USOO85O1463B2

(12) United States Patent (10) Patent No.: US 8,501,463 B2 Cox et al. (45) Date of Patent: Aug. 6, 2013

(54) ANAEROBC PRODUCTION OF HYDROGEN (56) References Cited AND OTHER CHEMICAL PRODUCTS U.S. PATENT DOCUMENTS (75) Inventors: Marion E. Cox, Morgan Hill, CA (US); 5,350,685 A 9/1994 Taguchi et al. Laura M. Nondorf, Morgan Hill, CA 5,464,539 A 11/1995 Ueno et al. 6,090,266 A 7/2000 Roychowdhury (US); Steven M. Cox, Morgan Hill, CA 6,251,643 B1 6/2001 Hansen et al. (US) 6,299,774 B1 * 10/2001 Ainsworth et al...... 210,603 6,342,378 B1 1/2002 Zhang et al. (73) Assignee: Anaerobe Systems, Morgan Hill, CA 6,569,332 B2 * 5/2003 Ainsworth et al...... 210,603 2004/0050778 A1 3/2004 Noike et al. (US) 2004/O115782 A1 6/2004 Paterek (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 WO WO-2006-119052 A2 11/2006 U.S.C. 154(b) by 1347 days. OTHER PUBLICATIONS (21) Appl. No.: 11/912,881 Liu et al., 2004. Effects of Culture and Medium Conditions on Hydro gen Production from Starch Using Anaerobic . Journal of (22) PCT Fled: Apr. 27, 2006 Bioscience and Bioengineering, vol. 98, No. 4, pp. 251-256.* Zhang et al., Distributed Computer Control of Penicillin Fermenta (86) PCT NO.: PCT/US2OO6/O16332 tion Industrial Production. Proceedings of the IEEE International Conference on Industrial Technology, 1996, pp. 52-56.* S371 (c)(1), New Brunswick, an eppenforf Company, pp. 1-3. http://www.nbsc. (2), (4) Date: Jun. 23, 2008 com/BioFlo-415-SIP-Fermentor.aspx Printed Mar. 26, 2012.* Prell et al. 2000. Lower layer control of the CSTR, a basic part of the bioreactor control system. 27th International Conference of Slovak (87) PCT Pub. No.: WO2O06/119052 Society of Chemical Engineering, Tatranske Matliare, Slovakia, PCT Pub. Date: Nov. 9, 2006 Proc. p. 30, Lecture No. R16.* Claassen, P.A.M. et al., “Utilisation of biomass for the supply of energy carriers.” Appl. Microbiol. Biotechnol. 52:741-755 (1999). (65) Prior Publication Data Gorman, J., “Hydrogen: The Next Generation, Cleaning up productionof a future fuel.” 8 pages retrieved from Internet on Oct. US 2008/0311640 A1 Dec. 18, 2008 27, 2004 http://www.sciencenews.org/articles/2w0021012/bob11. asp. Henley, M. et al., “Microbial Hydrogen Production.” 3 pages Related U.S. Application Data retrieved from Internet on Oct. 27, 2004 http://www.afrlhorizons. com/Briefs, Jun03/MLO227.html. (60) Provisional application No. 60/678,101, filed on May Levin, D.B. et al., “Biohydrogen production: prospects and limita 3, 2005, provisional application No. 60/677,856, filed tions to practical application.” Intl. J. Hydrogen Energy 29:173-185 (2004). on May 3, 2005, provisional application No. Logan, B. et al., "Biological Hydrogen Production as a Sustainable 60/678,077, filed on May 3, 2005, provisional GreenTechnology for Pollution Prevention.” 2 pages retrieved from application No. 60/678,100, filed on May 3, 2005, Internet on Oct. 27, 2004 http://www.engr/psu.edu/h2e/Pub/Logan provisional application No. 60/678,098, filed on May etal 1.htm. Logan, B. et al., “Biological Hydrogen Production in a Bioreactor” 1 3, 2005, provisional application No. 60/677,998, filed page retrieved from Internet on Oct. 27, 2004 http://www.engr/psu. on May 3, 2005. edu/h2e/Pub/Logan etal 1.htm. (51) Int. C. (Continued) CI2M I/07 (2006.01) CI2M I/2 (2006.01) Primary Examiner—Jon PWeber CI2P3/00 (2006.01) Assistant Examiner — Kailash C Srivastava CI2P I/04 (2006.01) (74) Attorney, Agent, or Firm — Wilson, Sonsini, Goodrich CI2N L/20 (2006.01) & Rosati CI2M I/36 (2006.01) CI2M 1/38 (2006.01) (57) ABSTRACT CI2M 1/107 (2006.01) Described herein are methods for producing chemical prod (52) U.S. C. ucts by anaerobically fermenting a particular biomass using USPC ...... 435/303.2; 435/168; 435/252.1; anaerobic bacteria. Such chemical products includehydrogen 435/286.1; 435/290.4 and other gases, acetic acid and other volatile organic acids, (58) Field of Classification Search Solvents, Solids, and salts of Volatile organic acids. USPC ...... 435/168,252.1, 286.1, 290.4, 303.2 See application file for complete search history. 5 Claims, 18 Drawing Sheets US 8,501.463 B2 Page 2

OTHER PUBLICATIONS Hussy et al., “Continuous Fermentative Hydrogen Production from a Wheat Starch Co-Product by Mixed Microfloara.” Biotech. Bioeng. Logan, B. et al., “Biological Hydrogen Production Measured in 84(6):619-626 (2003). Batch Anaerobic Respirometers.” Environ. Sci. Technol. 36:2530 Lay, J.J. et al., “Influence of chemical nature of organic wastes on 2535 (2002). Oh, S.E. et al., “The Relative Effectiveness of pH Control and Heat their conversion to hydrogen by heat-shock digested sludge.” Intl. J. Treatment for Enhancing Biohydrogen Gas Production.” Environ. Hydrogen Energy 28:1361-1367 (2003). Sci. Technol. 37:5186-5190 (2003). Taguchi et al., “Direct conversion of ceulolosic materials to hydro Raskin, S.S. et al., “Hydrogen Production by Anaerobic Microbial gen by Clostridium sp. Strain No. 2. Enzyme and Microbial Tech. Communities Exposed to Repeated Heat Treatments.” Proceedings 17: 147-150 (1995). of the 2002 U.S. DOE Hydrogen Program Review, NREL/CP-610 Yokol, H. et al., “Microbial production of hydrogen from starch 32405, 17 pages. manufacturing wastes.” Biomass and Bioenergy 22:389-395 (2002). Regan, J. et al., “Genetic Engineering of Chlostridium PCT/US06/16332 Search Report Dated Sep. 12, 2008. acetobutylicum for Enhanced Production of Hydrogen Gas, 1 page retrieved from Internet on Oct. 27, 2004 http://www.engrpsu.edu/ Abraham et al., "Commissioning and Re-Design of a Class A Ther h2e/Pub/Regan etal 1.htm. mal Hydrolysis Facility for Pre-Treatment of Primary and Secondary Ren, N.O. et al., “Hydrogen Production from Molasses by Anaerobic Sludge Prior to Anaerobic Digestion.” 76' Annual Water Environ Activated Sludge in Pilot-Scale Bioreactor.” Poster Presentation 2-27 ment Federation Technical Exposition and Conference, from 26' Symposium on Biotechnology for Fuels and Chemicals, XP002660698, pp. 1-14 (2003) Retrieved from the Internet: May 9-12, 2004, Chattanooga, TN USA, retrieved from Internet on URL:http://www.cambi.no/photoalbum/view2. Dec. 17, 2004, http://www.ct.ornil.gov/symposium/index files/2ab P3NpemU9b3JnJmlkPTIyMDAZMCZOeXB1PTE *abstract *p. 2, stracts/2 27.htm. last paragraph-p. 3, last paragraph. Taguchi, F. et al., “Continuous Hydrogen Production by Clostridium Wang et al., “Producing Hydrogen from Wastewater Sludge by sp. Strain No. 2 from Cellulose Harolysate in an Aqueous Two-Phase Clostridium Bifermentans,” Journal of Biotechnology, vol. 102, No. System.” J. Fermentation and Bioengineering 82(1):80-83 (1996). 1, pp. 83-92 (2003). Van Ginkel, S. et al., “Role of Initial Sucrose and pH Levels on EP06751822.5 Extended European Search Report mailed Oct. 18, Natural, Hydrogen-Producing, Anaerobe Germination.” Proceedings 2011. of the 2001 DOE Hydrogen Program Review, NREL/CP-570 3053.15 pages. * cited by examiner

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US 8,501,463 B2 1. 2 ANAEROBC PRODUCTION OF HYDROGEN into chemical products. In another aspect are assemblages AND OTHER CHEMICAL PRODUCTS which comprise sources of steam; turbines; and digesters. In one aspect is the use of bacteria for the production of CROSS REFERENCE hydrogen and other chemical products. In another aspect biomass prepared for use in hydrogen production has been This application claims the benefit of U.S. provisional sterilized, deoxygenated, concentrated, detoxificated, and/or application Ser Nos. 60/678,101, filed May 3, 2005; 60/677, pre-digested while optimal bacterial strains for anaerobic 856, filed May 3, 2005; 60/678,077, filed May 3, 2005; fermentation of the biomass have been selected by means of 60/678,100, filed May 3, 2005; 60/678,098, filed May 3, a Knowledge Management System, and selected and isolated 2005; and 60/677,998, filed May 3, 2005, all of which are 10 using an isolation/enrichment system. incorporated by reference in their entirety. Bacterial Strains In further or alternative embodiments of all aspects FIELD OF THE INVENTION described herein using bacterial strains to anaerobically fer ment biomass and obtain chemical products therefrom, the The methods and compositions described herein are 15 bacterial strains are substantially purified anaerobic bacteria directed to the production of hydrogen and other chemical and such substantially purified bacterial strains are selected to products via the anaerobic bacterial fermentation of biomass, anaerobically ferment the biomass, and in still further or and the production of hydrogen and other chemical products alternative embodiments of all aspects, the Substantially puri via bacterial conversion of products obtained from anaerobic fied bacterial strains are selected from the group consisting of fermentation. Acetivibrio cellulolyticus, Acetivibrio cellulosolvens, Acetiv ibrio ethanolgignens, Acetivibrio multivorans, Acetoanaero BACKGROUND OF THE INVENTION bium noterae, Acetofilamentum rigidium, Acetogenium kivui, Acetomicrobium faecale, Acetomicrobium flavidum, Aceto Hydrogen has high energy content, with water being the thermus paucivorans, Acidaminobacter hydrogenoformans, product resulting from combustion of hydrogen with oxygen. 25 Anaerobiospirillum succiniciproducens, Anaerobiospirillum AS Such, hydrogen represents a potentially ideal fuel source. ihomasii, Anaerorhabdus fircosa, Anaerovibrio burkinaben However, the use of hydrogen as a viable alternative energy sis, Anaerovibrio glycerini, Anaerovibrio lipolyticus, Atopo Source remains challenging, because many methods for pro biunfossor; Atopobium minutum, Atopobium parvulum, Ato ducing hydrogen, Such as water electrolysis, and petroleum pobium rimae, Atopobium vaginae, acidifaciens, reforming, are economically and energetically intense. 30 Bacteroides amylophilus, Bacteroides asaccharolyticus Bacteroides bivius, Bacteroides buccae, Bacteroides bucca SUMMARY OF THE INVENTION lis, Bacteroides caccae Bacteroides capillosus, Bacteroides capillus, Bacteroides cellulosolvens, Bacteroides coagulans, In one aspectare methods of preparing nutrients for use in Bacteroides corporis, Bacteroides denticola, Bacteroides anaerobic production of hydrogen. In another aspect are 35 disiens, Bacteroides distasonis, Bacteroides eggerthii, nutrients for use in the anaerobic production of hydrogen, Bacteroides endodontalis, Bacteroides forsythus, Bacteroi wherein the nutrients are prepared by methods, which des fragilis, Bacteroides fircosus, Bacteroides galacturoni include, but are not limited to concentrating, sterilization and cus, Bacteroides gingivalis, Bacteroides gracilis, Bacteroi deoxygenation. des helcogenes, Bacteroides heparinolyticus, Bacteroides In one aspect are isolation/enrichment systems for select 40 hypermegas, Bacteroides intermedius, Bacteroides levi, ing bacteria for use in anaerobic hydrogen production, Bacteroides loescheii, Bacteroides macacae, Bacteroides wherein the bacterial strains are selected for their ability to melaminogenicus, Bacteroides meloninogenicus Subsp. utilize biomass as nutrients and produce hydrogen gas and intermedius, Bacteroides melaminogenicus Subsp. macaca, other chemical products. In another aspect is an apparatus for Bacteroides melaminogenicus Subsp. melaninogenicus, selecting bacteria for use in anaerobic hydrogen production 45 Bacteroides merdae, Bacteroides microfitsus, Bacteroides comprising a sealed chamber capable of providing an appro multiacidus, Bacteroides nodosus, Bacteroides ochraceus, priate environment and growing conditions. Bacteroides oralis, Bacteroides Oris, Bacteroides Oulorum, In one aspect is a Knowledge Management System used to Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides identify bacteria for use in anaerobic production of hydrogen pentosaceus, Bacteroides pneumosintes, Bacteroides from using biomass, wherein bacteria are identified by col 50 polypragmatus, Bacteroides praeacutus, Bacteroides putre lecting appropriate information from various bacterial spe dinis, Bacteroides pyogenes, Bacteroides ruminicola, cies and deriving a probabilistic model from the information. Bacteroides ruminicola Subsp. brevis, Bacteroides rumini In another aspect is a computer program product to obtain cola Subsp. ruminicola, Bacteroides salivosus, Bacteroides candidate bacterial for use in the anaerobic produc splanchnicus, Bacteroides stercoris, Bacteroides succino tion of hydrogen and other chemical products from a particu 55 genes, Bacteroides suis, Bacteroides tectus, Bacteroides ter lar biomass. mitidis, Bacteroides thetaiotaOmicron, Bacteroides unifor In one aspect are chemical products produced by anaero mis, Bacteroides ureolyticus, Bacteroides veroralis, bically fermenting biomass with bacteria. In another aspect Bacteroides vulgatus, Bacteroides xylanolyticus, Bacteroides are chemical products formed by admixing hydrogen, a start zoogleoformans, Bifidobacterium adolescentis, Bifidobacte ing material and a catalyst, wherein the hydrogen is produced 60 rium angulatum, Bifidobacterium animalis, Bifidobacterium by anaerobically fermenting a biomass with bacteria. In animalis Subsp. animalis, Bifidobacterium animalis Subsp. another aspect are chemical products formed by admixing a lactis, Bifidobacterium asteroids, Bifidobacterium bifidum, mineral and a volatile organic acid, wherein the Volatile Bifidobacterium bourn, Bifidobacterium breve, Bifidobacte organic acid is produced by anaerobically fermenting a bio rium catenulatum, Bifidobacterium choerinum, Bifidobacte mass with bacteria. 