO (autotrophs) or from nas, Rhodospirillum and Rhodomicrobium. These bacteria preformed organic compounds (heterotrophs). Combining are photoheterotrophs, as they grow in anaerobic conditions, these categories, we get the four basic life strategies: photo gaining their energy from light reactions but using Volatile autotrophs (e.g. plants), chemoheterotrophs (e.g. animals, organic acids (e.g. acetic acid) as their carbon Source for fungi), photoheterotrophs and chemoautotrophs. Only in the cellular synthesis. The Volatile organic acids that they use are bacteria do we find all four basic life strategies. The prokary the fermentation products of other anaerobic bacteria (e.g. otic bacteria and archaea exhibit an astonishing metabolic Clostridium species), but the purple non-Sulphur bacteria are diversity, which far exceeds that of animals, plants, fungi and intolerant of high HS concentrations, so they occur above the other higher organisms, and Winogradsky columns also dem Zone where the green and purple Sulphur bacteria are found. onstrate how microorganisms occupy highly specific micro Nearer the top of the water column the purple non-sulphur US 2008/0311640 A1 Dec. 18, 2008

bacteria disappear due to the oxygenated water. A variety of these food sources at various locations throughout the isola microorganisms can grow in the oxygenated Zone at the top of tion/enrichment. In addition, any of the biomass known in the the water column; in particular Sulphur-oxidising bacteria, art may also be used. The anaerobe bacteria isolation/enrich and cyanobacteria. Any HS that diffuses into the aerobic ment system is filled with deoxygenated water, inoculated Zone can be oxidized to Sulphate by Sulphur-oxidising bacte with a mixture of bacteria species, and the top is then sealed. ria. These bacteria are chemosynthetic or chemoautotrophic The head space above the deoxygenated water is purged to organisms, since they gain energy from oxidation of H2S, and remove any oxygen present. The mixture of bacteria species they synthesize their own organic matter from CO. Similar used in the anaerobe bacteria isolation/enrichment system types of organism occur in Soils, gaining energy from the may be obtained from cattle rumen, a sample of soil, sludge, oxidation of ammonium to nitrate, which then leaches from the Soil and can accumulate in water Supplies. The photosyn an anaerobic bacteria culture, an aerobic bacteria culture, thetic cyanobacteria also grow in the aerobic Zones, and are anaerobic sediments from fresh or brackish waters, sewage, the only bacteria that have oxygen-evolving photosynthesis animal feces, hydrothermal soils and pools, deep water like that of plants. Once the cyanobacteria start to grow they hydrothermal vents or other potential source of anaerobic oxygenate the water, which forces the anaerobic bacteria bacteria. The bacteria in the isolation/enrichment system may toward the bottom, thereby allowing the cyanobacteria and thrive on a particular food source as they continue to grow other aerobes to occupy a larger portion of the Winogradsky under anaerobic conditions. The bacteria isolated in this sys column. tem may include, by way of example only, species such as Abiotrophia defectiva, Acidaminococcus fermentans, Actino 014.9 The Winogradsky column is essentially a miniature baculum Schalii, Actinomyces europaeus, Actinomyces ecosystem, wherein the metabolic diversity of bacteria allows finkei, Actinomyces georgiae, Actinomyces gerensceriae, for interdependent relationships to exist throughout the col Actinomyces graevenitzii, Actinomyces israelii, Actinomyces umn. The fermentation products of one species are consumed meyeri, Actinomyces naeslundi, Actinomyces neuii species by another, thereby cycling various elements, such as Sulphur, anitratus, Actinomyces neuii species neuii, Actinomyces Ond nitrogen, and carbon, and maintaining the life cycle. Without Ontolyticus, Actinomyces radicidentis, Actinomyces radin manipulating the growth of one species over another, the gae, Actinomyces turicensis, Actinomyces urogenitalis, Acti Winogradsky column demonstrates how microorganisms nomyces viscosis, Anaerorhabdus fircosa, Arcanobacterium occupy highly specific locations according to their environ bernardiae, Arcanobacterium hemolyticum, Arcanobacte mental tolerances and their carbon and energy requirements. rium pyogenes, Atopobium minutum, Atopobium parvulum, The initial food source is common throughout the initial Atopobium rimae, Atopobium vaginae, Bacteroides caccae, sediment, and the variety of species required for the develop Bacteroides capillosus, Bacteroides coagulans, Bacteroides ment of the “ecosystem” is initially present. distasonis, Bacteroides eggerthii, Bacteroides forsythus, 0150. The apparatus, methods and processes for bacterial Bacteroides fragilis, Bacteroides merdae, Bacteroides ova isolation and enrichment described herein include anaerobic tus, Bacteroides putredenis, Bacteroides pyogenes, Bacteroi bacteria isolation/enrichment systems designed to be ulti des splanchnicus, Bacteroides Stercoris, Bacteroides tectus, mately anaerobic and to optimize growth of bacteria species Bacteroides thetaiotaomicron, Bacteroides uniformis, depending on the food source provided. In this manner bac Bacteroides ureolyticus, Bacteroides vulgatus, Bifidobacte teria with optimal fermentation properties are selected out for rium adolescentis, Bifidobacterium angulatum, Bifidobacte maximum hydrogen production. In addition, the apparatus, rium bifidum, Bifidobacterium breve, Bifidobacterium methods and processes for bacterial isolation and enrichment catenulatum, Bifidobacterium denticolens, Bifidobacterium described herein may also allow for the growth and isolation dentium, Bifidobacterium infantis, Bifidobacterium inopina of anaerobic bacteria which utilize fermentation products of turn, Bifidobacterium longum, Bifidobacterium other species as food sources, thereby increasing hydrogen pseudocatenulatum, Bilophila wadsworthia, Bulleidia ext production. These features illustrate the difference between cructa, Campylobacter gracilis, Campylobacter hominis, the apparatus, methods and processes for bacterial isolation Campylobacter rectus, Campylobacter Showae, Capnocy and enrichment described herein, and a Winogradsky col tophaga granulosa, Capnocytophaga haemolytica, l, Clostridium argentinense, Clostridium baratii, Clostridium 0151. The isolation/enrichment system can take any bifermentans, Clostridium botulinum types A B F. shape, including, but not limited to, circular, cylindrical, Clostridium botulinum types B E F. Clostridium botulinum spherical, square, or rectangular form, and may have a Vol types C D. Clostridium butyricum, Clostridium cadaveris, ume ranging from 100 cm to 50,000 cm. One embodiment Clostridium carnis, Clostridium clostridioforme, of the anaerobic bacteria isolation/enrichment system Clostridium difficle, Clostridium glycolicurn, Clostridium described herein is shown in FIG. 9. Here, the isolation/ hastiforme, Clostridium histolyticum, Clostridium indolis, enrichment system resembles an ant farm or an aquarium, Clostridium innocuum, Clostridium limosum, Clostridium wherein two parallel glass plates with a spacer are sealed on malenominaturn, Clostridium novyi type A, Clostridium three sides to create an open trough. The top of the trough may paraputrificum, Clostridium perfingens, Clostridium pili eventually be closed and sealed to ensure anaerobic condi forme, Clostridium putrificurn, Clostridium rarnosurn, tions remain within the sealed chamber, although, venting Clostridium septicum, Clostridium sordellii, Clostridium systems may be used to prevent pressure build up due to gas sphenoides, Clostridium sporogenes, Clostridium subtermi evolution during fermentation. male, Clostridium symbiosum, Clostridium tertium, 0152 Preparation of the anaerobe bacteria isolation/en Clostridium tetani, Collinsella aerofaciens, Corynebacte richment system involves obtaining various potential food rium matriuchotii, Cryptobacterium curturn, Desulfomonas Sources, such as, by way of example only, vegetable materi pirgra, Dialister pneumosintes, Eggerthella lenta, Eikenella als, garlic material, shredded hay, grass clippings, shredded corrodens, Eubacteria sulci, Eubacteria tenue, Eubacterium newspaper, sawdust, corn starch, oatmeal, and arranging combesii, Eubacterium contortum, Eubacterium cylindroi US 2008/0311640 A1 Dec. 18, 2008 24 des, Eubacterium infirmum, Eubacterium limosum, Eubacte grown on agricultural waste, and then Subdividing the agri rium minutum, Eubacterium moniliforme, Eubacterium culture waste into specific crops, such as potatos, tomatos, modatum, Eubacterium rectale, Eubacterium saburreum, garlic, cranberries, and the like. Once it is evident that a Eubacterium saphenurn, Eubacterium WALI, Eubacterium bacterial colony is specific for a particular biomass type, the yuri, Filifactor alocis, Filifactor villosus, Finegoldia magna, bacteria are removed from the isolation/enrichment system, Fusobacterium gonidiaformans, Fusobacterium mortiferum, transferred for more controlled culture, grown, and the iso Fusobacterium naviforme, Fusobacterium necrophorum, lated bacteria species is harvested and stored for later use to Fusobacterium nucleatum, Fusobacterium russii, Fusobac inoculate and ferment a specific biomass in the digester. terium ulcerans, Fusobacterium varium, Gemella morbil Removal of bacteria from the isolation/enrichment system lorum, Granulicatella adiacens, Granulicatella elegans, may be done at any time period. By way of example only, Holdemaniafiliformis, Lactobacillus acidophilus, Lactoba bacteria may be removed after 1-7 days, or earlier, or after cillus brevis, Lactobacillus canteforme, Lactobacillus casei, 1-20 weeks, or longer. Bacteria may be removed from the Lactobacillus crispatus, Lactobacillus fermentum, Lactoba system by any means, for example, by use of a needle, Syringe cillus gasseri, Lactobacillus iners, Lactobacillus jensenii, O WaCUU. Lactobacillus leichmannii, Lactobacillus Oris, Lactobacillus paracasei subspecies paracasei, Lactobacillus paraplan Nutrient Selection and Preparation tarum, Lactobacillus plantarum, Lactobacillus rhamnosus, 0154 The various species of bacteria and various forms of Lactobacillus salivarius, Lactobacillus uli, Lactobacillus biomass described herein may be used in all aspects of vaginalis, Leptotrichia buccalis, Leptotrichia sanguinegens, anaerobic production of hydrogen and other, chemical prod Megasphaera elsdenii, Micromonas micros, Mitsuokella ucts presented herein. For example, the various species of multiacida, Mobiluncus curtisii species curtisii, Mobiluncus curtisii species holmesii, Mogibacterium pumilum, Mogibac bacteria may be used in the anaerobic fermentation appara terium timidum, Mogibacterium vescum, Peptococcus niger; tuses described herein, using any form of biomass presented Peptostreptococcus anaerobius, Peptostreptococcus asac as a food source. charolyticus, Peptosfeptococcus harei, Peptostreptococcus 0155 The methods described herein allows for the prepa hydrogenalis, Peptostreptococcus indolyticus, Peptostrepto ration of the biomass, which includes, but is not limited to, coccus ivorii, Peptostreptococcus lacrimalis, Peptostrepto harvesting or protecting the biomass quickly before spoilage, coccus lactolyticus, Peptostreptococcus Octavius, Pep sterilization (i.e. pasteurizing and/or acidifying), neutraliza to streptococcus prevOtii, Peptostreptococcus prevOtii/ tion (i.e. ammonium hydroxide), removal of oxygen (deoxy tetradius, Peptostreptococcus tetradius, Peptostreptococcus genation), and concentration adjustments, detoxification, trisimilis, Peptostreptococcus vaginallis, Porphyromonas and/or pre-digestion, prior to fermentation by isolated assacharolytica, Porphyromonas Cangingivalis, Porphy anaerobic bacteria. romonas canoris, Porphyromonas can sulci, Porphyromonas 0156 Harvesting or protecting the biomass before spoil cantoniae, Porphyromonas circumdentaria, Porphyromonas age or a state whereby the biomass is useless is a pre-condi crevioricanis, Porphyromonas endodontalis, Porphyromonas tion to optimize preparation of the biomass for bacterial con gingivalis, Porphyromonas gingivicanis, Porphyromonas Sumption. This is to ensure optimal hydrogen production guilae, Porphyromonas levi, Porphyromonas macacae, Pre from a biomass. An example of harvesting or protecting bio votella albensis, PrevOtella bivia, Prevotella brevis, Prevo mass before spoilage is when biomass is harvested and used tella bryantii, PrevOtella buccae, Prevotella buccalis, Prevo within a 24 hour period. An another example may include tella corporis, Prevotella dentalis, Prevotella denticola, refrigeration of the biomass to prevent spoilage. Prevotella disiens, Prevotella enoeca, Prevotella hep 0157 Pre-digestion includes the pre-treatment of biomass arinolytica, Prevotella intermedia, Prevotella loescheii, Pre to get it into a condition for the bacteria to feed upon. By way votella melanogenica, PrevOtella nigrescens, PrevOtella Ora of example only, pre-digestion may include addition of other lis, Prevotella Oris, Prevotella Oulorum, Prevotella pallens, insects, animals, enzymes, and/or solutions to modify the Prevotella ruminicola, Prevotella tannerae, PrevOtella ver biomass prior to introducing it to the bacteria. For example, oralis, PrevOtella zoogleoformans, Propionibacterium acnes, the breakdown of cellulose into glucose may be a pre-treat Propionibacterium avidum, Propionibacterium granulosum, ment/pre-digestion. Another example is the pre-treatment of Propionibacterium proionicus, Pseudoramibacter alactolyti manure into an anaerobic substrate feedstock for bacteria cus, Rothia dentocariosa, Ruminococcus hansenii, Rumino through use of an organism that breakdown bile/urea within coccus productus, Slakia exigua, Slakia heliontrinireducens, the manure. Staphylococcus saccharolytics, Streptococcus anginosus, 0158. The steps of sterilization, deoxygenation, concen Streptococcus contellatus, Streptococcus pleomorphus, tration adjustments, detoxification, and/or pre-digestion may Streptococcus intermedius, Sutterella wadsworthensis, Tis occur in a variety of orders such as deoxygenation of biomass sierella praeacuta, and Veillonella species, or genera contain first followed by concentration adjustments, sterilization, ing Such species. detoxification, and/or pre-digestion second or concentration 0153. The anaerobic bacteria isolation/enrichment system adjustments, sterilization, detoxification, and/or pre-diges may be large, allowing for increased available space for dif tion first followed by deoxygenation of biomass second or all ferent bacteria species which may utilize the same food steps simultaneously or other combinations thereof. An Source, and therefore minimizes competition between spe example of a process flow diagram is shown in FIG. 10: cies. Thus, it may be possible to isolate various high Volume double arrows display the order of deoxygenation of biomass hydrogen producing strains grown on a common food source before concentration adjustments, sterilization, detoxifica and then further differentiate them by subdividing the food tion, and/or pre-digestion and the singe arrow displays the Source into different categories. For instance, by way of order of concentration adjustments, sterilization, detoxifica example only, the isolation of high hydrogen producers tion, and/or pre-digestion before deoxygenation of biomass. US 2008/0311640 A1 Dec. 18, 2008