65 rium coryneforme, Bifidobacterium cuniculi, Bifidobacte In one aspect are anaerobic fermentation apparatuses rium denticolens, Bifidobacterium dentium, Bifidobacterium which use bacteria for anaerobically fermenting a biomass gallicum, Bifidobacterium gallinarum, Bifidobacterium glo US 8,501,463 B2 3 4 bosum, Bifidobacterium indicum, Bifidobacterium infantis, Leptotrichia Shahi, Leptotrichia trevisani, Leptotrichia Bifidobacterium inopinatum, Bifidobacterium lactis, Bifido wadei, Malonomonas rubra, Megamonas hypermegale, Mit bacterium longum, Bifidobacterium magnum, Bifidobacte suokella dentalis, Mitsuokella jalaludinii, Mitsuokella mult rium merycicum, Bifidobacterium minimum, Bifidobacterium acida, Oxalobacter formigenes, Oxalobacter vibrioformis, pseudocatenulatum, Bifidobacterium pseudolongum, Bifido 5 Pectinatus cerevisiphilus, Pectinatus frisingensis, Pectinatus bacterium pseudolongum Subsp. globosum, Bifidobacterium portalensis, Pelobacter acetylenicus, Pelobacter acidigallici, pseudolongum Subsp. pseudolongum, Bifidobacterium psy Pelobacter carbinolicus, Pelobacter massiliensis, Pelobacte chraerophilum, Bifidobacterium pullorum, Bifidobacterium rpropionicus, Pelobacter venetianus, Porphyromonas a sac ruminantium, Bifidobacterium saeculare, Bifidobacterium charolytica, Poiphyromonas Cangingivalis, Porphyromonas scardovii, Bifidobacterium subtile, Bifidobacterium suis, 10 canoris, Porphyromonas can sulci, Porphyromonas Catoniae, Bifidobacterium thermacidophilum, Bifidobacterium ther Porphyromonas circumdentaria, Porphyromonas creviorica macidophilum Subsp. porcinum, Bifidobacterium thermaci nis, Poiphyromonas endodontalis, Porphyromonas gingiva dophilum Subsp. thermacidophilum, Bifidobacterium ther lis, Porphyromonas gingivicanis, Porphyromonas guilae, Poi mophilum, Bilophila wadsworthia, Butyrivibrio crossotus, phyromonas levi, Porphyromonas macacae, Porphyromonas Butyrivibrio fibrisolvens, Butyrivibrio hungatei, Campylo 15 salivosa, Porphyromonas uenonis, PrevOtella albensis, Pre bacter butzleri, Campylobacter cinaedi, Campylobacter coli, votella bivia, Prevotella brevis, PrevOtella bryantii, Prevo Campylobacter concisus, Campylobacter Cryaerophilus, tella buccae, Prevotella buccalis, PrevOtella corporis, Prevo Campylobacter curvus, Campylobacterfennelliae, Campylo tella dentalis, Prevotella denticola, Prevotella disiens, bacter fetus, Campylobacter fetus Subsp. fetus, Campylo Prevotella enoeca, Prevotella heparinolytica, Prevotella bacter fetus Subsp. venerealis, Campylobacter gracilis, intermedia, PrevOtella loescheii, Prevotella melamino Campylobacter helveticus, Campylobacter horninis, Campy genica, Prevotella multiformis, Prevotella nigrescens, Prevo lobacter hyoilei, Campylobacter hyointestinalis, Campylo tella oralis, Prevotella Oris, Prevotella Oulorum, Prevotella bacter hyointestinalis Subsp. hyointestinalis, Campylobacter pollens, Prevotella ruminicola, Prevotella salivae, Prevotella hyointestinalis Subsp. lawsonii, Campylobacter insulaemi Shahi, Prevotella tannerae, Prevotella veroralis, Prevotella grae, Campylobacter jejuni, Campylobacter jejuni Subsp. 25 zoogleoformans, Propionibacterium acidipropionici, Propi doylei, Campylobacter jejuni Subsp. jejuni, Campylobacter Onibacterium acnes, Propionibacterium australiense, Propi lanienae, Campylobacter lari, Campylobacter mucosalis, Onibacterium avidum, Propionibacterium cyclohexanicum, Campylobacter mustelae, Campylobacter nitrofigilis, Propionibacterium feudenreichii, Propionibacterium Campylobacter pylori, Campylobacter pylori Subsp. muste freudenreichii subsp. Freudenreichii, Propionibacterium lae, Campylobacter pylori Subsp. pylori, Campylobacter rec 30 feudenreichi Subsp. Shermanii, Propionibacterium granu tus, Campylobacter Showae, Campylobacter sputorum, losum, Propionibacterium innocuum, Propionibacterium Campylobacter sputorum subsp. bubulus, Campylobacter jensenii, Propionibacterium lymphophilum, Propionibacte sputorum Subsp. mucosalis, Campylobactersputorum Subsp. rium microaerophilum, Propionibacterium propionicum, sputorum, Campylobacter upsaliensis, Catonella morbi, Propionibacterium thoenii, Propionigenium maris, Propi Centipeda periodontii, Dialister invisus, Dialister pneumos 35 Onigenium modestum, Propionispira arboris, Rikenella intes, Dichelobacter nodosus, Fervidobacterium gondwan microfitsus, Roseburia Cecicola, Roseburia intestinalis, ens, Fervidobacterium islandicum, Fervidobacterium Ruminobacter amylophilus, Sebaldella termitidis, Selenomo nodosum, Fervidobacterium pennivorans, Fibrobacter intes nas acidaminovorans, Selenomonas artemidis, Selenomonas tinalis, Fibrobacter succinogenes, Fibrobacter succinogenes dianae, Selenomonas flueggei, Selenomonas infelix, Seleno Subsp. Elongatus, Fibrobacter succinogenes Subsp. succino 40 monas lacticifex, Selenomonas lipolytica, Selenomonas genes, Fusobacterium alocis, Fusobacterium canifelinum, noxia, Setenomonas ruminantium, Selenormonas ruminan Fusobacterium equinum, Fusobacterium gonidiaformans, tium Subsp. lactilytica, Selenomonas ruminantium Subsp. Fusobacterium mortiferum, Fusobacterium naviforme, ruminantium, Selenomonas sputigena, Sporomusa aci Fusobacterium necrogenes, Fusobacterium necrophorum, dovorans, Sporomusa aerivorans, Sporomusa malonica, Fusobacterium necrophorum Subsp. funduliforme, Fusobac 45 Sporomusa ovata, Sporomusa paucivorans, Sporomusa sil terium necrophorum Subsp. necrophorum, Fusobacterium vacetica, Sporomusa sphaeroides, Sporomusa termitida, Suc nucleatum, Fusobacterium nucleaturn Subsp. animalis, cinimonas amylolytica, Sitccinivibrio dextrinosolvens, Syn Fusobacterium nucleatum Subsp. fusiforme, Fusobacterium trophobacterfiumaroxidans, Syntrophobacter pfennigii, nucleatum Subsp. nucleatum, Fusobacterium nucleatum Syntrophobacter wolinii, Syntrophomonas curvata, Syntro Subsp. polymorphum, Fusobacterium nucleatum Subsp. vin 50 phomonas erecta, Syntrophomonas Sapovorans, Syntroph centii, Fusobacterium perfoetens, Fusobacterium periodon Omonas wolfei, Syntrophomonas wolfei, Sutterella Stercori ticum, Fusobacterium plauti, Fusobacterium polysaccharo Canis, Sutterella Wadsworthensi, Saponavida, lyticum, Fusobacterium prausnitzii, Fusobacterium Thermobacteroides acetoethylicus, Thermobacteroides lep pseudonecrophorum, Fusobacterium russii, Fusobacterium to spartum, Thermobacteroides proteolyticus, Thermosipho simiae, Fusobacterium sulci, Fusobacterium ulcerans, Fuso 55 africanus, Thermosipho atlanticus, Thermosipho geolei, bacterium varium, Halanaerobacter chitinivorans, Hala Thermosiphojaponicus, Thermosipho melanesiensis, Ther naerobacter lacunarum, Halanaerobacter salinarius, Hala motoga elfi, Thermotoga hypogea, Thermotoga lettingae, naerobium acetethylicum, Halanaerobium alcaliphilum, Thermotoga maritima, Thermotoga naphthophila, Thermo Halanaerobium congolense, Halanaerobium fermentans, toga neapolitana, Thermotoga petrophila, Thermotoga Sub Halanaerobium kushneri, Halanaerobium lacusrosei, Hala 60 terranea, Thermotoga thermarum, Tissierella creatinini, Tis naerobium praevalens, Halanaerobium saccharolyticum, sierella creatinophila, Tissierella praeacuta, Wolinella curva, Halanaerobium saccharolyticum Subsp. Saccharolyticum, Wolinella recta, Wolinella succinogenes, Zymophilus pau Halanaerobium saccharolyticum Subsp. Senegalense, Hala civorans, Zymophilus raffinosivorans, Desulfobacter curva naerobium salsuginis, Ilyobacter delafieldii, Ilvobacter tus, Desulfobacter halotolerans, Desulfobacter hydrogeno insuetus, Ilvobacter polytropus, Ilvobacter tartaricus, 65 philus, Desulfobacter latus, Desulfobacterpostgatei, Johnsonella ignava, Lachnobacterium bovis, Leptotrichia Desulfobacter vibrioformis, Desulfobacterium anilini, Des buccalis, Leptotrichia goodfellowii, Leptotrichia hofstadii, ulfobacterium autotrophicum, Desulfobacterium catecholi US 8,501,463 B2 5 6 cum, Desulfobacterium cetonicum, Desulfobacterium indoli lonella rodentium, Coprococcus catu, Coprococcus comes, cum, Desulfobacterium macestii, Desulfobacterium Coprococcus eutactus, Peptococcus asaccharolyticus, Pepto phenolicum, Desulfobulbus elongatus, Desulfobulbus medi glycinophilus, Peptococcus heliotrinreducens, Pepto terraneus, Desulfobulbus propionicus, Desulfobulbus coccus indolicus, Peptococcus magnus, Peptococcus niger, rhabdoformis, Desulfococcus biacutus, Desulfococcus mul Peptococcus prevotii, Peptococcus saccharolyticus, Pep tivorans, Desulfomicrobium apsheronium, Desulfomicrobium to streptococcus anaerobius, Peptostreptococcus asaccharo baculatum, Desulfomicrobium escambiense, Desulfomicro lyticus, Peptostreptococcus barnesae, Peptostreptococcus bium macestii, Desulfomicrobium norvegicum, Desulfomi harei, Peptostreptococcus heliotrinreducens, Peptostrepto crobium oralem, Desulfomonas pigra, Desulfomonile limi coccus hydrogenalis, Peptostreptococcus indolicus, Pep maris, Desulfomonile tiedjei, Desulfonema ishimotonii, 10 to streptococcus ivorii, Peptostreptococcus lacrimalis, Pep Desulfonema limicola, Desulfonema magnum, Desulfosa to streptococcus lactolyticus, Peptostreptococcus magnus, rcina variabilis, Desulfotomaculum acetoxidans, Desulfoto Peptostreptococcus micros, Peptostreptococcus octavius, maculum aeronauticum, Desulfotomaculum alkaliphilum, Peptostreptococcus parvulus, Peptostreptococcus previotii, Desulfotomaculum antarcticum, Desulfotomaculum auripig Peptostreptococcus productus, Peptostreptococcus tetradius, mentum, Desulfotomaculum australicum, Desulfotomaculum 15 Peptostreptococcus vaginalis, Ruminococcus albus, Rumino geothermicum, Desulfotomaculum gibsoniae, Desulfoto coccus bromii, Ruminococcus callidus, Ruminococcus flave maculum guttoideum, Desulfotomaculum halophilum, Des faciens, Ruminococcus gnavus, Ruminococcus hansenii, ulfotomaculum kuznetsovii, Desulfotomaculum luciae, Des Ruminococcus hydrogenotrophicus, Ruminococcus lactaris, ulfotomaculum nigrificans, Desulfotomaculum Orientis, Ruminococcus luti, Ruminococcus obeum, Ruminococcus Desulfotomaculum putei, Desulfotomaculum ruminis, Des 20 palustris, Ruminococcus pasteurii, Ruminococcus produc ulfotomaculum sapomandens, Desulfotomaculum solfatari tus, Ruminococcus schinki, Ruminococcus torques, Sarcina cum, Desulfotomaculum thermoacetoxidans, Desulfoto maxima, Sarcina ventriculi, Clostridium absonium, maculum thermobenzoicum, Desulfotomaculum Clostridium aceticum, Clostridium acetireducens, thermobenzoicum Subsp. thermobenzoicum, Desulfotomacu Clostridium acetobutylicum, Clostridium acidisoli, lum thermobenzoicum Subsp. thermosyntrophicum, Desulfo 25 Clostridium acidurici, Clostridium aerotolerans, tomaculum thermocisternum, Desulfotomaculum thermosa Clostridium akagii, Clostridium aldrichii, Clostridium povorans, Desulfovibrio acrylicus, Desulfovibrio algidicarnis, Clostridium algidiyylanolyticum, Clostridium aespoeensis, Desulfo vibrio africanus, Desulfovibrio aminophilum, Clostridium aminovalericum, Clostridium alaskensis, Desulfo vibrio alcoholivorans, Desulfovibrio ami amygdalinum, Clostridium arcticum, Clostridium argentin nophilus, Desulfovibrio baarsii, Desidfovibrio baculatus, 30 ense, Clostridium aurantibutyricum, Clostridium baratii, Desulfovibrio bastinii, Desulfovibrio burkinensis, Des Clostridium barkeri, Clostridium bartlettii, Clostridium ulfovibrio carbinolicus, Desulfovibrio cuneatus, Des beijerinckii, Clostridium bifermentans, Clostridium bolteae, ulfovibrio dechloracetivorans, Desulfovibrio desulfuricans, Clostridium botulinum, Clostridium bowmanii, Clostridium Desulfovibrio desulfuricans Subsp. aestuarii, Desulfovibrio bryantii, Clostridium butyricum, Clostridium cadaveris, desulfuricans Subsp. desulfuricans, Desulfovibrio fructo 35 Clostridium caninithermale, Clostridium carnis, Sivorans, Desulfo vibriofurfuralis, Desulfovibriogabonensis, Clostridium cellatum, Clostridium celerecrescens, Desulfovibrio giganteus, Desulfo vibrio gigas, Desidfovibrio Clostridium cellobioparum, Clostridium