0159. After the concentration step, it may be necessary to digester. FIG. 13 is an example of a process flow diagram of adjust the concentration of the biomass depending on what is the concentration of biomass material. Concentration of bio used. For example, fats or meats which have high protein mass material or optional steps of concentration of biomass content would be diluted to an approximate 5% sugar con material can include, but are not limited to, centrifugation, centration equivalent before use. In addition, managing the reverse osmosis, filtering, boiling, removal of liquids, lyo proper rate at which the biomass is fed to the bacteria needs to philization, other methods known in the art of condensing be accounted. The dynamic feed rate should be balanced with material into a solid or semi-solid form or combinations the exponential growth rate of the bacteria as well as the rate thereof. of utilization. Oilier factors that should be monitored may include hydrogen production, pH levels and acid production. Fermentation Products 0160 Examples of biomass available for hydrogen pro 0164. Some anaerobic fermentation end products are ben duction via "dark fermentation' include, but are not limited eficial to humans and are the basis of a number of industries, to, mash of starched materials derived from corn, wheat, oats, Such as alternative energy, brewing industry, and the dairy and other grain materials, beet molasses, blackstrap molas industry. Examples of microorganisms and fermentation end ses, citrus molasses, invert Sugar, Sucrose, fructose, glucose, products include, but are not limited to: Streptococcus, which wood Sugar, Xylose, animal or fish tissue or parts, plant parts, produce lactate, formate, ethanol, and acetate when grown fruits, Sorghum, cheese whey, vegetables, plant processing under anaerobic conditions: Propionibacterium, produce waste, animal manure or urine, Solids isolated from fermen propionic acid acetic acid, and CO by fermentation of glu tation cultures, bovine manure, poultry manure, equine cose: Escherichia coli species cananaerobically produce ace manure, porcine manure, bovine urine, poultry urine, equine tic acid, lactic acid, succinic acid, ethanol, CO, and H urine, porcine urine, wood shavings or chips, slops, shredded Enterobacter species can anerobically produce formic acid, paper, cotton burrs, grain, chaff, seed shells, hay, alfalfa, ethanol. 2,3-butanediol, lactic acid, CO, and H. and grass, leaves, sea shells, seed pods, corn shucks, weeds, Clostridium species can anerobically produce butyric acid, aquatic plants, algae and fungus and combinations thereof. butanol, acetate, acetone, isopropyl alcohol, CO, and H. 0161 FIG. 11 illustrates an example of the preparation of 0.165. To obtain alternative energy sources such as hydro biomass for hydrogen production that may include but is not gen from anaerobic fermentation, it is evident that various limited to the removal of non-biomass material, an optional bacteria, Such as, Escherichia coli, Enterobacter and filtering or sifting of biomass material step, an optional pre Clostridia, are active hydrogen producers under anaerobic conditioning of biomass material step or combinations conditions. Clostridia are able to ferment various types of thereof. Biomass material or feed stock is selectively chosen biomass into hydrogen, carbon dioxide, and a variety of for optimal hydrogen production depending on the specific organic compounds. For instance, during fermentation of anaerobic bacteria used. Grit, Such as dirt, sand, Soil, stones, Sugars, hydrogen, carbon dioxide and Volatile organic acids, pebbles, rocks, feathers, hair and other such materials, may be Such as butyrate and acetate, are produced, whereas, during removed prior to addition of the feed stock to the anaerobic the fermentation of amino acids and fatty acids, a variety of digester; however, grit can be removed at any point along the foul Smelling compounds are formed. Clostridia also pro process. Equipment such as clarifiers, settling tanks, mul duce extracellular enzymes to degrade large biological mol tiphase tanks, and/or filters can be used to remove the grit. ecules in the environment into fermentable components. 0162 Deoxygenation of the biomass prior to introduction Hence, Clostridia play an important role in nature in biodeg to a digester, either as batch or continuous processes, ensures radation and many other nutrient cycles. viability of the anaerobic bacteria which may die on exposure 0166 Members of genus Clostridia are ubiquitous in to oxygen. Oxygen exposure also inactivates the enzyme nature and may be found in soil and in the gastrointestinal complex required for hydrogen production. Also, for continu tracts of animals and humans. Clostridia are Gram-positive, ous fermentation systems it rminimizes oxygen introduction spore-forming, mostly motile, rod shaped anaerobic bacteria. and limits the need to continuously purge the digester. FIG. 12 A Gram-stain is a good method for differentiating shows an example of a process flow diagram of the deoxy Clostridium from other genera, as the cell incorporates the genation of biomass material. Deoxygenation of biomass dye while the spore remains unstained. Most Clostridium material can include but are not limited to a single deoxygen species are strictly anaerobic, as their vegetative cells are ation step or optional multiple deoxygenation steps of biom killed by exposure to oxygen. However, they can Survive as ass material. Deoxygenation of biomass material can include spores in aerobic conditions, and therefore are able to wait for but are not limited to using pressurized steam, oxygen free gas anaerobic conditions required for active anaerobic bacteria Sparging, autoclaving, a reducing agent (Such as by way of growth. Clostridium butyricum, Clostridium acetobutylicum, example only, dithiothreitol, cysteine, thioglycolate, or Clostridium pasteurianum, and Clostridium barati produce sodium sulfide), or combinations thereof. The use of pressur hydrogen by anaerobic fermentation of glucose, with ized steam also serves to thoroughly sterilize the media and Clostridium butyricum being the most effective H producer. simultaneously convert any complex carbohydrates into a These bacteria may be utilized to obtain hydrogen for use as form more easily acted upon by the bacteria. fuel or as a chemical agent in the chemical industry. In addi 0163 The biomass concentration may have an effect on tion, Clostridium species may be referred to as solventogenic the bacteria metabolic pathways, which may affect the yield as they also are able to produce solvents as the result of of hydrogen. However, concentrating the biomass prior to continued fermentation of volatile organic acids (VOA) at the introduction to a digester, either as batch or continuous pro expense of hydrogen production, such as, by way of example cesses in combination with efficient removal of fermentation only, butyrate to butanol and/or acetate to acetone. products, such as hydrogen, carbon dioxide and volatile 0.167 Provided complete conversion occurs dark fermen organic acids, and control of the fermentation media pH tation of biomass comprising Sucrose produces eight moles of increases the yield of hydrogen per unit volume of the hydrogen per mole of Sucrose, or alternatively four moles of US 2008/0311640 A1 Dec. 18, 2008 26 hydrogen per mole of glucose. The other products of the dark natively the different bacteria species or genera may cohabi fermentation process are carbon dioxide and acetic acid, a tate as one colony, with bacteria feeding on the waste of precursor to acetone. If all of the substrate were converted to neighboring bacteria. butyric acid, then two moles of hydrogen may be produced 0171 Mimicking natural symbiotic type ecosystems for per mole of glucose. the anaerobic bacterial production of hydrogen is one aspect of the methods described herein. The initial bioprocess for 0168 FIG. 14 is a schematic showing the basic elements anaerobic production of hydrogen from carbohydrate based involved in the conversion of complex Substrates, through biomass is the heterotrophic fermentation (dark fermenta intermediate compounds, to hydrogen and carbon dioxide. tion) of carbohydrates to hydrogen, carbon dioxide and Vola First, complex organic compounds are hydrolyzed by extra tile organic acids (solvent precursors). The accumulation of cellular enzymes to generate free Sugars which are further these fermentation products decreases the pH, which can kill fermented to hydrogen, carbon dioxide, and Smaller organic the bacteria, or it can initiate changes in the bacteria metabo products, primarily VOAS. Such as, but not limited to, acetate, lism wherein continued fermentation (solventogenesis) of the formate, butyrate and propionate. Continued fermentation of Volatile organic acids to solvents occurs at the expense of the VOAS can produce the corresponding solvent, such as hydrogen yield. In addition, the accumulation of hydrogen acetone from acetate. Although acidogenic fermentation pro and carbon dioxide may affect hydrogen yield and bacteria duces acetate, other VOAS are formed, which can be further viability, respectively. A high hydrogen partial pressure may metabolized by acetic acid producing (acetogenic) bacteria to limit the rate of fermentation or may cause a metabolic shift, yield hydrogen, carbon dioxide and acetate. The acetate thereby increasing the conversion of Solvent precursors to formed may be subsequently converted into hydrogen and solvent, and severely affect the yield of hydrogen produced. carbon dioxide by photofermentation, or be isolated and puri The presence of a high concentration of carbon dioxide and fied for alternative uses such as industrial chemical applica Volatile organic acids may also decrease the pH of the fer tions. mentation media, resulting in bacteria death. In addition, solvent formation from reduction of volatile organic acids by 0169. Although hydrogen may be produced by acidogen Solventogenesis may also results in a decrease in pH, and esis and acetogenesis, the production of VOAS or solvents potential bacteria mortality. Similarly, anaerobic fermenta may be changed or manipulated depending on the composi tion of biomass containing Sulfur compounds, such as, by way tion of the fermentation medium and the Clostridia species of example only, proteins and hydrogen sulfide (H2S) which utilized. For instance, by way of example only, some cellu is converted into elemental sulfur by purple and green pho lose-degrading Clostridia species degrade cellulose to glu tosynthetic bacteria. High concentrations of HS are toxic to cose and then ferment the glucose, producing hydrogen, car bacteria and result in bacteria death. Therefore, in order to bon dioxide, and a range of VOAS. Such as formate, acetate, maintain optimal hydrogen production it is desirable to propionate, butyrate, and Succinate, as fermentation end remove the dark fermentation products, such as hydrogen, products. By way of example, Clostridium thermoaceticum, carbon dioxide, and volatile organic acids (VOAs) in order to Clostridium thermoautotrophicum, and Clostridium mag avoid these issues. Dark fermentation and removal of the num, convert glucose or Sucrose to acetate, whereas: heterotrophic fermentation products can proceed as either a Clostridium acetobutylicum, which is a saccharolytic bacte continuous process, or batch process, or a combination of rium, produces acetate, butyrate, carbon dioxide, hydrogen, both continuous and batch processes. and some lactate when grown in glucose-limited conditions at a neutral pH. In comparison, other nonpathogenic species Monitoring H, pH, Pressure and Temperature grown in glucose-limited media, such as Clostridium baratii, 0172. While direct and indirect photolysis systems pro produce large amounts of lactate in addition to acetate and duce pure H2, dark-fermentation processes produce a mixed butyrate, while Clostridium butyricum produces large biogas containing primarily H2 and carbon dioxide (CO), but amounts of formate in addition to acetate and butyrate. In which may also contain lesser amounts of methane (CH), contrast, Clostridium propionicum fermentation of lactate carbon monoxide (CO), and/or hydrogen sulfide (HS). The produces propionate, acetate, hydrogen and carbon dioxide. gas composition presents technical challenges as fuel Regardless of the solvents or VOAS produced, hydrogen and Sources, as it may require Scrubbing and purification before carbon dioxide may be obtained from anaerobic fermentation use in Some fuel cells. using any of these species with a variety of food sources. 0173 Anaerobic bacteria known to produce hydrogen 0170 The generation of large quantities of hydrogen is during dark-fermentation of carbohydrate-rich substrates one aspect of the methods described herein. The approach include, but are not limited to, species of Ruminococci, used to achieve efficient hydrogen production is analogous to Archaea, and Clostridia. Carbohydrates are a good Substrate Some wild ecosystems comprising various anaerobic bacteria for hydrogen-producing fermentations. Glucose, isomers of species of the same genus and/or bacteria of different genera: hexoses, or polymers in the form of starch or cellulose, yield wherein the fermentation waste (products) of one bacteria different quantities of H2 per mole of glucose, depending on colony is used as a food source for a different bacteria colony. the fermentation pathway and end-product(s). For instance, If waste is allowed to accumulate near the colony, the colony the highest theoretical yield of H is associated with acetate as may die or its metabolic pathways alter to metabolize the the fermentation end-product, wherein a theoretical maxi waste. This latter step may allow the colony to survive, how mum of 4 mole H per mole of glucose is obtained. Alterna everit may not flourish. The diffusion of waste away from one tively, when butyrate is the end-product, a theoretical maxi colony to a different colony, where it is consumed, creates a mum of 2 moles H. per mole of glucose is obtained. In concentration gradient which continually bleeds away waste. practice, however, high H yields may be associated with a This symbiotic type relationship allows both colonies to mixture of acetate and butyrate fermentation products, and flourish, without any changes in metabolic pathways. Alter low H yields are associated with propionate and reduced US 2008/0311640 A1 Dec. 18, 2008 27 end-products, such as alcohols (i.e. methanol, ethanol, pro 0176 The fermentation process and products may be panol, isobutanol, butanol, isopentanol, pentanol, acetone, affected by the pH of the fermentation media. During dark propanol, isopropanol, and 1.2-propanediol), solvents and fermentation at pH values greater than about 5.6 (depending lactic acid. Note that potential hydrogen molecules remain on the bacteria strain), the exponential growth of bacteria, contained in Volatile organic acid products including, but not Such as, by way of example only, Clostridium acetobutyli limited to acetate, butyrate, and the like. Thus, fermentation cum, generates large quantities of fermentation products, products produced by a bacterium depend on the environmen Such as Volatile organic acids (e.g. formic acid, acetic acid, tal conditions in which it grows. Reduced fermentation prod propionic acid, butyric acid, and Valeric acid), carbon dioxide ucts like ethanol, butanol, and lactate, contain hydrogen that and hydrogen. The formation of carbonic acid (from carbon has not been liberated as gas. For instance, the metabolism of dioxide) and Volatile organic acids causes the pH to decrease, Clostridium pasteurianum, which is a high Volume H2 and and as the acids accumulate, particularly in batch cultures, volatile organic acid (VOA) producer, can be shifted toward growth becomes linear and gradually stops. In addition, the Solvent production by having high glucose concentrations. To bacteria may die if the pH decreases significantly. Also, when maximize the yield of H, the metabolism of the bacterium the pH drops to 4.5-5.0, a shift in fermentation processes may may be directed away from solvents (ethanol, acetone and occur, resulting in solvent formation (Solventogenesis). The shift to solventogenesis is induced by high intracellular con butanol) and reduced acids (lactate), and directed more centrations of acids, low pH, a growth limiting factor (Such as towards Volatile organic acids. phosphate or Sulfate depletion) and high concentrations of 0.174. The concentration of hydrogen, carbon dioxide, car glucose and nitrogen compounds. Solvent formation, allows bon monoxide, and/or hydrogen sulfide, both in solution and for further metabolism by preventing excessive acidification, in the head space above the dark fermentation media, increase but this may be at the expense of hydrogen yield. Solvento as dark fermentation proceeds. The yield of hydrogen during genesis requires the induction of a new set of enzymes catal dark fermentation is dependent on process conditions such as ysing the formation of acetone, butanol and ethanol from pH, temperature, buffer capacity, hydraulic retention time glucose and assimilated acids. Also, Solventogenic (HRT), and gas partial pressure; each of which may affect Clostridia, depending on the strain and fermentation condi metabolic balance. To ensure optimal hydrogen production tions, can produce other alternative solvents such as propanol, occurs during anaerobic dark fermentation, these process isopropanol and 1.2-propanediol. parameters, as well as the concentration offermentation prod 0177 Optimal hydrogen production from dark fermenta ucts, such as Volatile organic acids, hydrogen, carbon dioxide, tion may be maintained and solventogenesis may be mini carbon monoxide and hydrogen sulfide, need to be monitored mized by monitoring of the fermentation media pH, either either continuously or at regular intervals. continuously or by periodic sampling. In-line and/or on-line 0175 Temperature is an important parameter in establish monitoring methods may be used for continuous pH moni ing the fermentation rate and determining the stability of the toring. In-line monitoring methods use instrumentation fermentation process. Digesters fermentation reactions can which can be directly inserted into the digester, whereas, be operated at mesophilic (25-40 C), thermophilic (40-65C), on-line monitoring involves diverting a small stream of media extreme thermophilic (65-80 C), or hyperthermophilic (>80 to a location where it is either continually sampled and ana C) temperatures. Higher gas yields may be obtained using lyzed by fixed instrumentation, or manually sampled and thermophilic conditions; however this requires energy to heat tested elsewhere. The stream of media is then either re-circu the digester. A 5 C to 10 C fluctuation in temperature may lating back into the digester, after appropriate sterilization, or result in an imbalance in fermentation and lead to digester taken off as waste. Examples of in-line pH monitoring instru instability for either temperature range, however thermo mentation are, but not limited to, glass membrane type pH philic digestion is more sensitive to Such fluctuations. There electrodes, Solid State type pH electrodes (e.g. silicon nitride fore, to ensure optimal anaerobic fermentation it is necessary based and metal oxide based), and optodes (optical type pH to monitor and control the digester temperature. The digester electrodes using fluorescence or absorbance measurements of temperature can be monitored using thermal sensors, such as, pH sensitive dyes). In-line methods requiresterilization of the by way of example only, thermometers, thermopiles, and measuring system and may have issues with electrode fouling thermocouples, which are placed either inside the digester or for particulates and bacteria. On-line monitoring methods do on the outside surface of the digester. Similarly, infra-red not require sterilization, provided no recirculation of the thermal sensors may be used as non-invasive thermal moni media occurs, plus it allows for sample cleanup by filtering of toring methods by measuring the radiative heat from the bacteria and particulates from the test stream. Examples of digester. However, using infra-red sensors, or monitoring the on-line pH monitoring instrumentation are, but not limited to, inside temperature by measuring the outside Surface tempera glass membrane type pH electrodes, Solid state type pH elec ture, requires calibration to correlate the outside temperature trodes (e.g. silicon nitride based and metal oxide based), with the inside temperature of the digester. Regardless of the optodes (optical type pH electrodes using fluorescence or method used to measure the digester temperature, the infor absorbance measurements of pH sensitive dyes), and flow mation can be used in a control loop as feedback to the system injection analysis (FIA). The same methodologies used for used to heat the digester, Such as, by way of example only, hot on-line monitoring may be used for periodic sampling. The plates, or heating mantles. The system used to heat the pH information obtained may be used for feedback in a con digester is adjusted manually, or may be automated by com trol loop to the system used to adjust the pH and thereby puter control, to maintain the optimal temperature needed. maintain the optimal pH for optimal growth. Examples of The heat required by these processes can be provided by the methods to adjust the pH include, but are not limited to, waste exhaustheat from power generating equipment. A non addition of deoxygenated Solutions of Sodium hydroxide to limiting example of such a process is the use of low pressure increase the pH and addition of deoxygenated solutions of steam exhausted from a back pressure turbine system. hydrochloric acid to decrease the pH. This control of the pH US 2008/0311640 A1 Dec. 18, 2008 28 adjustment system may be achieved manually, or may be digesters will be known to those skilled in the art, however, automated by computer control. one possible method is to controla Vacuum system attached to (0178. The partial pressure of H (pH2), is a parameter the digester. which may be monitored during continuous H. Synthesis as it 0180. Similarly, carbon dioxide in dark fermentation bio reflects the concentration of H. Hydrogen synthesis path gas mixtures may be monitored using measurement systems ways may be sensitive to H2 concentrations and which can know to one skilled in the art. However, some examples for then be subject to end-product inhibition. As H concentra detecting carbon dioxide are fiber optic sensors based fluo rescence and absorbance changes with pH sensitive dyes, tions increase, H. Synthesis decreases and metabolic path non-dispersive infra-red detection, field effect transistors ways shift to production of more reduced substrates Such as based on carbon nanotubes, pyroelectric detectors, thermal lactate, ethanol, acetone, butanol, or alanine. In addition, conductivity detectors, and thermal conductivity detectors temperature also affects the pH2 such that, by way of example coupled with gas chromatography. The carbon dioxide con only, for optimal H synthesis, pH-50 kPa at 60° C.; pH-20 centration information obtained may be used for feedback in kPa at 70° C.; pH2 and pH.