cellulofermentans, gracilis, Desulfovibrio halophilus, Desulfo vibrio hydrother Clostridium cellulolyticum, Clostridium cellulose, malis, Desulfovibrio indonesiensis, Desulfovibrio inopina Clostridium cellulovorans, Clostridium chartatabidum, tus, Desidfovibrio intestinalis, Desulfovibrio litoralis, Des 40 Clostridium chauvoei, Clostridium clostridioforme, ulfovibrio longreachensis, Desulfovibrio longus, Clostridium coccoides, Clostridium cochlearium, Desulfovibrio magneticus, Desulfovibrio mexicanus, Des Clostridium cocleatum, Clostridium colicanis, Clostridium ulfovibrio Oxyclinae, Desulfovibrio piger; Desulfovibrio pro colinum, Clostridium collagenovorans, Clostridium cylin findus, Desulfo vibrio putealis, Desulfovibrio salexigens, drosporum, Clostridium difficile, Clostridium dioli, Desulfovibrio Sapovorans, Desulfo vibrio Senezii, Des 45 Clostridium disporicum, Clostridium durum, Clostridium ulfovibrio simplex, Desulfovibrio sulfadismutans, Des estertheticum, Clostridium estertheticum subsp. estertheti ulfovibrio termitidis, Desulfovibrio thermophilus, Des cum, Clostridium estertheticum Subsp. laramiense, ulfovibrio vietnamensis, Desulfovibrio vulgaris, Clostridium fallax, Clostridium felsineum, Clostridium fervi Desulfovibrio vulgaris Subsp. oxamicus, Desulfovibrio vul dum, Clostridium fimetarium, Clostridium formicaceticum, garis Subsp. vulgaris, Desulfovibrio Zosterae, Desulfurella 50 Clostridium frigidicarnis, Clostridium frigoris, Clostridium acetivorans, Desulfitrella kamchalkensis, Desulfurella multi gasigenes, Clostridium ghonii, Clostridium glycolicum, potens, Desulfurella propionica, Desulfuromonas acetexi Clostridium grantii, Clostridium haemolyticum, Clostridium gens, Desulfuromonas acetoxidans, Desulfuromonas chloro halophilum, Clostridium hastiforme, Clostridium hathewayi, ethenica, Desulfuromonas paimitatis, Desulfuromonas Clostridium herbivorans, Clostridium hiranonis, tkiophila, Thermodesulfobacterium commune, Thermodes 55 Clostridium histolyticum, Clostridium homopropionicum, ulfobacterium hyeragerdense, Thermodesulfobacterium Clostridium hungatei, Clostridium hydroxybenzoicum, hydrogeniphilum, Thermodesulfobacterium thermophilum, Clostridium hylemonae, Clostridium jejuense, Clostridium Acidaminococcus fermentans, Megasphaera cerevisiae, indolis, Clostridium innocuum, Clostridium intestinale, Megasphaera elsdenii, Megasphaera micronuciformis Syn Clostridium irregulare, Clostridium isatidis, Clostridium trophococcus sucromutans, Veillonella alcalescens, Veil 60 josui, Clostidium kluyveri, Clostridium lactatifermentans, lonella alcalescens Subsp. alcalescens, Veillonella alcale Clostridium lacusfiyxellense, Clostridium laramiense, scens Subsp. Criceti, Veillonella alcalescens Subsp. dispar; Clostridium lentocellum, Clostridium lentoputrescen, Veillonella alcalescens subsp. ratti, Veillonella atypica, Veil Clostridium leptum, Clostridium limosum, Clostridium lito lonella caviae, Veillonella criceti, Veillonella dispar, Veil rale, Clostridium lituseburense, Clostridium ljungdahli, lonella montpellierensis, Veillonella parvula, Veillonella par 65 Clostridium lortetii, Clostridium magnum, Clostridium vula Subsp. atypical, Veillonella parvula Subsp. parvula, malenominatum, Clostridium mangenotii, Clostridium may Veillonella parvula subsp. rodentium, Veillonella ratti, Veil ombei, Clostridium methoxybenzovorans, Clostridium meth US 8,501,463 B2 7 8 ylpentosum, Clostridium neopropionicum, Clostridium next Julia, Rhodopseudomonas marina, Rhodopseudomonas ille, Clostridium novyi, Clostridium oceanicum, Clostridium palustris, Rhodopseudomonas rhenobacensis, Orbiscindens, Clostridium Oroticum, Clostridium oxalicum, Rhodopseudomonas rosea, Rhodopseudomonas rutila, Clostridium papyrosolvens, Clostridium paradoxum, Rhodopseudomonas sphaeroides, Rhodopseudomonas sulfi Clostridium paraperfringens, Clostridium paraputrificum, dophila, Rhodopseudomonas sulfoviridis, Rhodopseudomo Clostridium pasculi, Clostridium pasteurianum, Clostridium nas viridis, Rhodospirillum centenum, Rhodospirillum full peptidivorans, Clostridium perenne, Clostridium perfirin vum, Rhodospirillum molischianum, Rhodospirillum gens, Clostridium pfennigii, Clostridium phytofermentans, photometricum, Rhodospirillum rubrum, Rhodospirillum Clostridium piliforme, Clostridium polysaccharolyticum, salexigens, Rhodospirillum salinarum, Rhodospirillum Sodo Clostridium populeti, Clostridium propionicum, Clostridium 10 mense, Rhodospirillum tenue, Erythrobacter aquimaris, proteoclasticum, Clostidium proteolyticum, Clostridium Erythrobacter citreus, Erythrobacterflavus, Erythrobacter psychrophilum, Clostridium puniceum, Clostridium purini gaetbuli, Erythrobacter litoralis, Erythrobacter longus, lyticum, Clostridium putrefaciens, Clostridium putrificum, Erythrobacter Seohaensis, Methanobacterium aarhusense, Clostridium quercicolum, Clostridium quinii, Clostridium Methanobacterium alcaliphilum, Methanobacterium arbo ramosum, Clostridium rectum, Clostridium roseum, 15 philicum, Methanobacterium beijingense, Methanebacte Clostridium saccharobutylicum, Clostidium saccharolyti rium bryantii, Methanobacterium Congolense, Methanobac cum, Clostridium saccharoperbutylacetonicum, Clostridium terium defluvii, Methanobacterium espanolae, Sardiniense, Clostridium Sartagoforme, Clostridium scatolo Methanobacterium formicicum, Methanobacterium ivanovii, genes, Clostridium scindens, Clostridium septicum, Methanobacterium mobile, Methanobacterium Oryzae, Clostridium sordellii, Clostridium sphenoides, Clostridium Methanobacterium ruminantium, Methanobacterium subter spiroforme, Clostridium sporogenes, Clostridium raneum, Methanobacterium thermaggregains, Methanobac sporosphaeroides, Clostridium Stercorarium, Clostridium terium thermalcaliphilum, Methanobacterium thermau Stercorarium Subsp. leptospartum, Clostridium Stercorarium totrophicum, Methanobacterium thermoflexum, Subsp. Stercorarium, Clostridium stercorarium Subsp. ther Methanobacterium thermoformicicum, Methanobacterium molacticum, Clostridium Sticklandii, Clostridium Stramini 25 thermophilum, Methanobacterium uliginosum, Methanobac solvens, Clostridium subterminale, Clostridium symbiosum, terium wolfei, Methanobrevibacter acididurans, Methano Clostridium termitidis, Clostridium tertium, Clostridium brevibacter arboriphilus, Methanobrevibacter curvatus, tetani, Clostridium tetanomorphum, Clostridium thermace Methanobrevibacter cuticularis, Methanobrevibacter filifor ticum, Clostridium thermautotrophicum, Clostridium ther mis, Methanobrevibacter gottschalkii, Methanobrevibacter moalcaliphilum, Clostridium thermobutyricum, Clostridium 30 oralis, Methanobrevibacter ruminantium, Methanobrevi thermocellum, Clostridium thermocopriae, Clostridium ther bacter Smithii, Methanobrevibacter thaueri, Methanobrevi mohydrosulfuricum, Clostridium thermolacticum, Closti bacter woesei, Methanobrevibacter wolinii, Methanococcus idium thermopalmarium, Clostridium thermopapyrolyticum, delta, Methanococcus fervens, Methanococcus frisius, Clostridium thermosuccinogenes, Clostidium thermosulfil Methanococcus halophilus, Methanococcus igneus, Metha rigenes, Clostridium thiosulfatireducens, Clostridium 35 nococcus infernus, Methanococcus jannaschii, Methanococ tyrobutyricum, Clostridium uliginosum, Clostridium ultun cus maripaludis, Methanococcus mazei, Methanococcus ense, Clostridium villosum, Clostridium vincentii, thermolithotrophicus, Methanococcus vannieli, Methano Clostridium viride, Clostridium Xylanolyticum, Clostridium coccus voltae, Methanococcus vulcanius, Methanococcoides xylanovorans, Amoebobacterpedioformis, Amoebobacter burtonii, Methanococcoides methylutens, Methanolobus pendens, Amoebobacterpurpureus, Amoebobacter roseus, 40 bombayensis, Methanolobus Oregonensis, Methanolobus buderi, Chromatium glycolicum, Chromatium Siciliae, Methanolobus taylorii, Methanolobus tindarius, gracile, Chromatium minus, Chromatium minutissimum, Methanolobus vulcani, Methanolacinia paynteri, Metha Chromatium Okenii, Chromatium purpuratum, Chromatium nomicrobium mobile, Methanomicrobium paynteri, Metha salexigens, Chromatium tepidum, Chromatium vinosum, nogenium aggregains, Methanogenium bourgense, Meihano Chromatium violascens, Chromatium warmingii, Chroma 45 genium cariaci, Methanogenium frigidium, Methanogenium tium weissei, Lamprobacter modestohalophilus, Lamprocys frittonii, Methanogenium liminatans, Methanogenium mari tis purpurea, Lamprocystis roseopersicina, Thiocapsa halo num, Methanogenium marisnigri, Methanogenium Olen phila, Thiocapsa litoralis, Thiocapsa marina, Thiocapsa tangyi, Methanogenium Organophilum, Methanogenium penden, Thiocapsa rosea, Thiocapsa roseopersicina, Thio tationis, Methanogenium thermophilicum, Methanospirillum cystis gelatinosa, Thiocystis minor. Thiocystis violacea, Thio 50 hungatei, Methanoplanus endosymbiosus, Methanoplanus cystis violascens, Thiodictyon bacillosum, Thiodictyon limicola, Methanoplanus petrolearius, Methanothrix con elegans, Thiopedia rosea, Thiospirillum jenense, Ectothior cilii, Methanothrix soehngenii, Methanothrix thermoaceto hodospira abdelmalekii, Ectothiorhodospira haloalka phila, Methanothrix thermophila, Methanothermus fervidus, liphila, Ectothiorhodospira halochloris, Ectothiorhodospira Methanothermus sociabilis, Methanocorpusculum halophila, Ectothiorhodospira marina, Ectothiorhodospira 55 aggregains, Methanocorpusculum bavaricum, Methanocor marismortui, Ectothiorhodospira mobilis, Ectothiorho pusculum labreanum, Methanocorpusculum parvum, Metha dospira Shaposhnikovii, Ectothiorhodospira vacuolata, nocorpusculum sinense, Methanoculleus bourgensis, Metha Rhodobacter adriaticus, Rhodobacter azotoformans, Rhodo noculleus chikugoensis, Methanoculleus marisnigri, bacter blasticus, Rhodobacter capsulatus, Rhodobacter Methanoculleus oldenburgensis, Methanoculleus Olentangyi, euryhalinus, , Rhodobacter sidfido 60 Methanoculleus palmolei, Methanoculleus submarinus, philus, Rhodobacter veldkampii, Rhodocyclus gelatinosus, Methanoculleus thermophilus, Methanohalobium evestiga Rhodocyclus purpureus, Rhodocyclus tenuis, Rhodomicro tum, Methanohalophilus halophilus, Methanohalophilus bium vannieli, Rhodopilla globiformis, Rhodopseudomonas mahi, Methanohalophilus Oregonensis, Methanohalophilus acidophila, Rhodopseudomonas adriatica, Rhodopseudomo portucalensis, Methanohalophilus Zhilinae, Methanosarcina FCS blastica, Rhodopseudomonas capsulata, 65 acetivorans, Methanosarcina baltica, Methanosarcina bark Rhodopseudomonas faecalis, Rhodopseudomonas gelati eri, Methanosarcina frisia, Methanosarcina lacustris, nosa, Rhodopseudomonas globiformis, Rhodopseudomonas Methanosarcina methanica, Methanosarcina semesia, US 8,501,463 B2 9 10 Methanosarcina Siciliae, Methanosarcina thermophila, lus helveticus, Lactobacillus heterohiochi, Lactobacillus hil Methanosarcina vacuolata, Methanosphaera cuniculi, gardii, Lactobacillus homohiochii, Lactobacillus iners, Lac Methanosphaera stadtmanae, Eubacterium acidaminophi tobacillusinglu viei, Lactobacillus intestinalis, Lactobacillus lum, Eubacterium aerofaciens, Eubacterium aggregans, jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, Eubacterium alactolyticum, Eubacterium angustum, Eubac Lactobacillus kandleri, Lactobacillus kefiranofaciens, Lac terium barkeri, Eubacterium biforme, Eubacterium brachy, tobacillus kefiranofaciens subsp. kefiranofaciens, Lactoba Eubacterium budayi, Eubacterium callanderi, Eubacterium cillus kefiranofaciens subsp. kefirgranum, Lactobacillus cellulosolvens, Eubacterium combesii, Eubacterium contor kefirgranum, Lactobacillus kefir, Lactobacillus kimchi, Lac tum, Eubacterium coprostanoligenes, Eubacterium cylin tobacillus kitasatonis, Lactobacillus kunkeei, Lactobacillus droids, Eubacterium desmolans, Eubacterium dolichum, 10 Eubacterium eligens, Eubacterium exiguum, Eubacterium lactis, Lactobacillus leichmannii, Lactobacillus lindneri, fissicatena, Eubacterium formicigenerans, Eubacterium fos Lactobacillus malefermentans, Lactobacillus mali, Lactoba Sor, Eubacterium hadrum, Eubacterium hallii, Eubacterium cillus maltaronicus, Lactobacillus manihotivorans, Lacto infinnum, Eubacterium lentum, Eubacterium limosum, mindensis, Lactobacillus minor; Lactobacillus Eubacterium minutum, Eubacterium moniliforme, Eubacte 15 mucosae, Lactobacillus murinus, Lactobacillus nagelii, Lac rium multiforme, Eubacterium nitritogenes, Eubacterium tobacillus Oris, Lactobacillus panis, Lactobacillus pantheris, modatum, Eubacterium oxidoreducens, Eubacterium plautii, Lactobacillus parabuchneri, Lactobacillus paracasei, Lacto Eubacterium plexicaudatum, Eubacterium pyruvativorans, bacillus paracasei Subsp. paracasei, Lactobacillus paracasei Eubacterium