<2 kPa at 98°C. The increase in a control loop to the system used to control the removal of temperature is aimed at better performance of the hyperther fermentation gases from the digester. In addition, the gas flow mophiles hydrogenases, because the affinity for hydrogen rate may be monitored to assist in controlling the removal of decreases and the thermodynamic equilibrium of hydrogen gases from the digester. Typical methods for measuring flow formation from acetate, or other volatile organic acids, is rate are thermal based detectors, or hot wire anemometers. favored at higher temperatures. In addition, at large hydrogen The system used for gas removal may be manually controlled, and carbon dioxide concentrations there is potential for the or may be automated using computer control. Methods for onset of acetogenesis, resulting in the formation of acetate removal of fermentation gases from digesters will be known from hydrogen and carbon dioxide. Although this conserves to those skilled in the art, however, one possible method is to the hydrogen in a different chemical form, it decreases the control a vacuum system attached to the digester. potential dark fermentation yield of hydrogen. In addition, 0181 Carbon monoxide in dark fermentation biogas mix the rate of fermentative hydrogen production may be inhib tures may be monitored using measurement systems know to ited by high partial pressures (i.e. concentration) of hydrogen. one skilled in the art. However, Some examples for detecting 0179 The measurement of hydrogen is achieved using carbon monoxide are electrolytic sensors, colorimetric sen techniques known in the art, and may be carried out in the sor, MOS detectors (Metal Oxide Semiconductor Sensor), head space inside the digester or externally in the pipe/tube thermal conductivity detectors, and thermal conductivity system used remove the biogas from the digester. Described detectors coupled with gas chromatography. The carbon below are examples of hydrogen gas measurement systems, monoxide concentration information obtained may be used however, any hydrogen sensing or measurement system for feedback in a control loop to the system used to control the known in the art may be used. As with pH monitoring, any of removal of fermentation gases from the digester. In addition, the hydrogen measurements may be used either continuously the gas flow rate may be monitored to assist in controlling the or by periodic sampling, and may used as in-line and/or removal of gases from the digester. Typical methods for mea on-line monitoring methods. Hydrogen gas measurement suring flow rate are thermal based detectors, or hot wire may be achieved using fiber optic sensors based on the anemometers. The system used for gas removal may be chemochromic reaction of certain transition metal oxides, manually controlled, or may be automated using computer such as tungsten oxide or W03 with hydrogen in air, wherein, control. Methods for removal of fermentation gases from the reaction is catalyzed by palladium or platinum, and the digesters will be known to those skilled in the art, however, color change of the film in the presence of hydrogen is one possible method is to controla Vacuum system attached to detected by reflectance spectroscopy. Another method is to the digester. use thermal conductivity detectors (TCD), in which the ther 0182 Hydrogen sulfide in dark fermentation biogas mix mal conductivity of the digester gas is compared to that of a tures may be monitored using measurement systems know to reference gas, and the difference in conductivity can then be one skilled in the art. However, Some examples for detecting calibrated to give a hydrogen concentration value. Thermal HS are conductiometric sensors (CuO SnO2) based on conductivity detection may also be used in conjunction with resistance changes upon exposure to HS, thermal conductiv gas chromatography (GC) as a method to identify the various ity detectors, and thermal conductivity detectors coupled with components of the fermentation gas mixture and obtain their gas chromatography. The hydrogen Sulfide concentration respective concentrations. The application of GC with TCD information obtained may be used for feedback in a control detection for gas analysis is know to one skilled in the art. loop to the system used to control the removal offermentation Sensing is based on resistance changes upon hydrogen gases from the digester. In addition, the gas flow rate may be adsorption to platinum and/or palladium, field effect transis monitored to assist in controlling the removal of gases from tors based on carbon nanotubes are other approaches used to the digester. Typical methods for measuring flow rate are monitoring hydrogen concentrations in digesters. The hydro thermal based detectors, or hot wire anemometers. The sys gen concentration information obtained may be used for feed tem used for gas removal may be manually controlled, or may back in a control loop to the system used to control the be automated using computer control. Methods for removal removal of fermentation gases from the digester. In addition, of fermentation gases from digesters will be known to those the gas flow rate may be monitored to assist in controlling the skilled in the art, however, one possible method is to control removal of gases from the digester. Typical methods for mea a vacuum system attached to the digester. suring flow rate are thermal based detectors, or hot wire 0183 Gas partial pressure may affect the metabolic bal anemometers. The system used for gas removal may be ance and therefore affects the hydrogen yield obtained from manually controlled, or may be automated using computer dark fermentation. The digester pressure may be monitored control. Methods for removal of fermentation gases from using methods and techniques known to one skilled in the art. US 2008/0311640 A1 Dec. 18, 2008 29

However, examples of pressure sensing methods used to ing a valve, either manually or computer controlled, to monitor the digester pressure are solid state pressure sensors increase or decrease addition of deoxygenated nutrient solu or manometers, either attached to the digester or incorporated tions into the digester. with the outlet pipe/tube. The pressure information obtained 0186. In addition, monitoring of the volatile organic acid may be used for feedback in a control loop to the system used fermentation products, either continuously or by periodic to control the removal of fermentation gases from the sampling, is needed for optimal hydrogen production from digester. In addition, the gas flow rate may be monitored to dark fermentation. In-line and/or on-line monitoring methods assist in controlling the removal of gases from the digester. may be used for Volatile organic acid monitoring. Examples of on-line Volatile organic acid monitoring instrumentation Typical methods for measuring flow rate are thermal based are, but not limited to, Fluorescence Immunoassay (FIA) detectors, or hot wire anemometers. The system used for gas methods, high performance liquid chromatography (HPLC), removal may be manually controlled, or may be automated high performance liquid chromatography coupled with Mass using computer control. Methods for removal offermentation Spectrometry (HPLC-MS), capillary electrophoresis (CE), gases from digesters will be known to those skilled in the art, and ion chromatography. The same methodologies used for however, one possible method is to control a vacuum system on-line monitoring may be used for periodic sampling. The attached to the digester. Volatile organic acid concentration information may be used 0184 In dark-fermentation processes, the gas produced is for feedbackina control loop to the system used to remove the a mixture of primarily H and CO, but may also contain other Volatile organic acid from the digester, and thereby maintain gases such as CO, methane, and H2S, depending on the bio the minimal volatile organic acid concentration necessary for mass and bacteria present. If the digester becomes contami optimal growth. Volatile organic acids may be removed by nated with undesirable bacteria it is likely that other fermen cross flow dialysis, with the rate of removal dependent on the tation gases, such as methane (CH) from methanogenesis flow rate of the media on the opposite side of the dialysis and ammonia (NH) from nitrate or nitrite reduction may be membrane. The control of flow rate may involve adjusting a present. Early detection of these contamination gases may pump, either manually or computer controlled, to increase or allow for quick containment and remedy of the contamina decrease the flow rate of the deoxygenated media used to remove the Volatile organic acid from the digester as they tion. One method for detection and quantification of contami diffuse across the dialysis membrane. nation gases is, but not limited to, thermal conductivity detec 0187 Changes in the fermentation buffer capacity may be tion in conjunction with gas chromatography (GC). This obtained with knowledge of the digester pH and the concen method allows identification of the various components of the trations of the volatile organic acids. If the buffer capacity is fermentation gas mixture and obtains their respective concen too low, such that a slight increase in fermentation rate may trations. cause a rapid decrease in pH, then fresh deoxygenated media 0185. Optimal hydrogen production from dark fermenta may be added. The buffer capacity information may be used tion may be maintained and solventogenesis may be mini for feedback in a control loop to the system used to add mized by monitoring, either continuously or by periodic Sam deoxygenated media into the digester, and thereby maintain pling, the nutrient concentration in the fermentation media. the optimal buffer capacity. The control of media addition In-line and/or on-line monitoring methods may be used for may involve adjusting a valve, either manually or computer nutrient monitoring. In-line monitoring methods use instru controlled, to increase or decrease addition of deoxygenated mentation which can be directly inserted into the digester, media into the digester. whereas, on-line monitoring involves diverting a small 0188 Removal of the dark fermentation products, such as stream of media to a location where it is either continually hydrogen, carbon dioxide, hydrogen sulfide, carbon monox sampled and analyzed by fixed instrumentation, or manually ide and the Volatile organic acids during fermentation creates sampled and tested elsewhere. The stream of media is then conditions for use of concentrated nutrients and the ability to either re-circulating back into the digester, after appropriate maintain optimal hydrogen production. Without this removal process hydrogen production would be diminished due to sterilization, or taken off as waste. Examples of in-line nutri initiation of alternate metabolic pathways at high concentra ent monitoring instrumentation are, but not limited to, glu tion of fermentation products. Therefore, separation of cose sensors (optical or amperometric). In-line methods microbial growth and product formation improves hydrogen require sterilization of the measuring system and may have production yields, however optimizing the process condi issues with electrode fouling from particulates and bacteria. tions, such as pH, and temperature also improve hydrogen On-line monitoring methods do not require Sterilization, pro yield. vided no recirculation of the media occurs, plus it allows for sample clean up by filtering of bacteria and particulates from the test stream. Examples of on-line nutrient monitoring Removal of Hydrogen, Carbon Dioxide and Hydrogen Sul instrumentation are, but not limited to, glucose sensors (opti fide cal or amperometric), flow injection analysis (FIA) methods, 0189 Dark-fermentation systems have great potential as high performance liquid chromatography (HPLC), high per practical biohydrogen systems by incorporating rapid gas formance liquid chromatography coupled with Mass Spec removal and separation, rapid Volatile organic acid removal trometry (HPLC-MS), and capillary electrophoresis (CE). and separation, and bioreactor design. In dark-fermentation The same methodologies used for on-line monitoring may be processes, the gas produced in the anaerobic digester is a used for periodic sampling. The nutrient concentration infor mixture of H and CO, but may also contain other gases such mation may be used for feedback in a control loop to the as CO, CH, HS, or ammonia (NH), which is be present in system used to add nutrients into the digester, and thereby fermentation media and headspace above the reaction solu maintain the optimal nutrient concentration for optimal tion. The content of each gas in the headspace and the reaction growth. The control of nutrient addition may involve adjust Solution varies according to the conditions, feed stock and/or US 2008/0311640 A1 Dec. 18, 2008 30 anaerobic microbe present within the anaerobic digester. and carbon dioxide, then compressing and cooling the mix Rapid removal and purification of the H and removal of ture to transform at least one of the gases into a liquid form. diluting (CO, CH4) and/or contaminating (CO, HS) gases The remaining gaseous products may then be compressed and allows for maintaining continuous H synthesis. cooled at a different location using different conditions, thus 0190. As discussed above, the pH2 is a parameter which making the process a differential compression process. The may be monitored during continuous H. Synthesis, since with conditions used for compressing and cooling vary depending increasing H concentrations, H. Synthesis decreases and on the gas mixture. By way of example only, the removal and metabolic activity shifts to pathways that synthesize more purification of hydrogen from carbon dioxide may use a pres reduced Substrates. Thus, efficient gas removal bypasses sure of 58 bars at a temperature of 15° C. to liquefy the carbon these pathways, thereby increasing H2 production. The con dioxide allowing for easy separation of the gaseous hydrogen. centration of CO., also affects the rate of synthesis and final 0.195 Once separated, the hydrogen may be optionally yield of H due to Succinate and formate synthesis using CO, processed with one or more dehydration stages, then com pyruvate and nicotinamide adenine dinucleotide (NADFf) via pressed and alternatively stored in pressurized storage vessels the hexose monophosphate pathway. This pathway competes or tanks, or used directly as a fuel to generate electricity via with reactions in which H is synthesized by NADH-depen fuel cells or combustion/turbine type systems. The generated dent hydrogenases (which oxidize NADH to NAD+). Thus, electricity may then be used to operate the anaerobic digester efficient removal of CO from the fermentation system and associated facility, or can be added to the external power reduces competition for NADH, and results in increased H. system via connection to the external grid. If the purified synthesis. hydrogen is stored, it can be used later as a fuel source, or used 0191 Methods of removal of fermentation gases include, as a chemical component in an industrial process. Similarly, but are not limited to, sparging with N or argon (Ar) gas. the purified CO can be directly used as feed stock for alter applying a vacuum to the digester head space, or using mem nate fermentation processes, it can be stored and used later as brane technologies, such as, by way of example only, hollow feed stock, it can be used as a greenhouse Supplement to fiber/silicone rubber membranes and non-porous, synthetic