ramulus, Eubacterium rectale, Eubacterium Subsp. tolerans, Lactobacillus paracollinoides, Lactobacil ruminantium, Eubacterium saburreum, Eubacterium saphe lus parakefiri, Lactobacillus parallimentarius, Lactobacillus num, Eubacterium Siraeum, Eubacterium suis, Eubacterium paraplantarum, Lactobacillus pentosus, Lactobacillus pero sulci, Eubacterium tarantellae, Eubacterium tardum, Eubac lens, Lactobacillus piscicola, Lactobacillus plantarum, Lac terium tenue, Eubacterium timidum, Eubacterium tortuosum, tobacillus pontis, Lactobacillus psittaci, Lactobacillus reu Eubacterium uniforme, Eubacterium ventriosum, Eubacte teri, Lactobacillus rhamnosus, Lactobacillus rimae, rium xylamophilum, Eubacterium yuri, Eubacterium yuri 25 Lactobacillus rogosae, Lactobacillus rossii, Lactobacillus Subsp. margaretiae, Eubacterium yuri Subsp. schtitka, ruminis, Lactobacillus saerimneri, Lactobacillus sakei, Lac Eubacterium yuri Subsp. Yurii, Abiotrophia adiacens, tobacillus sakei Subsp. carnosus, Lactobacillus sakei Subsp. Abiotrophia balaetiopterae, Abiotrophia defectiva, Abiotro sakei, Lactobacillus salivarius, Lactobacillus salivarius phia elegans, Atopobium fossor, Atopobium minutum, Atopo Subsp. salicinius, Lactobacillus salivarius Subsp. salivarius, bium parvulum, Atopobium rimae, Atopobium vaginae, 30 Lactobacillus Sanfranciscensis, Lactobacillus satsumensis, Gemella bergeri, Gemella cuniculi, Gemella haemolysans, Lactobacillus sharpeae, Lactobacillus spicheri, Lactobacil Gemella morbillorum, Gemella palaticanis, Gemella San lus Suebicus, Lactobacillus Suntoryeus, Lactobacillus ther guinis, Granulicatella adiacens, Granulicatella bal motolerans, Lactobacillus trichodes, Lactobacillus uli, Lac aenopterae, Granulicatella elegans, Finegoldia magna, Lac tobacillus ultunensis, Lactobacillus vaccinostercus, tobacillus acetotolerans, Lactobacillus acidifarinae, 35 Lactobacillus vaginalis, Lactobacillus versmoldensis, Lacto Lactobacillus acidipiscis, Lactobacillus acidophilus, Lacto bacillus viridescens, Lactobacillus vitulinu, Lactobacillus bacillus acidophilus, Lactobacillus agilis, Lactobacillus xylosus, Lactobacillus vananashiensis, Lactobacillus yama algidus, Lactobacillus alimentarius, Lactobacillus amy nashiensis Subsp. mali, Lactobacillus vananashiensis Subsp. lolyticus, Lactobacillus amylophilus, Lactobacillus amylo yamanashiensis, Lactobacillus zeae, Lactobacillus zymae, vorus, Lactobacillus animalis, Lactobacillus antri, Lactoba 40 Actinomyces bernardiae, Actinomyces bovis, Actinomyces cillus arizonensis, Lactobacillus aviarius, Lactobacillus bowdenii, Actinomyces canis, Actinomyces cardiffensis, Acti aviarius Subsp. Ardfinosus, Lactobacillus aviarius Subsp. nomyces catuli, Actinomyces Coleocanis, Actinomyces denta Aviarius, Lactobacillus bavaricus, Lactobacillus bifermen lis, Actinomyces denticolens, Actinomyces europaeus, Acti tans, Lactobacillus brevis, Lactobacillus buchneri, Lactoba nomyces funkei, Actinomyces Georgia, Actinomyces cillus bulgaricus, Lactobacillus carnis, Lactobacillus casei, 45 gerencseriae, Actinomyces graevenitzii, Actinomyces Lactobacillus casei Subsp. alactosus, Lactobacillus casei hongkongensis, Actinomyces hordeovulneris, Actinomyces Subsp. casei, Lactobacillus casei Subsp. pseudoplantarum, howellii, Actinomyces humiferus, Actinomyces hyovaginalis, Lactobacillus casei Subsp. rhamnosus, Lactobacillus casei Actinomyces israelii, Actinomyces marinammalium, Actino Subsp. tolerans, Lactobacillus catenaformis, Lactobacillus myces meyeri, Actinomyces naeslundii, Actinomyces nasi cellobiosus, Lactobacillus Coleohominis, Lactobacillus col 50 cola, Actinomyces neuii, Actinomyces neuii Subsp. anitratus, linoide, Lactobacillus confuses, Lactobacillus coryniformis, Actinomyces neuii Subsp. neuii, Actinomyces Odontolyticus, Lactobacillus coivniformis Subsp. coryniformis, Lactobacil Actinomyces Oricola, Actinomyces pyogenes, Actinomyces lus coryniformis Subsp. torquens, Lactobacillus crispatus, pyogenes, Actinomyces radicidentis, Actinomyces radingae, Lactobacillus curvatus, Lactobacillus curvatus Subsp. curva Actinomyces slackii, Actinomyces suinastitidis, Actinomyces tus, Lactobacillus curvatus Subsp. melibiosus, Lactobacillus 55 suis, Actinomyces turicensis, Actinomyces urogenitalis, Acti Cypricasei, Lactobacillus delbrueckii, Lactobacillus del nomyces vaccinaxillae, Actinomyces viscosus, Arcanobacte brueckii Subsp. bulgaricus, Lactobacillus delbrueckii Subsp. rium bernardiae, Arcanobacterium haemolyticum, Arcano delbrueckii, Lactobacillus delbrueckii Subsp. indicus, Lacto bacterium hippocoleae, Arcanobacterium phocae, bacillus delbrueckii Subsp. lactis, Lactobacillus diolivorans, Arcanobacterium pluranimalium, Arcanobacterium pyo Lactobacillus divergens, Lactobacillus durianis, Lactobacil 60 genes, Actinobaculum Schaali, Actinobaculum suis, Actino lus equi, Lactobacillus farciminis, Lactobacillus ferintoshen baculum urinale, Bulleidia extructa, Collinsella aerofaciens, sis, Lactobacillus fermentuni, Lactobacillus formicalis, Lac Collinsella intestinalis, Collinsella stercoris, Cryptobacte tobacillus fructivorans, Lactobacillus fructosus, rium curtum, Holdemania filiformis, Rothia aeria, Rothia Lactobacillus frumenti, Lactobacillus fitchuensis, Lactoba amarae, Rothia dentocariosa, Rothia mucilaginosa, Rothia cillus gallinarum, Lactobacillus gasseri, Lactobacillus gas 65 nasimurium, Pseudoramibacter alactolyticus, Mogibacte tricus, Lactobacillus gramini, Lactobacillus halotolerans, rium diversum, Mogibacterium neglectum, Mogibacterium Lactobacillus hammesii, Lactobacillus hamsteri, Lactobacil pumilum, Mogibacterium timidum, Mogibacterium vescum, US 8,501,463 B2 11 12 Slackia exigua, Slackia heliotrinireducens, and Eggerthella ous processes. In still further or alternative embodiments of lenta (hereinafter referred to as “anaerobic bacteria' or a all aspects, the processes of anaerobically fermenting biom similar term). asS are batch processes. In further or alternative embodiments of all aspects using Chemical Products and Uses of Chemical Products substantially purified anaerobic bacterial strains, the bacteria In further or alternative embodiments of all aspects in may be collected from bovine rumen, Soil samples, sludge, which chemical products are produced by anaerobically fer anaerobic bacteria cultures, aerobic bacteria cultures, anaero menting biomass, the chemical products may be gaseous, bic sediments from fresh or brackish waters, sewage, animals, non-gaseous, or combinations thereof. In further or alterna animal feces, insect digestive tract, dental samples, hydro tive embodiments of such aspects, the non gaseous chemical thermal soils, hydrothermal pools and deep water hydrother 10 products may be solids, solvents, Volatile organic acids, salts of volatile organic acids, or combinations thereof. In further mal vents. or alternative embodiments of Such aspects, the gaseous In further or alternative embodiments of all aspects using chemical products may be hydrogen, carbon dioxide, carbon substantially purified anaerobic bacterial strains, the bacteria monoxide, methane, hydrogen Sulfide, ammonia, nitrogen, may be obtained by genetic modification of known bactial 15 and combinations thereof. In further or alternative embodi strains. In further or alternative embodiments of all aspects ments of such aspects, the gaseous chemical products may be using Substantially purified anaerobic bacterial strains, the hydrogen, carbon dioxide, carbon monoxide, hydrogen Sul genetic modification results from transformation procedures, fide, ammonia, nitrogen, and combinations thereof. In further bacterial conjugation, transduction, interaction of bacterial or alternative embodiments of all aspects, the solid chemical strains with mutagens, and combinations thereof. In further or products may be solids which comprises Sulfur, and in further alternative embodiments, the mutagens include, but not lim or alternative embodiments of such aspects, the Solid chemi ited to, chemicals, ultraviolet light, and radioactive elements. cal products may be elemental sulfur. In still further or alter In further or alternative embodiments of all aspects using native embodiments of Such aspects, the chemical products substantially purified anaerobic bacteria cultures, the sub may be solvents, such as, but not limited to, acetone, butanol, stantially purified anaerobic bacteria cultures incorporate, 25 propanol, isopropanol, 1,2-propanediol, ethanol, methanol, along with appropriate growth media, Substantially purified and combinations thereof. In further or alternative embodi single bacterial strains. In further or alternative embodiments ments of Such aspects, the chemical products are volatile of all aspects utilizing Substantially purified anaerobic bacte organic acids, such as, but not limited to formic acid, acetic rial strains, the substantially purified anaerobic bacterial acid, propionic acid, butyric acid, Valeric acid, and combina strains are part of inoculants. In further or alternative embodi 30 tions thereof. In further or alternative embodiments of such ments of all aspects using Substantially purified anaerobic aspects, the chemical products are salts of Volatile organic bacterial strains, the substantially purified single bacterial acids in which the anion include, but are not limited to, strains are at least 95% purified. In still further or alternative formate, acetate, propionate, butyrate, Valerate, and combi embodiments of all aspects using Substantially purified nations thereof. In further or alternative embodiments of such anaerobic bacterial strains, the Substantially purified single 35 aspects, the chemical products are salts of the Volatile organic bacterial strains are at least 99% purified. In even further or acids the cations may alkali metal ions, alkaline earth ions, alternative embodiments of all aspects using Substantially ammonium ion, or combinations thereof. In still further or purified anaerobic bacterial strains, the substantially purified alternative embodiments of Such aspects, cations of the salts single bacterial strains are at least 99.5% purified. of volatile organic acids may be Na", K", Ca", Mg", NH.", Biomass 40 and combinations thereof. In further or alternative embodiments of all aspects using In one aspect are compositions which include at least one biomass, the biomass comprises material obtained from chemical product produced by anaerobically fermenting bio energy crops, Surplus agricultural products, waste from Sugar mass using Substantially purified anaerobic bacteria cultures. production and processing facilities, animal waste from Zoos, In an embodiment of this aspect, the substantially purified waste from fruit processing industries, waste from pulp and 45 anaerobic bacteria cultures have not been subjected to heat paper mills, silvaculture residues, waste from wood process shocking processes, and in further or alternative embodi ing, waste from agricultural product processing, food waste, ments, the substantially purified anaerobic bacteria cultures Solids isolated from fermentation cultures, municipal sewer are used as inoculants in anaerobic fermentation apparatuses. waste, animal manure, animal urine, animal parts, fish parts, In another aspect are feedstocks for chemical industries and combinations thereof. In further or alternative embodi 50 which are chemical products produced by anaerobically fer ments of all aspects utilizing a biomass, the biomass may be menting biomass. In an embodiment of this aspect, the chemi material Such as, but not limited to, glucose, beet Sugar, Sugar cal industries utilizing such feedstocks include polymer beet molasses, Sugar beet syrup, Sugar beetjuice, Sugar cane industries, industrial synthesis industries, photographic molasses, cane Sugar, Sugarcane Syrup, Sugarcane juice, corn industries, coatings industries, fertilizer industries, printing syrup, cereal grains, oat flour, rice flour, corn flour, wheat 55 industries, and combinations thereof. flour, potatoes, tomatoes, potato juice, tomato pulp, tomato In another aspectare energy sources for a power generation juice, potato pulp, cheese whey, Sorghum, corn mash, wheat systems which are gaseous chemical products and solvents mash, oat mash, blackstrap molasses, citrus molasses, invert produced by anaerobically fermenting biomass. In an Sugar, Sucrose, fructose, glucose, wood Sugar, cellulose, embodiment of this aspect, the power generation systems Xylose, plant parts, fruit, vegetable, bovine manure, poultry 60 may be fuel cells, internal combustion generators, turbine manure, equine manure, porcine manure, bovine urine, poul generators, Stirling engines, and combinations thereof. In try urine, equine urine, porcine urine, wood shavings, wood further or alternative embodiments, the power generation sys chips, shredded paper, cotton burrs, grain, chaff, seed shells, tems are part of power stations. In further or alternative hay, alfalfa, grass, leaves, seed pods, corn shucks, weeds, embodiments, the power stations are decentralized power aquatic plants, algae, fungus, and combinations thereof. 