enhance greenhouse plant, fruit and vegetable production, it polyvinyltrimethylsilane (PVTMS) membranes. can be used as a refrigerant in the anaerobic fermentation 0.192 The hydrogen and carbon dioxide produced by dark facility, it can be used as a fire-extinguishing material, or it fermentation in the anaerobic digester may be cleaned or can be used as chemical component in an industrial process, purified by a scrubber to remove moisture, vapor, droplets, Such as Soda ash production. In addition, collected carbon suspended solids or other such contaminants. The scrubber dioxide may be used commercially such as, but are not limited can comprise one or more of a filter, desiccant, Zeolite, acti to, in the production of carbonated beverages, in water soft vated carbon, fiber, countercurrent wash solution, mixer, ening, in the manufacture of aspirin, used as a refrigerating homogenizer, or other such components typically used in agent, used in the Solvay process for the preparation of association with or comprised within gas scrubbers. Such Sodium carbonate, used to provide an inert atmosphere for components are well known to those of ordinary skill in the packaging and storage of foods, and used as pressurizing art of gas processing. In general, hydrogen sulfide (H2S) is an medium and propellant in aerosol cans of food, fire extin undesired by-product or off-gas, which is removed from the guishers, target pistols, and for inflating life rafts. desired hydrogen gas; however it may be used as feed Stock (0196) Substantial gains in H, production can also be for bacteria Such as, by way of example only, purple Sulfur achieved through optimization of bioreactor designs. By way bacteria, to obtain more hydrogen and elemental Sulfur. of example only, fixed-bed bioreactors enhance H produc 0193 The gases which exit the anaerobic digester or the tion by using activated carbon as a Support matrix, plus a scrubber are then optionally separated into their individual membrane filter system for removal of the biogas. The Sup components using conventional gas separation equipment, port matrix allows for retention of the H producing bacteria which is known to those of ordinary skill in the art for sepa within the bioreactor, and the membrane system maintains rating gas mixtures. By way of example only, H2S and CO low gas partial pressures. can be removed from the gas product stream with the use of membranes which are permeable to HS or CO and not Removal of Volatile Organic Acids permeable to hydrogen. Alternatively, palladium-silver alloy/ 0197) The volatile organic acids generated during dark ceramic composite membranes with high selectivity and flux fermentation are removed as their accumulation may have an rate for hydrogen may be used for separating hydrogen from effect on the anaerobic bacteria's ability to produce hydrogen. CO, and HS. Thus, CO, and HS may be selectively stripped In particular, as fermentation proceeds the pH decreases, and from the gas product stream yielding purified H. In addition, continued fermentation may yield pH-neutral components the, CO may be selectively stripped from HS using a variety Such as acetone and butanol at the expense of hydrogen pro of methods for HS removal, such as, by way of example only, duction. Removal of these products can be either continu a monoethanolamine Girbotol type process. The CO may ously or in batch mode, wherein both continuous and batch then be isolated and purified for later use. Alternatively, the removal methods include, but are not limited to, dead-end CO, and H.S mixture may be used as feed stock for purple filtration, cross-flow filtration, ultrafiltration, pervaporation, sulfur bacteria, wherein the HS is converted into hydrogen and dialysis. Dead-end filtration involves the use of a filter and elemental Sulfur, and the CO is Subsequently removed element through which the media is passed while leaving the from the H to yield purified CO and H. bacteria and other solid particulates behind. The use of a 0194 Alternatively, the gases which exit the anaerobic dead-end filter can be continuous, with fresh media being digester are separated and purified using a differential com introduced to replenish the quantity filtered, alternatively pression system. The differential compression process dead-end filtration can be a batch process. One issue encoun involves initially drying the mixture of gaseous anaerobic tered with dead-end filtration is clogging and potential block fermentation products, such as, but not limited to, hydrogen, age of the filter element, which can limit the through put of a US 2008/0311640 A1 Dec. 18, 2008

continuous system. To overcome clogging issues a cross flow fluid passes through the membrane the rest continues down filtration system can be used, wherein tangential flow of stream, Sweeping the rejected species away from the mem media, bacteria and other particulates occurs across the filter brane. Reverse osmosis is capable of rejecting bacteria, salts, element minimizing the potential for clogging. Cross-flow Sugars, proteins, particles, dyes, and other constituents that filtration can also be used in a batch mode approach. Ultra have a molecular weight of greater than 150-250 daltons (Da). filtration can be used for bacteria not filterable using other The separation of ions with reverse osmosis is aided by pore sizes. The presence offilter systems effectively removes charged membranes. Thus, dissolved ions that carry a charge, the Volatile organic acids from the fermentation culture, and such as salts, are more likely to be rejected by the membrane generally, dead-end filtration, cross-flow filtration and ultra filtration use positive pressure to force fluid through the filter than those that are not charged. Such as organics. In this element. Alternatively; methods without the use of positive manner, any solvents produced, such as, by way of example pressure to force fluid through a filter element, such as reverse only, acetone, may be removed while maintaining the Volatile osmosis, dialysis or electro-dialysis, may be used for the organic acids in the flow stream. The larger the charge and the removal of the volatile organic acids from the dark fermen larger the particle, the more likely it will be rejected. tation culture. 0201 The volatile organic acids may be removed from the (0198 The use of dialysis for the removal of volatile anaerobic fermentation system and then purified by osmosis organic acids from the bacteria used for dark fermentation as a salt. The Volatile organic acids anions, including, but not relies on diffusion of the Volatile organic acids across a semi limited to, formate, acetate, propionate, butyrate, and Valer permeable membrane and leaving the bacteria behind. A con ate, may be combined with alkali metal cations, alkaline earth centration gradient is needed for the process to occur and this cation, ammonium ion, and combinations thereof to form a can be achieved continuously or in a batch mode. In the batch salt. By way of example only, the cations may be Na', K", mode the dark fermentation culture is poured into a dialysis Ca", Mg", NH, or combinations thereof. bag (constructed from semi-permeable membrane material) and the filled bag is placed into a container of fresh fermen Convert Volatile Organic Acids to H/CO, tation media or water. A concentration gradient is present between the inside of the bag and the outside, and diffusion of 0202 The filtrate obtained from dead-end filtration, cross soluble ions and molecules across the semi-permeable mem flow filtration, ultrafiltration, or dialysis of the dark fermen brane. Large molecules, particles and bacteria which are tation stage may contain other Volatile organic acids than unable to pass through the semi-permeable membrane remain acetic acid. Therefore, the filtrate may be fed into separate inside the bag. This process continues until equilibrium is anaerobic digesters and further metabolized by acetic acid reached, after which a fresh container of fresh fermentation producing (acetogenic) bacteria to yield hydrogen, carbon media or water is used for thorough removal of volatile dioxide and acetate. A close monitoring of hydrogen concen organic acids. In a continuous process a semi-permeable trations is necessary for acetogenic bacteria conversion of membrane may be used to separate the dark fermentation fatty acids (e.g., formic, acetic, propionic, isobutyric, butyric, digester from a system in which fresh fermentation media or isovaleric, Valeric, isocaproic, caproic, and heptanoic acids) water is flowing past, thereby creating a concentration gradi and alcohols into acetate, hydrogen, and carbon dioxide ent and continuously removing Volatile organic acids as they because acetate formation may be reduced under relatively diffuse into the flowing stream. high H. partial pressure. The use of acetogenic bacteria for 0199 Electro-dialysis is a membrane process, during generation of acetate not only increases hydrogen output, but which ions are transported through semi permeable mem it is an alternative approach to increasing the acetate concen brane, under the influence of an electric potential. The mem tration in the filtered dark fermentation media. The acetate branes are cation- or anion-selective, allowing either positive generated during acetogenic bacteria fermentation may be ions or negative ions to flow through. Cation-selective mem used as feed stock for other fermentation stages in separate branes may be negatively charged polyelectrolytes, which anaerobic digesters, such as, by way of example only, photo rejects negatively charged ions and allows positively charged fermentation (FIG. 14). With light, and under anaerobic con ions to flow through. Cation-selective membranes may be ditions, reduced organic compounds, like acetic acid, can be Sulphonated polystyrene, while anion-selective membranes converted to hydrogen and carbon dioxide by certain micro may be polystyrene with quaternary ammonia functional organism, such as, by way of example only, purple bacteria groups. Particles and bacteria are too large to pass through the (Chromatium, Rhodospirillium, Rhodomicrobium). There membrane and are not removed. Electro-dialysis can also be fore, photo-fermentation may be added as another fermenta used in a continuous or batch process as described above for tion stage to obtain more hydrogen from the photohet dialysis. erotrophic fermentation of acetate into hydrogen and carbon 0200. The filtrate obtained from the above methods may dioxide. The hydrogen and carbon dioxide can be separated be further purified with the use of reverse osmosis. Reverse and purified. osmosis, also known as hyperfiltration, process uses a mem 0203 Alternatively, the filtrate obtained from dead-end brane that is semi-permeable, allowing the fluid to pass filtration, cross-flow filtration, ultrafiltration, or dialysis of through it, while rejecting ions from crossing. This in effect is the dark fermentation stage, or the media, concentrated by a method to concentrate and purify the Volatile organic acids reverse osmosis, may be further purified to isolate the various obtained from dark fermentation. The process of reverse Volatile organic acids. Isolation and purification methods can osmosis requires a driving force to push the fluid through the include distillation, adsorption chromatography, ion membrane, and the most common force is pressure from a exchange chromatography, and affinity chromatography. pump. Most reverse osmosis technology uses a process Such purified Volatile organic acids may then be used as either known as cross flow to allow the membrane to continually commercial products, or as precursors or reactants for Syn clean itself, analogous to cross flow filtration. As some of the thesis of commercial products. US 2008/0311640 A1 Dec. 18, 2008 32