65 stations. In further or alternative embodiments, the fuel cells In further or alternative embodiments of all aspects, the may be alkaline fuel cells, phosphoric acid fuel cells, molten processes of anaerobically fermenting biomass are continu carbonate fuel cells, Solid oxide fuel cells, proton exchange US 8,501,463 B2 13 14 membrane fuel cells, and combinations thereof. In further or anaerobically fermenting a biomass using Substantially puri alternative embodiments, the fuel cells are used to generate fied anaerobic bacteria cultures. Hydrogenation occurs by electricity for residential consumption, commercial con admixing starting materials and catalysts, with hydrogen. In Sumption, motor vehicle consumption, or combinations an embodiment of this aspect, the substantially purified thereof. In still further or alternative embodiments, the elec anaerobic bacteria cultures have not been subjected to heat tricity generated by such fuel cells may be used locally, added shocking processes. In further or alternative embodiments, to the grid, or combinations thereof. In further or alternative the chemical product is aniline and the starting material is embodiments, the turbine generators are used to generate nitrobenzene. In further or alternative embodiments, the cata electricity from steam created by heating water, wherein the lysts may be NiS/CuS catalysts or Cu catalysts. In further or heat is generated by combustion of compositions comprising 10 alternative embodiments, the aniline may be used in the Syn chemical products produced by anaerobically fermenting thesis of pharmaceuticals, polymers, dyes, or solvents. biomass. In further or alternative embodiments, the electricity In another aspectare compositions which contain chemical generated by Such turbines may be used for residential con products formed by admixing minerals and Volatile organic Sumption or commercial consumption. In still further or alter acids, wherein the Volatile organic acids are produced by native embodiments, the electricity generated by Such tur 15 anaerobically fermenting a biomass using Substantially puri bines may be used locally, added to the grid, or combinations fied anaerobic bacteria cultures. In an embodiment of this thereof. aspect, the Substantially purified anaerobic bacteria cultures In another aspectare compositions which contain chemical have not been Subjected to heatshocking processes. In further products formed by admixing carbon oxides, catalysts, and or alternative embodiments, the Volatile organic acid is acetic hydrogen, in which the hydrogen has been produce by acid. In further or alternative embodiments, the mineral is anaerobically fermenting biomass using Substantially puri dolomite and the chemical product is calcium magnesium fied anaerobic bacteria cultures. In an embodiment of this acetate. In further or alternative embodiments, the calcium aspect, the Substantially purified anaerobic bacteria cultures magnesium acetate may be used as?in anti-freeze agents, have not been Subjected to heatshocking processes. In further deicing agents, or anti-icing agents. or alternative embodiments, the chemical product is methane. 25 In another aspectare compositions which contain chemical In further or alternative embodiments, the carbon oxide may products formed by admixing oxides, liquid ammonia and be carbon monoxide, carbon dioxide, or combination thereof. Volatile organic acids, wherein the Volatile organic acids are In further or alternative embodiments, the catalysts contain produced by anaerobically fermenting biomass using Sub activated nickel, and in still further or alternative embodi stantially purified anaerobic bacteria cultures. In an embodi ments the catalyst is NiO/MgO. In further or alternative 30 ment of this aspect, the Substantially purified anaerobic bac embodiments, are energy sources for a power generation teria cultures have not been subjected to heatshocking systems which contain such chemical products. In still further processes. In further or alternative embodiments, the volatile or alternative embodiments, the chemical product of such organic acid is acetic acid. In further or alternative embodi energy sources is methane. In further or alternative embodi ments, the oxide is Zinc oxide and the chemical product is Zinc ments, the power generation system may be fuel cells, inter 35 ammonium acetate. In further or alternative embodiments, nal combustion generators, turbine generators, and combina the calcium magnesium acetate may be used as/infertilizer or tions thereof. In further or alternative embodiments, the seed germination enhancers. power generation systems are part of power stations. In fur Anaerobic Fermentation Apparatuses ther or alternative embodiments, the power stations are In one aspect are anaerobic fermentation apparatuses decentralized power stations. In further or alternative 40 which use populations of Substantially purified anaerobic embodiments, the fuel cells may molten carbonate fuel cells, bacterial strains for anaerobically fermenting biomass into solid oxide fuel cells, and combinations thereof. In further or chemical products. The aforementioned anaerobic fermenta alternative embodiments, the fuel cells are used to generate tion apparatuses include (a) Sterilization and deoxygenation electricity for residential consumption, commercial con systems, (b) anaerobic digesters, each of which contains a Sumption, motor-vehicle consumption, or combinations 45 population of Substantially purified anaerobic bacteria and thereof. In still further or alternative embodiments, the elec are equipped with anaerobic digester control systems, (c) tricity generated by such fuel cells may be used locally, added plurality of pipelines and pumps for introducing and re-cir to the grid, or combinations thereof. In further or alternative culating biomass, and (d) removal pipelines connected to the embodiments, the turbine generators are used to generate anaerobic digesters for removing chemical products from the electricity from steam created by heating water, wherein the 50 anaerobic digesters. In addition to this aspect, the population heat is generated by combustion of Such compositions com of substantially purified anaerobic bacterial strain has not prising chemical products produced by anaerobically fer been Subjected to a heatshocking process. menting biomass. In further or alternative embodiments, the In an embodiment of the aforementioned aspect, the electricity generated by such turbines may be used for resi anaerobic fermentation apparatuses further include pipelines dential consumption or commercial consumption. In still fur 55 in communication with the sterilization and deoxygenation ther or alternative embodiments, the electricity generated by systems having pumps which are used for transferring steril Such turbines may be used locally, added to the grid, or ized and deoxygenated biomass to concentrating systems. In combinations thereof. In further or alternative embodiments, further or alternative embodiments, the anaerobic fermenta the chemical product may be used as an energy source for heat tion apparatuses also incorporate pipelines in communication generation systems. In still further or alternative embodi 60 with concentrating systems and have pumps for transferring ments, the heat generation systems may be furnaces, stove deoxygenated, concentrated, sterilized, detoxified, and/or tops, ovens, barbeques, and driers. In still further or alterna pre-digested biomass to at least one anaerobic digester. tive embodiments, such an energy source may be a In further or alternative embodiments, the anaerobic fer replacement for, or a compliment to, natural gas and methane. mentation apparatuses may incorporate anaerobic digesters, In another aspectare compositions which contain chemical 65 each of which may contain a population of substantially puri products formed by hydrogenation of starting materials using fied anaerobic photo-fermentation bacteria and are equipped hydrogen and catalysts, wherein the hydrogen is produced by with anaerobic digester control systems. In further or alter US 8,501,463 B2 15 16 native embodiments, the anaerobic fermentation apparatuses storage tank. In further or alternative embodiments, non may incorporate anaerobic digesters, each of which may con gaseous chemical products may be removed from anaerobic tain a population of substantially purified anaerobic acetoge digesters using at least one output pipeline, and in further or nic bacteria and are equipped with anaerobic digester control alternative embodiments, the non-gaseous chemical products systems. In further or alternative embodiments, the anaerobic are volatile organic acids which are removed from output fermentation apparatuses may incorporate anaerobic digest pipelines by reverse osmosis systems which are coupled to ers, each of which contain may a population of Substantially the output pipelines and to at least one storage tank. Similarly, purified anaerobic solventogenic bacteria and are equipped in further or alternative embodiments, the non-gaseous with anaerobic digester control systems. In still further or chemical product may be salts of volatile organic acids which alternative embodiments, the anaerobic fermentation appara 10 may be removed from output pipelines by reverse osmosis tuses may incorporate anaerobic digesters, each of which may systems which are coupled to output pipelines and at least one contain a population of Substantially purified anaerobic pro storage tank. In still further or alternative embodiments, the teolytic bacteria and are equipped with anaerobic digester Solid non-gaseous chemical products may be removed from control systems. output pipelines by filtration systems which are coupled to the In further or alternative embodiments, the anaerobic fer 15 output pipeline and to at least one storage tank. In addition, in mentation apparatuses may be stand alone systems for further or alternative embodiments the solid chemical product anaerobically fermenting biomass into chemical products, or is elemental sulfur which may be removed from output pipe in still further or alternative embodiments, the anaerobic fer lines by filtration systems which are coupled to the output mentation apparatuses may be components of assemblages pipelines and at least one storage tank. In further or alternative for generating power. embodiments, such filtration systems are cross-flow filtration In further or alternative embodiments, the sterilization and systems. In still further or alternative embodiments, the non deoxygenation systems of the anaerobic fermentation appa gaseous chemical products may be solvents which are ratuses are used to sterilize and deoxygenate biomass by removed from the output pipelines by osmosis systems which means of steam treatment processes, wherein the steam treat are coupled to the output pipelines and to at least one storage ment processes may be pressurized Steam treatment pro 25 tank. cesses which use low pressure steam or high pressure steam. In further or alternative embodiments, the gaseous chemi In further or alternative embodiments, the sterilization and cal products which have been removed from the anaerobic deoxygenation systems are steam heat exchanger systems. In digester via output pipelines may be isolated and purified by still further or alternative embodiments, the sterilization and means of a differential compression process. In further or deoxygenation systems are autoclaves. 30 alternative embodiments, the Volatile organic acids which In further or alternative embodiments, the sterilization and have been removed from the anaerobic digester via output deoxygenation systems of the anaerobic fermentation appa pipelines may be isolated and purified using distillation. In ratuses are assemblages which incorporate at least one vat, at still further or alternative embodiments, the solvents which least one source of steam, and at least one turbine. The have been removed from the anaerobic digester via output Sources of steam may be sources of high pressure steam, 35 pipelines may be isolated and purified using distillation. In which include, but are not limited to, boilers. In further or further or alternative embodiments, the salts of volatile alternative embodiments, the turbines of the sterilization and organic acids which have been removed from the anaerobic deoxygenation systems may be used to produce low pressure digester via output pipelines may be isolated and purified by steam and to generate electricity, and in still further or alter reverse osmosis. native embodiments, such turbines include, but are not lim 40 In further or alternative embodiments, anaerobic digester ited to, backpressure turbine generators which generate low control systems combine at process control tools, metrology pressure steam to sterilize and deoxygenate biomass and pro tools to acquire metrology data relating to anaerobic fermen duce electricity as power for local energy consumer, or for tation parameters; and process controllers operatively addition to grid systems of centralized power stations. Such coupled to the process control tools and the metrology data, local energy consumer may include, but are not limited to, 45 wherein the process controllers consists of decision making facilities for anaerobic fermentation, factories, buildings, units, input/output boards, and database units to store the facilities for processing farm produce, and combinations metrology data. Such anaerobic digester control systems may therein. be used to optimally operate anaerobic digesters. In further or alternative embodiments, the concentrating In further or alternative embodiments, process control systems of the anaerobic fermentation apparatuses are cen 50 tools are used to adjust operating parameters of the anaerobic trifugation systems used to concentrate biomass by means of digester, wherein the operating parameters are related to centrifugation processes. In further or alternative embodi anaerobic fermentation parameters. In further or alternative ments, the concentrating systems are reverse osmosis systems embodiments, such process control tools are valves or any used to concentrate biomass by means of reverse osmosis means for adjusting the temperature of the digester. processes. 55 In further or alternative embodiments, the decision making In further or alternative embodiments, the removal pipe units are used in feedback control processes to acquire lines connected to anaerobic digesters have at least one output metrology data from the input/output boards, then determine pipeline for gaseous chemical products and at least one output control adjustments to maintain anaerobic fermentation pipeline for non-gaseous chemical products. In further or parameters within defined operational ranges, then modify alternative embodiments, gaseous chemical products may be 60 the magnitude of the control adjustments, and return the removed from anaerobic digesters using at least one output modified control adjustments to the input/output boards, pipeline, and in still further or alternative embodiments, gas whereby by the modified control adjustments are sent to eous chemical products are removed from anaerobic digest process control tools to adjust operating parameters, which ers by means of a vacuum applied to at least one output are related to anaerobic fermentation parameters of the pipeline. In further or alternative embodiments, gas scrubbers 65 anaerobic fermentation digester. In further or alternative are connected to at least one output pipeline and gas compres embodiments, the feedback control processes are continuous, sors are connected to the scrubbers, and at least one gas automatic feedback control loops, while in still further or US 8,501,463 B2 17 18 alternative embodiments, the feedback control processes are includes growing various bacterial species on particular Sub intermittent, manual feedback control loops. In still further or strates, under various growth conditions to optimize the bac alternative embodiments, the process controller decision terial hydrogen production. In further or alternative embodi making units may be computers, PROMs, EPROMs, ments, the various growth conditions are selected from the EEPROMs, and combinations thereof. group consisting of variation in temperature, pH, fermenta In further or alternative embodiments, the anaerobic fer tion products, exposure to various natural gases or com mentation parameters may be temperature, pH, pressure, gas pounds, antibiotic efficacy, antibiotic resistance, compound flow, biomass concentration, and chemical products concen stimulation-digestion, compound toxicity or Survivability. In tration, wherein the metrology tool used to monitor Such further or alternative embodiments, the collection of appro parameters may be pH sensors, stirrers, oxidation-reduction 10 priate information comprises selecting response data from the monitors, nutrient concentration, pressure sensors, gas flow scientific literature. In further or alternative embodiments, the sensors, gas sensors, temperature sensors, gas chromato appropriate information comprises characteristics, wherein graphic systems, flow injection analysis systems, High Per Such characteristics are bacteria growth on various Substrates, formance Liquid Chromatographic systems, Mass specro bacteria sensitivity to various conditions and/or bacteria pro photometry, and combinations thereof. In further or 15 duction of various metabolites. In still further or alternative alternative embodiments, the pH sensors are selected from embodiments, such characteristics are categorized into traits glass membrane pH electrodes, Solid state pH electrodes, which include, but are not limited to, acetic acid major meta optode pH sensors, and combinations thereof. In further or bolic product, acetic acid minor metabolic product, acetone, alternative embodiments, the temperature sensors selected ADH, ALP, alpha-fucosidase, alpha-galactosidase, ammonia from thermometers, thermopiles, thermocouples, and combi production, alpha-glucosidase, arabinose, ArgA, bacillus, nations thereof. In further or alternative embodiments, the beta-galactosidase, beta-glucosidase, beta-glucuronidase, metrology tools may be located on-line, or in further or alter beta-NAG, beta-xylosidase, box car shape, butyric acid major native embodiments, the metrology tools may be located metabolic product, butyric acid minor metabolic product, in-line. Butanol, CAMP. caproic acid major metabolic product, car In further or alternative embodiments, the anaerobic fer 25 bon dioxide production, catalase, cellobiose, cellulose, char mentation apparatuses may be incorporated into centralized treuse fluorescence, chymotrypsin, CO. growth, coccus, des power generation facilities, and in still further or alternative ulfoviridin, double Zone beta-hemolysis, esculin hydrolysis, embodiments, the anaerobic fermentation apparatuses may ethanol production, F/Frequired, fructose, gelatin hydrolysis, be incorporated into decentralized power generation facili glucose, glycogen, gram reaction, growth in bile, His A, ties. 30 hydrogen production, hydrogen Sulfide production, 1-arabi In another aspect are assemblages combining sources of nose, indole, isobutyric acid major metabolic product, isobu steam, turbines, and digesters. In an embodiment of this tyric acid minor metabolic product, isocapronic acid major aspect, the Sources of steam may be sources of high pressure metabolic product, isocapronic acid minor metabolic prod steam. In further or alternative embodiments, the sources of uct, isovaleric acid major metabolic product, isovaleric acid high pressure steam may be boilers. In still further or alter 35 minor metabolic product, lactate converted to propionate, native embodiments, the sources of high pressure steam are lactic acid major metabolic product, lactic acid minor meta boilers heated by combustion of hydrogen, wherein the bolic product, lactose, leithinase, LeuA, lipase, maltose, man hydrogen has been produced by anaerobically fermenting nitol, mannose, melezitose, melibiose, methane production, biomass. In further or alternative embodiments, the digester milk clot formed, milk digested, motile, N-Acetyl-beta-glu may beat least one anaerobic digester. In further or alternative 40 cosaminidase, nitrate, ONPG(Beta-galactosidase), oxygen embodiments, the turbines of the assemblages may be used to tolerance, Phe A, phenylacetic acid minor metabolic product, produce low pressure steam and to generate electricity. In pigment, pitting of agar, ProA, propionic acid major meta further or alternative embodiments, the low pressure steam bolic product, Pyra, raffinose, red fluorescence, reverse produced by such turbines may be used for biomass steriliza CAMP test, rhamnose, ribose, salicin, sensitive to colistin, tion and deoxygenation. In still further or alternative embodi 45 sensitive to kanamycin, sensitive to SPS, sensitive to Vanco ments, the electricity generated by Such turbines may be used mycin, Sorbitol, spore former, starch hydrolysis, strictly as power for local energy consumers or for addition to grid anaerobic, Subterminal spore location, Succinic acid major systems of centralized power stations. In even further or alter metabolic product, Succinic acid minor metabolic product, native embodiments, the local energy consumers may be Sucrose, terminal spore location, threonine converted to pro facilities for anaerobic fermentation, factories, buildings, 50 pionate, trehalose, trypsin, Tyra, urease, Valeric acid major homes, facilities for processing farm produce, and combina metabolic product, Valeric acid minor metabolic product, tions thereof. Xylan, and Xylose. Such categorization in the case of infor Knowledge Management System mation regarding bacteria growth on various Substrates may In another aspect are methods for identifying bacterial allow identification of bacteria strains which utilize specific species for use in the anaerobic production of hydrogen from 55 Substances as food sources, or it may allow the identification a particular biomass. The methods involve collecting appro of optimal food sources for specific bacterial strains. In addi priate information from various bacterial species and deriving tion, categorization of the information regarding bacteria sen probabilistic models from the information, the probabilistic sitivity to various conditions may allow for identification of models being indicative of identification of bacterial species optimal conditions for the anaerobic fermentation of various for use in the anaerobic production of hydrogen and other 60 Substances, while categorization of the information regarding chemical products from particular biomass. In an embodi bacteria production of various metabolites may allow for ment of this aspect, the identification of bacterial species for identification the metabolites produced by various bacterial use in the anaerobic production of hydrogen and other chemi strains using various food sources. cal products from particular biomass includes use of cultiva In further or alternative embodiments, the appropriate tion systems to identify various bacterial species and collect 65 information may be bacterial response which may be posi ing appropriate information from the cultivation system. In tive, negative, a variable response, or non responsive. In still further or alternative embodiments, the cultivation systems further or alternative embodiments, the bacterial response is US 8,501,463 B2 19 20 measured by Smell, color, growth, non-growth, death, sym cinic acid minor metabolic product, Sucrose, terminal spore biosis, inhibition, and non-symbiosis. In further or alternative location, threonine converted to propionate, trehalose, embodiments, the appropriate information is collected in trypsin, Tyr A, urease, Valeric acid major metabolic product, computer readable media. In further or alternative embodi Valeric acid minor metabolic product, Xylan, and Xylose. ments, the probabilistic models are derived using minimizing In further or alternative embodiments, the appropriate parameters based on the appropriate information. information may be bacterial response which may be posi In another aspectare program products for use in comput tive, negative, or non responsive. In further or alternative ers that executes program instructions recorded in a com embodiments, the program product may measures bacterial puter-readable media to produce candidate bacterial species response by Smell, color, growth, non-growth, death, symbio for use in the anaerobic production of hydrogen and other 10 sis, and non-Symbiosis. In further or alternative embodi chemical products from particular biomass, the program ments, the program product may collect the appropriate infor products comprising recordable media and a plurality of com mation in computer readable media. puter-readable program instructions on the recordable media In another