Convert HS with Appropriate Bacteria to H/CO and Sulfur This behavior may be utilized to obtain elemental sulfur from 0204 Proteobacteria or “purple bacteria' may be the larg oxidation of HS, which has been obtained from certain types estand most physiologically diverse group of bacteria, which of biomass during anaerobic dark fermentation. exhibit a wide variety of types of metabolism: aerobic, 0208 Lithoautotrophic sulfur oxidizers are found in envi anaerobic, heterotrophic, autotrophic, phototrophic, and ronments rich in H.S. Such as Volcanic hot springs and fuma lithotrophic. The use of carbon dioxide as the carbon source roles, and deep-sea thermal vents. Some are found as Sym and an inorganic compound (water, or H2S) as the source of bionts and endosymbionts of higher organisms. Since they reducing power is reflected in the terms autotroph and can generate energy from an inorganic compound and fix CO lithotroph (respectively) which are used for an organism per as autotrophs, they may play a fundamental role in primary forming this reaction. With light as the ultimate source of production in environments that lack Sunlight. In addition, energy, the organism would also be termed a phototroph. Some are hyperthermophiles that grow at temperatures of Thus, in addition to protobacteria being anoxygenic photo 115°C., while some are halophilic (salt loving), such as the heterotrophs, proteobacteria include chemolithotrophs and genus Halothiobacillus, which can be found in Soda lakes and chemoorganotrophs, wherein lithotrophs are able to fix car salterns. bon, while organotrophs require fixed carbon as a nutrient. 0209 Most purple non-sulfur photosynthetic bacteria are Thus, purple bacteria may be used for H and CO, production able to grow as photoheterotrophs, photoautotrophs or from acetate by a photoheterotrophic process, or alterna chemoheterotrophs. A few species are capable of anaerobic tively, purple bacteria may be used to obtain hydrogen and growth though most species are aerobic. The mode of growth elemental sulfur from HS in an anaerobic lithotrophic pro is determined by the available conditions, such as, availability CCSS, of light (needed for phototrophic growth), the degree of 0205 Proteobacteria are all gram negative, but otherwise anaerobiosis (oxygen level), the availability of CO as a car represent a diverse range of organisms such as the purple bon source for autotrophic growth, and the availability of phototrophic, nitrifying bacteria and enteric bacteria, as well organic compounds (such as simple Sugars, Volatile organic as the bacteria responsible for animal bioluminescence. They acids, and aromatic compounds) for heterotrophic growth. can be divided into five sections (depending on their RNA), Therefore, photo-fermentation may be used to obtain more referred to by the Greek letters alpha, beta, gamma, delta and hydrogen from the photoheterotrophic fermentation of epsilon. Photosynthetic protobacteria (anoxygenic photoau acetate into hydrogen and carbon dioxide by purple non totrophy) are found in alpha, beta, and gamma groups, in Sulfur. Typical purple non-sulfur bacteria genera are Rho particular purple sulfur bacteria are generally beta or gamma dospirillum and Rhodopseudomonas and Rhodobacter. proteobacteria, and purple non-Sulfur bacteria are primarily 0210 Chemotrophic growth for the purple non-sulfur bac alpha proteobacteria. Alpha, beta, and gamma groups also teria is achieved by respiration, although there are some include chemolithotrophs and chemoorganotrophs. In con exceptional Strains and species which can obtain energy by trast, the delta and epsilon proteobacteria are not photosyn fermentation or anaerobic respiration. In addition, it was thetic and are all chemoorganotrophs. thought that purple non-sulfur bacteria could not use hydro 0206 Most purple bacteria are strict anaerobes and live in gen Sulfide as an electron donor for the reduction of carbon the sediment of ponds and lakes. Purple phototrophic bacteria dioxide when growing photoautotrophically, hence the use of use energy from Sunlight in a process known as anoxygenic “non-sulfur in their group name. Sulfide can be used if photosynthesis and can be divided into two groups depending present in a low concentration, however, higher concentra whether or not hydrogen sulfide (HS) is used as an electron tions of HS (in which the purple sulfur bacteria and green donor for carbon dioxide reduction. If HS is used as an sulfur bacteria can thrive) are toxic. electron donor then they are called purple sulfur bacteria, if HS is not used as an electron donor, then they are known as Use of Fermentation Products purple non-sulfur bacteria. For purple non-sulfur bacteria, 0211 FIG. 15 is a schematic showing one approach hydrogen can be used as the reducing agent, although some toward producing and processing various fermentation prod purple non-sulfur bacteria may use other compounds. Purple ucts for their use. The fermentation products, also referred to sulfur bacteria fix CO to live, whereas non-sulfur purple as chemical products, may include, but are not limited to, bacteria can grow aerobically in the dark by respiration on an gases, Volatile organic acids, Solids and solvents. These organic carbon Source. chemical products may be obtained from dark fermentation, 0207 Purple sulfur bacteria normally are anaerobic or acetogenesis, photo-fermentation, Solventogenesis, fermen microaerophilic, and are often found in Sulfur springs or tation with proteolytic bacteria, or combinations thereof. The stagnant water. An environment they can be found in is the gases produced include, but are not limited to, hydrogen, illuminated but anoxic Zones of these aquatic environments. carbon dioxide, carbon monoxide, hydrogen sulfide, meth The presence of oxygen hinders their growth. Purple sulfur ane, ammonia, and nitrogen; which are removed from the bacteria oxidize HS or S as an electron donor for carbon various digesters, separated and purified for use as feedstock dioxide reduction during the dark reaction of photosynthesis. for chemical industry, as energy sources for power produc The HS is oxidized to produce granules of elemental sulfur, tion, or as products themselves. The Volatile organic acids which in turn may be oxidized to form sulfuric acid and include, but are not limited to, formic acid, acetic acid, pro therefore acidifies the environment. Some of the sulfur oxi pionic acid, butyric acid, and Valeric acid; which are removed dizers are acidophiles that are able to grow at a pH of 1 or less. from the various digesters, separated and purified for use as The photosynthetic proteobacteria may be placed into two feedstock for chemical industry, feedstock for other fermen families, the Chromatiaceae and the Ectothiorhodospiraceae. tation processes, or as products themselves. In addition, the Ectothiorhodospiraceae are gamma-proteobacteria, but are Volatile organic acids may be removed from the digester and distinctive because they deposit sulfur on the outside of their converted to the respective salts of the Volatile organic acids, cells, whereas Chromatiaceae deposit sulfur inside their cells. which comprise at least one cation. The anions of Such salts US 2008/0311640 A1 Dec. 18, 2008

include, but are not limited to, formate, acetate, propionate, butyrate, and Valerate, while the at least one cation includes, -continued but is not limited to, alkali metal ions (such as, by way of O example only Na" and K"), an alkaline earth ions (such as, by Ca(Ac)2Mg(Ac)2 wherein Acis CH-C-O- way of example only Ca" and Mg"), and ammonium ion (NH). The solvents produced include, but are not limited, to Calcium Magnesium Acetate acetone, butanol, propanol, isopropanol. 1.2-propanediol. and the Solid may be compounds which comprise Sulfur, or 0214. The calcium magnesium acetate formed in this may be elemental sulfur. The solvents and solids are removed manner may be used as an anti-freeze agent, a deicing from the various digesters, separated and purified and may be agent, or an anti-icing agent. In addition, glacial acetic acid may be used as described above, wherein the glacial used as feedstock for chemical industry, as energy sources for acetic acid is formed by concentrating the acetic acid power production, or as products themselves. obtained from anaerobic fermentation of a biomass. 0212. The fermentation products, such as gases, volatile 0215 Similarly, volatile organic acids may be admixed organic acids, salts of Volatile organic acids, Solids and Sol with an oxide and liquid ammonia and reacted to form a vents may be used as feedstock in other industries, such as, composition. This is demonstrated by an exemplary embodi but not limited to, polymer industry, industrial synthesis ment in which acetic acid, obtained from anaerobic fermen industry, photographic industry, coatings industry, fertilizer tation of a biomass, is admixed and reacted with Zinc oxide to industry, printing industry, and combinations thereof. In addi form Zinc ammonium acetate (reaction Scheme 2). tion, the Volatile organic acids may also be used as feedstock for other fermentation processes, such as, but not limited to, Scheme 2 acetogenesis (to produce more acetic acid from other Volatile organic acids), photo-fermentation (to produce more hydro O gen and other fermentation gases, such as, but not limited to, ZnO + NH4OH -- ch--on Her carbon dioxide) and solventogenesis (to produce solvents and Zine Ammonium Acetic acid more hydrogen and other fermentation gases, such as, but not oxide hydroxide limited to, carbon dioxide). By way of example only, acetic O acid obtained from the dark fermentation and acetogenic conversion can be purified, and optionally concentrated into ZnNH4 (Ac)3 wherein Ac is CH3 O glacial acetic acid (99.5% pure acetic acid), and used as Zinc Ammonium Acetate feedstock for the chemical industry. Non-limiting examples of the use of acetic acid include the production of fibers and 0216. The zinc ammonium acetate formed in this man resins such as cellulose acetate used in making acetate rayon, ner may be used as a fertilizer or a seed germination the production of pharmaceuticals, bleaches, preservatives enhancer. Again the glacial acetic acid may also be used, and photographic chemicals, fertilizers, plastics, nonflam wherein the glacial acetic acid is formed by concentrat mable motion-picture film, photographic film, lacquers, and ing the acetic acid obtained from anaerobic fermentation paint solvents. In addition, the acetic acid obtained by anaero of a biomass. bically fermenting a biomass, as described herein, may be 0217 Hydrogen plus catalysts are used extensively in the used to make acetate esters which may be used as solvents for chemical industry to hydrogenate a variety of starting mate various resins in protective coatings and for formulating inks. rials to create chemical products for further chemical synthe sis use. The hydrogen obtained from anaerobic fermentation Such acetate esters include, but are not limited to, amyl, butyl, of a biomass, as described herein, may be used for hydroge ethyl, methyl, and propyl acetates which may be used as nation processes in the chemical industry. Such processes Solvents in quick-drying lacquers, cements and adhesives. include, but are not limited to, the following examples: 0213. The volatile organic acids obtained from anaerobic 0218 a) Hydrogenation of unsaturated hydrocarbons fermentation, as described herein, may be used in the synthe and aromatics sis of other chemical products. By way of example only, a 0219 e.g. benzene to cyclohexane using Ni /Pt— Volatile organic acid and a mineral may be admixed and Al2O3 catalyst, wherein the cyclohexane, is used as reacted to form a composition. This is demonstrated by an starting material for nylon production or as a solvent, exemplary embodiment in which acetic acid, obtained from 0220 b) Hydrogenation of ketones and aldehydes anaerobic fermentation of a biomass, is admixed and reacted 0221 e.g. acetone to mesityl oxide to methyl isobutyl with dolomite (calcium magnesium carbonate) to form cal ketone (MIBK) for use as a solvent 0222 c) Hydrogenation of nitrogen containing com cium magnesium acetate (reaction Scheme 1). pounds 0223 e.g. nitrobenzene to aniline using a Cu cata Scheme 1 lysts such as NiS/CuS, wherein the aniline is used as a starting material for dyes, pharmaceuticals, poly O mers, and solvents. (CaCO3/MgCO3 + ch--on --- 0224 Hydrogen gas is an environmentally friendly energy Source, as it can be used generate electricity without produc Dolomite Acetic acid ing greenhouse gas emissions. Thus, a hydrogen based sys tem offers totally clean energy Supplies with no pollution. US 2008/0311640 A1 Dec. 18, 2008 34

Hydrogen may be produced by a number of processes, includ generating electricity in the MW range. Solid oxide fuel cells ing electrolysis of water, thermocatalytic reformation of (SOFC) utilize oxygen radicals (O) as the mobile ion and hydrogen-rich organic compounds such as methane, and bio operate between 500° C. and 1000° C. Like MCFC, SOFC logical processes. Biological production of hydrogen pro can utilize H, CO, and/or CH as fuel, which means they can vides a wide range of approaches to generate hydrogen, use methane, coal gas, or biogas as fuel Sources. Carbon including direct biophotolysis, indirect biophotolysis, photo dioxide is not utilized as a fuel and is discharged as a waste fermentations, and dark-fermentation. The fermentation gas. Like other high-temperature fuel cell systems (PAFC and products, such as gases and solvents, produced as described MCFC) SOFC systems are used as stationary power sources, herein, may be used as fuel and/or energy sources for power generating electricity from the low kW to the MW range. generation using power generation systems. Such energy and Proton exchange membrane fuel cells (PEMFC) utilize power production applications, include, but are not limited to, hydrogen protons (H) as the mobile ion, operate in the powering motor vehicles, running turbines or fuel cells to 50-100° C. range, require pure H, and are sensitive to the produce electricity at centralized power stations, running tur presence of CO. Of all fuel cell systems available, PEMFC bines or fuel cells to produce electricity at localized decen systems are especially Suitable for mobile and transportation tralized power stations, and generating heat by direct com applications, such as engines for cars. Small PEMFC's, in the bustion. The power generation systems include, but are not 1-10 kW range, may be useful for electrical applications for limited to, internal combustion generators, turbine genera homes. tors, fuel cells, and Such systems may be part of a centralized power station or a decentralized power station. In addition, Anaerobic Fermentation System the electricity obtained locally from the decentralized power 0227. The anaerobic fermentation system described station may be used for residential consumption or commer herein is an assemblage of components, combined to produce cial consumption, or the electricity may be added to the grid chemical products by anaerobically fermenting biomass. and then used residentially or commercially. FIG. 16 is a schematic representation of a non-limiting 0225. The gaseous fermentation products used as fuel or example of the components of Such an assemblage. The energy sources include, but are not limited to hydrogen, car arrows between