aspectare program products for use in comput that are executable by the computer to perform a method ers that executes program instructions recorded on computer comprising receiving a plurality of bacterial species, where 15 readable media to obtain candidate bacterial species for use in relationships between species are defined, receiving a plural the anaerobic production of hydrogen from a particular bio ity of appropriate information for the plurality of bacteria mass, the program products comprise recordable media; and species, and generating the candidate bacterial species for use a plurality of computer-readable program instructions on the in the anaerobic production of hydrogen from a particular recordable media that are executable by the computer to biomass by traversing the plurality of information received hi perform a method comprising: a) determining a bacterial order to find the optimal match. species of interest; b) determining the appropriate informa In further or alternative embodiments, the program prod tion for the bacterial species of interest; and c) repeating steps ucts may collect appropriate information by conducting cul a-b for other bacterial species; and d) comparing appropriate tivation systems to identify various bacterial species or by information collected from step c to assess optimal candidate selecting response data from the Scientific literature. In fur 25 bacterial species for use in the anaerobic production of hydro ther or alternative embodiments, the program product may gen from a particular biomass. have cultivation systems comprises growing various bacterial In an embodiment of the aforementioned aspect, the pro species on particular Substrates, under various growth condi gram product may collect appropriate information by con tions to optimize the bacterial hydrogen production. In further ducting cultivation systems to identify various bacterial spe or alternative embodiments, the program product may have 30 cies or selecting response data from the scientific literature, various growth conditions selected from the group consisting wherein the appropriate information comprises information of variation in temperature, pH, fermentation products, expo regarding bacteria growth on a substrate, bacteria sensitivity Sure to various natural gases or compounds, drug efficacy, to a condition and/or bacteria production of metabolites, compound toxicity or Survivability. which may be further categorized into traits which include, In further or alternative embodiments, the program product 35 but are not limited to, acetic acid major metabolic product, may collect appropriate information involving bacteria acetic acid minor metabolic product, ADH, ALP, alpha-fu growth on a Substrate, bacteria sensitivity to a condition and/ cosidase, alpha-galactosidase, alpha-glucosidase, arabinose, or bacteria production of metabolites, wherein the appropri ArgA, bacillus, beta-galactosidase, beta-glucosidase, beta ate information is categorized into traits which include, but glucuronidase, beta-NAG, beta-xylosidase, box car shape, are not limited to, acetic acid major metabolic product, acetic 40 butyric acid major metabolic product, butyric acid minor acid minor metabolic product, ADH, ALP alpha-fucosidase, metabolic product, CAMP. caproic acid major metabolic alpha-galactosidase, alpha-glucosidase, arabinose, ArgA, product, catalase, cellobiose, chartreuse fluorescence, chy bacillus, beta-galactosidase, beta-glucosidase, beta-glucu motrypsin, CO. growth, coccus, desulfoviridin, double Zone ronidase, beta-NAG, beta-xylosidase, box car shape, butyric beta-hemolysis, esculin hydrolysis, F/F required, fructose, acid major metabolic product, butyric acid minor metabolic 45 gelatin hydrolysis, glucose, glycogen, gram reaction, growth product, CAMP. caproic acid major metabolic product, cata in bile, His A, I-arabinose, indole, isobutyric acid major meta lase, cellobiose, chartreuse fluorescence, chymotrypsin, CO bolic product, isobutyric acid minor metabolic product, iso growth, coccus, desulfoviridin, double Zone beta-hemolysis, capronic acid major metabolic product, isocapronic acid esculin hydrolysis, F/F required, fructose, gelatin hydrolysis, minor metabolic product, isovaleric acid major metabolic glucose, glycogen, gram reaction, growth in bile, His A, 50 product, isovaleric acid minor metabolic product, lactate con 1-arabinose, indole, isobutyric acid major metabolic product, Verted to propionate, lactic acid major metabolic product, isobutyric acid minor metabolic product, isocapronic acid lactic acid minor metabolic product, lactose, leithinase, major metabolic product, isocapronic acid minor metabolic LeuA, lipase, maltose, mannitol, mannose, melezitose, meli product, isovaleric acid major metabolic product, isovaleric biose, milk clot formed, milk digested, motile, N-Acetyl acid minor metabolic product, lactate converted to propi 55 beta-glucosaminidase, nitrate, ONPG(Beta-galactosidase), onate, lactic acid major metabolic product, lactic acid minor oxygen tolerance, Phea, phenylacetic acid minor metabolic metabolic product, lactose, leithinase, LeuA, lipase, maltose, product, pigment, pitting of agar, ProA, propionic acid major mannitol, mannose, melezitose, melibiose, milk clot formed, metabolic product, Pyra, raffinose, red fluorescence, reverse milk digested, motile, N-Acetyl-beta-glucosaminidase, CAMP test, rhamnose, ribose, salicin, sensitive to colistin, nitrate, ONPG(Beta-galactosidase), oxygen tolerance, Phe A. 60 sensitive to kanamycin, sensitive to SPS, sensitive to Vanco phenylacetic acid minor metabolic product, pigment, pitting mycin, Sorbitol, spore former, starch hydrolysis, strictly ofagar, ProA, propionic acid major metabolic product, Pyra, anaerobic, Subterminal spore location, Succinic acid major raffinose, red fluorescence, reverse CAMP test, rhamnose, metabolic product, Succinic acid minor metabolic product, ribose, salicin, sensitive to colistin, sensitive to kanamycin, Sucrose, terminal spore location, threonine converted to pro sensitive to SPS, sensitive to Vancomycin, sorbitol, spore 65 pionate, trehalose, trypsin, Tyra, urease, Valeric acid major former, starch hydrolysis, strictly anaerobic, Subterminal metabolic product, Valeric acid minor metabolic product, spore location, Succinic acid major metabolic product, Suc Xylan, and Xylose. US 8,501,463 B2 21 22 In further or alternative embodiments, the program product alternative embodiments, the biomass material is deoxygen may involve a cultivation system which comprises growing ated by autoclaving. In further or alternative embodiments, various bacterial species on particular Substrates, under Vari the deoxygenated biomass material is further deoxygenated ous growth conditions to optimize the bacterial hydrogen by adding a reducing agent. In further or alternative embodi production. In further or alternative embodiments, the various 5 ments, the reducing agent is dithiothreitol, cysteine, thiogly growth conditions are selected from the group consisting of collate, or sodium sulfide. In further or alternative embodi variation in temperature, pH, fermentation products, expo ments, the biomass material is sterilized and deoxygenated Sure to various natural gases or compounds, drag efficacy, with pressurized steam. In further or alternative embodi compound toxicity or survivability. In further or alternative ments, the biomass material is sterilized and deoxygenated by embodiments, the appropriate information may be bacterial 10 autoclaving. In further or alternative embodiments, the ster response that may be positive, negative, or non responsive, ilized and deoxygenated biomass material is further deoxy and which may be measured by Smell, color, growth, non genated by adding a reducing agent. growth, death, symbiosis, and non-Symbiosis. In further or In further or alternative embodiments, the deoxygenated alternative embodiments, the appropriate information is col biomass material is suitable for producing hydrogen and lected in computer readable media. 15 other chemical products using isolated non-heatshocked Nutrient/Biomass Preparation anaerobic bacteria. In further or alternative embodiments, the In another aspect are methods of anaerobically producing biomass material is deoxygenated with oxygen scavengers. In hydrogen and other chemical products comprising obtaining further or alternative embodiments, the biomass material is biomass material Suitable as nutrients for isolated non-heat deoxygenated with oxygen scavenger microorganisms. In shocked anaerobic bacteria; concentrating or diluting the bio further or alternative embodiments, the biomass material is mass material by at least a factor of 2; Sterilizing the biomass deoxygenated with a cellular membrane preparation of oxy material, deoxygenating the biomass material; and ferment gen Scavenger microorganisms. In further or alternative ing the concentrated (or diluted), Sterilized, deoxygenated, embodiments, the deoxygenated biomass material contains detoxified, and/or pre-digested biomass material with the iso less than about 100 parts per million of oxygen. In further or lated non-heatshocked anaerobic bacteria under anaerobic 25 alternative embodiments, the deoxygenated biomass material conditions so as to produce hydrogen and other chemical contains less than about 50 parts per million of oxygen. In products. In one embodiment, the biomass material is con further or alternative embodiments, the deoxygenated biom centrated by at least a factor of 2; in an alternative embodi ass material contains less than about 20 parts per million of ment, the biomass material is diluted by at least a factor of 2. oxygen. In further or alternative embodiments, deoxygenated In an embodiment of this aspect, the hydrogen is produced 30 water is added to the concentrated, sterilized, deoxygenated, continuously. In further or alternative embodiments, the fer detoxified, and/or pre-digested biomass material to facilitate mentation occurs in anaerobic digesters, landfills, trenches, growth of the isolated non-heatshocked anaerobic bacteria. In or combinations thereof. In further or alternative embodi further or alternative embodiments, the isolated non-heat ments, the concentration step is completed before initiating shocked anaerobic bacteria are mixed with deoxygenated the sterilization step and/or the deoxygenation step. In further 35 water prior to the fermentation step. or alternative embodiments, the dilution step is completed In another aspect are methods of anaerobically producing before initiating the sterilization step and/or the deoxygen hydrogen which comprise (a) obtaining biomass material ation step. In further or alternative embodiments, the concen suitable as nutrients for isolated non-heatshocked bacteria tration step is initiated after completion of the sterilization selected from the group consisting of the following bacteria step and/or the deoxygenation step. In further or alternative 40 genera Acetivibrio, Acetoanaerobium, Acetofilamentum, embodiments, the concentration, Sterilization and deoxygen Acetogenium, Acetothermus, Acidaminobacter, Anaerobio ation steps overlap. In further or alternative embodiments, the spirillum, Anaerorhabdus, Anaerovibrio, Atopobium, sterilization step is completed before initiating the concentra Bacteroides, Bifidobacterium, Bilophila, Butyrivibrio, tion step and/or the deoxygenation step. In further or alterna Campylobacter, Catonella, Centipeda, Dialister; Dichelo tive embodiments, the sterilization step is initiated after 45 bacter, Fervidobacterium, Fibrobacter, Fusobacterium, completion of the concentration step and/or the deoxygen Halanaerobacter, Halanaerobium, Ilvobacter, Johnsonella, ation step. In further or alternative embodiments, the biomass Lachnobacterium, Leptotrichia, Malonomonas, Megamonas, material is concentrated by at least a factor of 4. In further or Mitsuokella, Oxalobacter, Pectinatus, Pelobacter, Porphy alternative embodiments, the dilution step is initiated after romonas, PrevOtella, Propionibacterium, Propionigenium, completion of the sterilization step and/or the deoxygenation 50 Propionispira, Rikenella, Roseburia, Ruminobacter, step. In further or alternative embodiments, the dilution, ster Sebaldella, Selenomonas, Sporomusa, Succinimonas, Suc ilization and deoxygenation steps overlap. In further or alter cinivibrio, Syntrophobacter, Syntrophomonas, Sutterella, native embodiments, the sterilization step is completed Saponavida, Thermobacteroides, Thermosipho, Thermo before initiating the dilution step and/or the deoxygenation toga, Tissierella, Wolinella, Zymophilus, Desulfobacter, Des step. In further or alternative