components are used to illustrate the trans bon dioxide, carbon monoxide, hydrogen Sulfide and meth portation of material from one component to another, con ane, wherein the methane may be produced by anaerobic tinuous movement of material from one component to fermentation or may be formed by methanation using the another, depiction of the next step in the process or combina hydrogen and carbon oxides produced by anaerobic fermen tions thereof. In addition, each component represented may tation. Methane formation by methanation results by mixing incorporate a single process, multi step processes, a single hydrogen and carbon oxides, such as, by way of example device, multiple devices, or combinations thereof. only, carbon dioxide and carbon monoxide, and a catalyst 0228. In FIG. 16 the biomass is pretreated prior to being Such as, by way of example only activated nickel catalysts and transported to the anaerobic fermentation digester. The pre NiO/MgO. The methane formed either by methanation or by treatment process involves concentration adjustment, detoxi anaerobic fermentation may be used as a fuel or energy source fication, sterilization, deoxygenation, and/or pre-digestion, to generate electricity from fuel cells or to generate heat by not necessarily in this order. By way of example only, the combustion in a heat generation system. The heat generation biomass may be sterilized and deoxygenated prior to concen system includes, but is not limited to, furnaces to heat build tration adjustment, detoxification, and/or pre-digestion steps: ings, stove-top, ovens, and barbeques for cooking, and dryers the biomass may be concentrated prior to sterilization, detoxi for drying clothes or commercial products Such as plastics, fication, deoxygenation, and/or pre-digestion; the biomass polymers, plywood, paper, and pharmaceutical products. may be sterilized, then deoxygenated, then detoxified, then Combustion of solvents obtained as fermentation products concentrated, and then pre-digested; the biomass may be may be used to generate steam to run turbines, used in internal detoxified, deoxygenated, then sterilized, and then concen combustion generators and turbine generators. Such solvent trated; the biomass may be concentrated, then sterilized, then include, but are not limited to, acetone, butanol, ethanol, deoxygenated, and then detoxified; or the biomass may be propanol, isopropanol. 1.2-propanediol and or other solvents. concentrated, then deoxygenated, then detoxified, and then 0226. There are various types of fuel cells, each with par sterilized. The concentration adjustment step may be accom ticular operating conditions and fuel requirements. The plished using methods which separate solids from liquids, hydrogen produced as described herein may be used in any of Such as, but not limited to, centrifugation and filtration, or the them. Alkaline fuel cells (AFC) utilize hydroxyl ions (OH) concentration adjustment step may be accomplished using as the mobile ion (derived from potassium hydroxide, KOH), methods which separate Solution phase chemicals from liquid operate in the 50 to 200° C. range, and are sensitive to the Such as, but not limited to, reverse osmosis, dialysis and presence of CO. Phosphoric acid fuel cells (PAFC) utilize osmosis. The components used to concentrate biomass using protons (H) as the mobile ion and operate at approximately Such techniques are known to one skilled in the art. 200° C. PAFC systems were the first fuel cells produced 0229. In the operation of the anaerobic fermentation sys commercially and are used as stationary power sources, gen tem described hereinsterilization can be accomplished using erating up to 200 kW of electricity. The high operating tem pasteurization and/or acidification. Other methods known in perature and corrosive nature of the electrolyte makes them the art may also be used to sterilize the biomass. unsuitable for use in mobile and transportation applications. 0230. In the operation of the anaerobic fermentation sys Molten carbonate fuel cells (MCFC) utilize carbonate ions tem described herein deoxygenation can be accomplished (CO) as the mobile ion, operate at approximately 650° C. using high pressure and low pressure steam. In general high and can take H2, CO, CO, and/or CH as fuel, which means pressure steam is generated with the use of a boiler, although they can use natural gas, coal gas, or biogas as fuel Sources. any means to generate high pressure steam may be used. This Like PAFC, MCFC are used as stationary power sources, high pressure steam may be used for deoxygenation, or a US 2008/0311640 A1 Dec. 18, 2008

pressure reducing valve may be used to lower the pressure, control tool and at least one metrology data, and wherein the and the low pressure Steam may be used for deoxygenation. In process controller comprises decision making units, input/ another embodiment turbines, including, but not limited to, output boards, and database units to store at least one metrol backpressure turbine generators, are used to both create low ogy data. The decision making unit is used in a feedback pressure Steam by reducing the pressure of high pressure control process to acquire metrology tool data from the input/ steam and generate electricity. The advantage associated with output board and to determine control adjustments based on the use of a turbine to generate low pressure steam is that in the metrology tool data and defined operational ranges for the addition to the low pressure steam being made available for desired anaerobic fermentation parameters. This maintains deoxygenation, the turbine can be used to generate electricity the anaerobic fermentation parameters within operational which can be used to operate the facility containing the ranges and allows for optimal operation of the anaerobic anaerobic digesters. FIG. 17 is a schematic demonstrating the digester. The magnitude of the control adjustments, are modi use of a turbine system to generate low pressure steam for fied and returned to the input/output board, whereby by the sterilization and deoxygenation of a biomass, in conjunction modified control adjustments are sent to process control tools to the generation of electricity for use by the facility, or any to adjust operating parameter of the anaerobic fermentation other electricity consumer. As seen in FIG. 17, a heat source digester and thereby maintain the digester at optimal condi (i.e. commerically bought or from an anerobic substrate tions as defined by the operational ranges. Source/reaction) heats the boiler. High pressure steam enters 0233. The type of decision making units incorporated into the turbine which causes the blades to turn and thereby turn the anaerobic fermentation systems described herein include, ing a generator and creating electricity. Thus the steam tur but are not limited to, computers, PROM’s (Programmable bine generator makes electricity by converting a steam pres Read-Only Memory), EPROM's (Erasable Programmable Sure drop into mechanical power to spin a generator. As the Read-Only Memory) and EEPROM's (Electrically Erasable high-pressure steam enters the turbine and drives the genera Programmable Read-Only Memory). The types of metrology tor, the lower pressure steam exhausts from the turbine and is tool incorporated into the anaerobic fermentation systems used to either heat the facility, Sterilize and deoxygenate a described herein include, but are not limited to, at least one biomass, or combinations thereof. In addition, single back pH sensor, at least one pressure sensor, at least one gas flow pressure turbine generators may be used, or multistage back sensor, at least one temperature sensor, at least one GC, at pressure turbine generators may be used to handle different least one HPLC, at least one FIA, and combinations thereof. steam paths form different high pressure steam sources. In addition, the type of process control tool incorporated into 0231. Afterpretreatment the biomass is transported to the the anaerobic fermentation systems described herein anaerobic fermentation digester (FIG. 16), where it is fer includes, but is not limited to, a valve, a hotplate, or a heating mented using anaerobic bacteria chosen for the particular mantle. biomass present. The conditions inside the anaerobic digester 0234 FIG. 18 is a schematic of an embodiment of the may vary according to the anaerobic bacteria being used, the anaerobic digester control system, wherein, by example only, configuration of the anaerobic digester, the feedstock being the metrology tool is a pH sensor and the operating parameter converted, the desired productivity of the anaerobic digester, to be controlled is the pH of the anaerobic fermentation the chemical product being produced, and the form of bacte media. Adjustment of the pH is by addition of acid or base ria (immobilized or free-flowing) used. Mobilized bacteria from appropriate tanks by control of valves which couple the can be prepared using any methods known by the artisan of acid and base tanks to the anaerobic digester. Adjustment ordinary in the arts. In order to optimally operate the anaero occurs to maintain the pH in a defined operational range. The bic digester the conditions used to ferment the biomass are pH sensor information is acquired by the I/O board which monitored, and the fermentation parameter are then con sends the information to the decision making unit. Depending trolled to achieve optimal production of chemical product and on the pH value and the resulting calculated control adjust the maintenance of anaerobic bacteria viability. The fermen ment, the valve, to either the acid or base tanks, is opened and tation parameters include, but are not limited to, Solids con a predetermined aliquot is added to the digester. The valve is tent, nutrient Solution composition, temperature, gas content, then closed and the process repeats to maintain the digester at digestion rate, anaerobic bacteria content, agitation, feed and optimal pH as defined by the operational range. effluent rates, gas production rate, carbon/nitrogen ratio of the feed Stock, pressure, pH, and retention time in the digester. ILLUSTRATIVE EXAMPLES These parameters may be monitored continuously or inter 0235. The following examples are provided for illustrative mittently, using pH sensors, gas sensors, GC's, HPLC's, purposes only and not to limit the scope of the claims pro FIA's, pressure sensors, temperature sensors, optical densito vided herein. meters, refractometers, and gas flow sensors 0232. The operation of the anaerobic fermentation sys Example 1 tems described herein may be a continuous process or a batch process. In either approach the anaerobic fermentation Batch Anaerobic Fermentation of Plant Material parameters. Such as, by way of example only, temperature, Stem, Leaf, Seed and Vegetable Parts of Bell Pepper pH, pressure, gas flow, biomass concentration, and fermen Plants tation chemical product concentration, may be monitored and the information used to optimally operate the anaerobic fer 0236 Parts of bell pepper plants, including leaves, stems, mentation digester system. The anaerobic digester control Vegetables and seeds, is obtained from a bell pepper farm and system may comprise at least one process control tool; at least is shredded using a mulching machine. The shredded material one metrology tool to acquire at least one metrology data and water are placed into a vat, whereby the mixture is treated relating to at least one anaerobic fermentation parameter; a with low pressure steam for sterilization and deoxygenation. process controller operatively coupled to at least one process The steam treatment also assists in degrading cellulosic mate US 2008/0311640 A1 Dec. 18, 2008 36 rial for easier fermentation. The sterilized/deoxygenated carrier gas and a fused silica capillary column 0.1 umx0.53 mixture is then concentrated by removing a portion of the mmx15 m MXT-WAX (Restek, Bellefonte, Pa.) water using a centrifuge system. After centrifugation the con centrated/sterilized/deoxygenated material is pumped into an Example 3 anaerobic digester which has been deoxygenated by sparging Utilizing the Enrichment/Isolation System and with nitrogen. The anaerobic digester is a modified commer Knowledge Management System to Identify Optimal cial digester (DCI Inc., St. Cloud, Minn.). After addition of Biomass for Bacterial Strains in Producing Hydro the treated bell pepper material the anaerobic digester is gen Anaerobically inoculated with a bacterial culture of Clostridium butyricum. 0238. Using two parallel glass plates approximately During fermentation the temperature, pH, pressure, concen 10,000 cm involume, a sealed chamber is formed to house an tration of anaerobic fermentation products and optical density enrichment/isolation system. In very concentrated spots are monitored and changes to the digester operating param nutrients are placed within the sealed chamber either by direct eters are adjusted accordingly. Throughout the fermentation placement or metering in with a hose. Different types of process the anaerobic fermentation gas products are col nutrients are placed away from each other a distance so that lected, separated and purified. Upon completion of the fer different spatial Zones of bacteria around the nutrients may mentation process the digester is emptied and the slurry is form. Nutrients used are organic acids, diesel, food waste fats filtered to obtain the liquid which is further processed to and cellulose. Bacteria used in the sealed chamber are har obtain the Volatile organic acid produced. Vested from decomposing bell peppers. The bacteria are introduced into sealed chamber so that they use the nutrients Example 2 as a food source. After 3-4 weeks bacterial samples around the nutrients are taken out using a syringe or tool which is Continuous Anaerobic Fermentation of Plant Mate non-evasive. The bacterial samples are tested to determine rial whether the bacteria are primary or secondary users of the food source. The bacteria are isolated, put on defined medium Stem, Leaf, Seed and Vegetable Parts of Bell Pepper to test what they use as nutrients. Two particular indicator tests that are preformed are the nitrate and catalase tests. The Plants bacteria are also assayed for their production of hydrogen at different growth conditions involving variation in tempera 0237 Parts of bell pepper plants, including leaves, stems, ture, pressure, pH, fermentation products, exposure to various Vegetables and seeds, is obtained from a bell pepper farm and natural gases or compounds, drug efficacy, compound toxic is shredded using a mulching machine. The shredded material ity or survivability. and water are placed into a vat, whereby the mixture is treated 0239 Information regarding which types of bacteria spe with low pressure steam for sterilization and deoxygenation. cies uses what particular type of nutrients as a food source and The steam treatment also assists in degrading cellulosic mate optimal bacterial growth conditions for producing hydrogen rial for easier fermentation. The sterilized/deoxygenated are entered into the Knowledge Management System data mixture is then concentrated by pumping the slurry through a base. The Knowledge Management System houses data that continuously fed reverse osmosis system (GE Osmonics can be quickly accessed and manipulated into a user friendly Corp., Kent, Wash.); thereby a portion of the water is form such as a graph or spreadsheet. In particular, the Knowl removed. The concentrated/sterilized/deoxygenated slurry is edge Management System can narrow down which bacteria pumped into an anaerobic digester which is continuously uses particular nutrients for producing a specific amount of anaerobically fermenting biomass and contains a population hydrogen. of Clostridium butyricum. The anaerobic digester is a modi 0240 All references cited herein, including patents, patent fied commercial digester (DCI Inc., St. Cloud, Minn.). Dur applications, and publications, are hereby incorporated by ing fermentation the temperature, pH, pressure, concentra reference in their entirety. All of the methods disclosed and tion of anaerobic fermentation products and optical density claimed herein can be made and executed without undue are constantly monitored using a digester control system and experimentation in light of the present disclosure. It will be changes to the digester operating parameters are adjusted apparent to those of skill in the art that variations may be continuously by the digester control system. The digester applied to the methods and in the steps or in the sequence of control system is a custom built system, wherein pH sensors, steps of the method described herein without departing from pressure sensors, temperature sensors, and gas sensors are the concept, spirit and scope of the invention. More specifi monitored using Lab VIEW NIDAQ software (National Instruments Corporation, Austin, Tex.), running a DAQPad cally, it will be apparent that certain agents that are both 6508 National Instruments I/O board. Any adjustments chemically and physiologically related may be substituted for needed to the operating parameters are calculated using a the agents described herein while the same or similar results laptop computer based on the sensor signals. Throughout the would be achieved. All such similar substitutes and modifi fermentation process the anaerobic fermentation gas products cations apparent to those skilled in the art are deemed to be are continuously removed from the digester using a vacuum. within the spirit, scope and concept of the invention as defined The gaseous products are separated and purified using differ by the appended claims. ential compression. The fermenting slurry is recycled through What is claimed: a reverse osmosis system (GEOsmonics Corp., Kent, Wash.) 1. A method of anaerobically producing hydrogen com to remove the Volatile organic acids produced. The concen prising: obtaining a biomass material Suitable as a nutrient for tration of the organic acids are measured using a gas chro isolated non-heatshocked bacterial species from the matograph (Model 8610C: SRI Instruments, Torrance, Calif.) Clostridium genus; concentrating the biomass material by at equipped with a flame ionization detector, using nitrogen as least a factor of 2; Sterilizing the biomass; deoxygenating the US 2008/0311640 A1 Dec. 18, 2008 37 biomass material; and fermenting the concentrated, sterilized wherein the population of substantially purified anaerobic and deoxygenated biomass material with the isolated non bacterial strain has not been Subjected to a heatshocking heatshocked bacterial species from the Clostridium genus process. under anaerobic conditions So as to produce hydrogen. 44. (canceled) 2. (canceled) 45. (canceled) 3. The method of claim 1, wherein the concentration, ster 46. (canceled) ilization, and the deoxygenation steps overlap. 47. (canceled) 4. (canceled) 48. (canceled) 5. (canceled) 49. (canceled) 6. (canceled) 50. (canceled) 7. (canceled) 51. The anaerobic fermentation apparatus of claim 43, 8. (canceled) wherein the anaerobic fermentation apparatus is a component 9. (canceled) of an assemblage for generating power. 10. (canceled) 52. (canceled) 11. (canceled) 53. (canceled) 12. (canceled) 54. (canceled) 13. (canceled) 55. (canceled) 14. (canceled) 56. The anaerobic fermentation apparatus of claim 43, 15. (canceled) wherein the chemical product is selected from the group 16. The method of claim 1, wherein the biomass material consisting of a gaseous chemical product, a non-gaseous comprises molasses, raw paper, agricultural waste, and/or chemical product, and combinations thereof. mulch. 57. (canceled) 17. (canceled) 58. (canceled) 18. (canceled) 59. (canceled) 60. (canceled) 19. (canceled) 61. (canceled) 20. (canceled) 62. (canceled) 21. The method of claim 1, wherein the substantially puri 63. (canceled) fied anaerobic bacteria culture comprises a substantially puri 64. (canceled) fied single bacterial strain. 65. (canceled) 22. (canceled) 66. (canceled) 23. (canceled) 67. (canceled) 24. (canceled) 68. (canceled) 25. (canceled) 69. (canceled) 26. The method of claim 1, further comprising collecting a 70. (canceled) chemical product is selected from the group consisting of a 71. The anaerobic fermentation apparatus of claim 43, gas, a Solid, a solvent, a Volatile organic acid, a salt of a wherein the anaerobic digester control system is used to opti Volatile organic acid, and combinations thereof. mally operate the anaerobic digester and comprises: an at 27. (canceled) least one process control tool; an at least one metrology tool 28. (canceled) to acquire at least one metrology data relating to at least one 29. (canceled) anaerobic fermentation parameter; an a process controller 30. (canceled) operatively coupled to the at least one process control tool and 31. (canceled) the at least one metrology data, wherein the process controller 32. (canceled) comprises a decision making unit, an input/output board, and 33. (canceled) a database unit to store the at least one metrology data. 34. (canceled) 72. (canceled) 35. (canceled) 73. (canceled) 36. (canceled) 74. (canceled) 37. (canceled) 75. (canceled) 38. (canceled) 76. (canceled) 39. (canceled) 77. (canceled) 40. (canceled) 78. (canceled) 41. (canceled) 79. (canceled) 42. (canceled) 80. (canceled) 43. An anaerobic fermentation apparatus using a popula 81. (canceled) tion of substantially purified anaerobic bacterial strain for 82. (canceled) anaerobically fermenting a biomass into a chemical product 83. (canceled) comprising: (i) a sterilization and deoxygenation system; (ii) 84. The anaerobic fermentation apparatus of claim 43, an anaerobic digester containing a population of substantially wherein the biomass comprises a material selected from the purified anaerobic bacteria and equipped with an anaerobic group consisting of energy crops, Surplus agricultural prod digester control system; (iii) a plurality of pipelines and ucts, waste from Sugar production and processing facilities, pumps for introducing and re-circulating biomass, and (iv) waste from fruit processing industries, waste from pulp and removal pipelines connected to the anaerobic digester for paper mills, Silvaculture residues, waste from wood process removing a chemical product from the anaerobic digester, ing, waste from agricultural product processing, food waste, US 2008/0311640 A1 Dec. 18, 2008

Solids isolated from fermentation cultures, municipal sewer 102. The method of claim 101, wherein the appropriate waste, animal manure, animal urine, animal parts, fish parts, information is collected by conducting a cultivation system to and combinations thereof. identify various bacterial species. 85. (canceled) 103. The method of claim 102, wherein the cultivation system comprises growing various bacterial species on par 86. (canceled) ticular substrates, under various growth conditions to opti 87. An apparatus for selecting bacteria for use in anaerobic mize the bacterial hydrogen production. hydrogen production comprising a sealed chamber capable 104. The method of claim 103, wherein the various growth for providing an anaerobic environment and a multiplicity of conditions are selected from the group consisting of variation growth conditions. in temperature, pH, fermentation products, exposure to vari 88. The apparatus of claim 87, further comprising bacteria, ous natural gases or compounds, drag efficacy, compound collected from bovine rumen, Soil samples, sludge, anaerobic toxicity or survivability. bacteria cultures, aerobic bacteria cultures, anaerobic sedi 105. The method of claim 101, wherein the bacteria are ments from fresh or brackish waters, sewage, animals, animal collected from bovine rumen, Soil samples, sludge, anaerobic feces, insect digestive tract, dental samples, hydrothermal bacteria cultures, aerobic bacteria cultures, anaerobic sedi soils, hydrothermal pools or deep water hydrothermal vents. ments from fresh or brackish waters, sewage, animals, animal 89. (canceled) feces, insect digestive tract, dental samples, hydrothermal 90. The apparatus of claim 87, wherein the multiplicity of soils, hydrothermal pools and deep water hydrothermal vents. growth conditions are selected from the group consisting of 106. (canceled) variation in temperature, pressure, pH, fermentation prod 107. (canceled) ucts, exposure to various natural gases of compounds, drug 108. (canceled) efficacy, compound toxicity or Survivability. 109. (canceled) 110. (canceled) 91. (canceled) 111. (canceled) 92. (canceled) 112. (canceled) 93. (canceled) 113. (canceled) 94. The apparatus of claim 87, further comprising a par 114. (canceled) ticular biomass sample the bacteria utilize as nutrients for 115. (canceled) producing hydrogen gas. 116. A bacterial culture comprising a purified bacterial 95. The apparatus of claim 94, wherein the biomass sample strain, wherein the bacterial strain produces hydrogen by comprises a material selected from the group consisting of anaerobically fermenting a biomass without heatshocking the energy crops, Surplus agricultural products, waste from Sugar bacterial strain and wherein the biomass has been deoxygen production and processing facilities, waste from fruit pro ated. cessing industries, waste from pulp and paper mills, silvacu 117. (canceled) lture residues, waste from wood processing, waste from agri 118. The bacterial culture of claim 116, wherein the bio cultural product processing, food waste, Solids isolated from mass comprises material from at least one member of the fermentation cultures, municipal sewer waste, animal genus Capsicum. manure, animal urine, animal parts, fish parts, and combina 119. (canceled) tions thereof. 120. The bacterial culture of claim 116, wherein the bio 96. (canceled) mass comprises material from at least one member of the 97. (canceled) genus Allium. 98. (canceled) 121. (canceled) 99. (canceled) 122. The bacterial culture of claim 116, wherein the bio 100. (canceled) mass comprises material from at least one member of the 101. A program product for use in a computer that executes genus Saccharum. program instructions recorded in a computer-readable media 123. (canceled) to obtain candidate bacterial species for use in the anaerobic 124. The bacterial culture of claim 116, wherein the bio production of hydrogen from a particular biomass, the pro mass is a species selected from the group consisting of gram product comprising: a recordable medium; and a plu Solanum Esculentum, Solanum melongena, Solanum tubero rality of computer-readable program instructions on the sum, Lycopersicon esculentum, Beta vulgaris, or combina recordable media that are executable by the computer to tions thereof. perform a method comprising: 125. (canceled) 126. (canceled) a) determining a bacterial species of interest; 127. (canceled) b) determining the appropriate information for the bacte 128. (canceled) rial species of interest; and 129. (canceled) c) repeating steps a-b for other bacterial species; and 130. (canceled) d) comparing appropriate information collected from step 131. (canceled) c to assess optimal candidate bacterial species for use in 132. (canceled) the anaerobic production of hydrogen from a particular biomass.