embodiments, the sterilization 55 ulfobacterium, Desulfobulbus, Desulfococcus, Desulfomi step is initiated after completion of the dilution step and/or the crobium, Desulfomonas, Desulfomonile, Desulfonema, deoxygenation step. In further or alternative embodiments, Desulfosarcina, Desulfotomaculum, Desulfo vibrio, Des the biomass material is diluted by at least a factor of 4. ulfitrella, Desulfuromonas, Thermodesulfobacterium, In further or alternative embodiments, the biomass mate Acidaminococcus, Megasphaera, Syntrophococcus, Veil rial is concentrated by centrifugation. In further or alternative 60 lonella, Coprococcus, Peptococcus, Peptostreptococcus, embodiments, the biomass material is concentrated by Ruminococcus, Sarcina, Clostridium, Amoebobacter; Chro reverse osmosis. matium, Lamprobacter; Thiocapsa, Thiocystis, Thiodictyo, In further or alternative embodiments, the biomass mate Thiopedia, Thiospirillum, Ectothiorhodospira, Rhodobacter, rial is sterilized with pressurized steam. In further or alterna Rhodocyclus, Rhodomicrobium, Rhodopilla, tive embodiments, the biomass material is sterilized by auto 65 Rhodopseudomonas, Rhodospirillum, Erythrobacter, Metha claving. In further or alternative embodiments, the biomass nobacterium, Methanobrevibacter, Methanococcu, Metha material is deoxygenated with pressurized steam. In further or nococcoides, Methanolobus, Methanolacinia, Methanomi US 8,501,463 B2 23 24 crobium, Methanogenium, Methanospirillum, mass material with the listed isolated non-heatshocked Methanoplanus, Methanothrix, Methanothermus, Methano anaerobic bacteria under anaerobic conditions so as to pro corpusculum, Methanoculleus, Methanohalobium, Methano duce hydrogen. halophilus, Methanosarcina, Methanosphaera, Eubacte In an embodiment of this aspect, the listed bacteria are rium, Abiotrophia, Atopobium, Gemella, Granulicatella, collected from bovine rumen, Soil samples, sludge, anaerobic Finegoldia, Lactobacillus, Actinomyces, Arcanobacterium, bacteria cultures, aerobic bacteria cultures, anaerobic sedi Bulleidia, Collinsella, Cryptobacterium, Holdemania, ments from fresh or brackish waters, sewage, animals, animal Rothia, Pseudoramibacter, Mogibacterium, Slackia, and feces, insect digestive tract; dental samples, hydrothermal Eggerthella; (b) concentrating the biomass material by at soils, hydrothermal pools or deep water hydrothermal vents. 10 In further or alternative embodiments, the hydrogen is pro least a factor of 2; (c) Sterilizing the biomass, (d) deoxygen duced continuously. In further or alternative embodiments, ating the biomass material; and (e) fermenting the concen the fermentation occurs in anaerobic digesters, landfills, trated, sterilized, detoxified, deoxygenated and/or pre-di trenches, or combinations thereof. In further or alternative gested biomass material with Such isolated non-heatshocked embodiments, the concentration step occurs prior to, after, or anaerobic bacteria under anaerobic conditions so as to pro 15 simultaneously with the sterilization and/or deoxygenation duce hydrogen. In an embodiment of this aspect, the hydro steps. In further or alternative embodiments, the biomass gen is produced continuously. In further or alternative material is concentrated by at least a factor of 4. In further or embodiments, the fermentation occurs in anaerobic digesters, alternative embodiments, the biomass material is deoxygen landfills, trenches, or combinations thereof. In further or ated by a method selected from pressurized steam, autoclav alternative embodiments, the concentration step is completed ing, addition of reducing agents, or combinations thereof. In before initiating the sterilization step and/or the deoxygen further or alternative embodiments, the reducing agent is ation step. In further or alternative embodiments, the concen dithiothreitol, cysteine, thioglycollate, or sodium sulfide. In tration step is initiated after completion of the sterilization further or alternative embodiments, the resulting deoxygen step and/or the deoxygenation step. In further or alternative ated biomass material is suitable for producing hydrogen embodiments, the concentration, Sterilization and deoxygen 25 using the listed isolated non-heatshocked bacteria of this ation steps overlap. In further or alternative embodiments, the aspect. sterilization step is completed before initiating the concentra In further or alternative embodiments, the biomass mate tion step and/or the deoxygenation step. In further or alterna rial is deoxygenated using an agent selected from the group tive embodiments, the sterilization step is initiated after consisting of oxygen scavengers, oxygen scavenging micro 30 organisms, cellular membrane preparation of oxygen Scav completion of the concentration step and/or the deoxygen enging microorganisms, and combinations thereof. In further ation step. In further or alternative embodiments, the biomass or alternative embodiments, the deoxygenated biomass mate material is concentrated by at least a factor of 4. rial contains less than about 20 parts per million of oxygen. In In further or alternative embodiments, the biomass mate further or alternative embodiments, the deoxygenated water rial is concentrated by a method selected from centrifugation, 35 is added to the concentrated, sterilized, detoxified, deoxygen reverse osmosis, drying or a combination thereof. In further ated, and/or pre-digested biomass material to facilitate or alternative embodiments, the biomass material is sterilized growth of the listed isolated non-heatshocked bacteria of this by a method selected from pressurized steam or autoclaving. aspect. Infurther or alternative embodiments, the biomass material is In another aspect are methods of anaerobically producing deoxygenated by a method selected from pressurized steam, 40 hydrogen comprising obtaining a biomass material Suitable autoclaving, addition of a reducing agent, or a combination as a nutrient for isolated non-heatshocked Clostridia, concen thereof. In further or alternative embodiments, the reducing trating the biomass material by at least a factor of 2 via agent is dithiothreitol, cysteine, thioglycollate, or sodium centrifugation, reverse osmosis or a combination thereof. Sulfide. In the resulting deoxygenated biomass material is sterilizing the biomass material by a method selected from Suitable for producing hydrogen using the listed isolated non 45 pressurized steam or autoclaving, deoxygenating the biomass heatshocked bacteria species of this aspect. material by a method selected from pressurized steam, auto In further or alternative embodiments, the biomass mate claving, addition of reducing agents, or combinations thereof, rial is deoxygenated using an agent selected from the group wherein the deoxygenated biomass material contains less consisting of oxygen scavengers, oxygen scavenging micro than about 20 parts per million of oxygen; and fermenting the organisms, cellular membrane preparation of oxygen Scav 50 concentrated, deoxygenated, detoxified, and/or pre-digested enging microorganisms, and combinations thereof. In further biomass material with the isolated non-heatshocked anaero or alternative embodiments, the deoxygenated biomass mate bic bacteria under anaerobic conditions in an anaerobic rial contains less than about 20 parts per million of oxygen. In digestor So as to produce hydrogen. further or alternative embodiments, the deoxygenated water In an embodiment of this aspect, the hydrogen is produced is added to the concentrated, sterilized, detoxified, deoxygen 55 continuously. In further or alternative embodiments, the fer ated, and/or pre-digested biomass material to facilitate mentation occurs in anaerobic digester, landfills, trenches, or growth of the listed isolated non-heatshocked bacteria of this combinations thereof. In further or alternative embodiments, aspect. the concentration step occurs priorto, after, or simultaneously In another aspect are methods of anaerobically producing with the sterilization and/or deoxygenation steps. In further or hydrogen comprising (a) obtaining a biomass material Suit 60 alternative embodiments, the biomass material is concen able as a nutrient for isolated non-heatshocked bacteria trated by at least a factor of 4. In further or alternative embodi selected from the group consisting of anaerobic bacteria (see ments, the reducing agent is dithiothreitol, cysteine, thiogly above); (b) concentrating the biomass material by at least a collate, or sodium sulfide. In the resulting deoxygenated factor of 2 using centrifugation, reverse osmosis or a combi biomass material is suitable for producing hydrogen using nation thereof; (c) Sterilizing the biomass, (d) deoxygenating 65 isolated non-heatshocked Clostridia. the biomass material; and (e) fermenting the concentrated, In further or alternative embodiments, the biomass mate sterilized, detoxified, deoxygenated and/or pre-digested bio rial is further deoxygenated using an agent selected from the US 8,501,463 B2 25 26 group consisting of oxygen Scavengers, oxygen Scavenging order to anaerobically produce hydrogen and other chemical microorganisms, cellular membrane preparation of oxygen products from the biomass. In an embodiment of their aspect, Scavenging microorganisms, and combinations thereof. In the bacteria are placed into a sealed chamber with substan further or alternative embodiments, the deoxygenated water tially deoxygenated water and various types of biomass. In is added to the concentrated, sterilized, deoxygenated, further or alternative embodiments, the sealed chamber ulti detoxified, and/or pre-digested biomass material to facilitate mately hosts an anaerobic environment. In further or alterna growth of the isolated non-heatshocked Clostridia. tive embodiments, the sealed chamber can provide a multi In another aspect are methods of anaerobically producing plicity of growth conditions. In further or alternative hydrogen comprising obtaining a biomass material Suitable embodiments, the multiplicity of growth conditions are as a nutrient for isolated non-heatshocked Clostridia, 10 selected from the group consisting of variation in tempera wherein the biomass material comprises molasses, raw paper, ture, pressure, pH, fermentation products, exposure to various agricultural waste, or mulch; concentrating the biomass mate natural gases or compounds, drug efficacy, drug resistance, rial by at least a factor of 2 via centrifugation, reverse osmosis compound toxicity or survivability. In further or alternative or a combination thereof. Sterilizing the biomass material by embodiments, the sealed chamber has a shape selected from a method selected from pressurized steam or autoclaving, 15 the group consisting of a circular, cylindrical, spherical, deoxygenating the biomass material by a method selected square, or rectangular form. In further or alternative embodi from pressurized steam, autoclaving, addition of reducing ments, the sealed chamber has a volume of 100 cm to 50,000 agents, or combinations thereof, wherein the deoxygenated cm. biomass material contains less than about 20 parts per million In further or alternative embodiments, isolated non-heat of oxygen; and fermenting the concentrated, sterilized, shock anaerobic bacteria are taken from the sealed chamber deoxygenated, detoxified, and/or pre-digested biomass mate and further selected by cultivation systems. In further or rial with the isolated non-heatshocked anaerobic bacteria alternative embodiments, the cultivation systems comprise under anaerobic conditions in an anaerobic digester so as to growing various bacterial species on particular substrates, produce hydrogen. In an embodiment of this aspect, the under various growth conditions to optimize the bacterial hydrogen is produced continuously. In further or alternative 25 hydrogen production. In further or alternative embodiments, embodiments, the fermentation occurs in anaerobic digesters, the isolated non-heatshock anaerobic bacteria are selected by landfills, trenches, or combinations thereof. In further or its production of hydrogen, including by way of example alternative embodiments, the concentration step occurs prior only, at least 5%, at least 10%, at least 15%, at least 20%, at to, after, or simultaneously with the sterilization and/or least 25%, or at least 30% of the total theoretical amount of deoxygenation steps. In further or alternative embodiments, 30 hydrogen that could be produced from a particular biomass. the biomass material is concentrated by at least a factor of 4. In further or alternative embodiments, the isolated non-heat Infurther or alternative embodiments, the reducing agentis shock anaerobic bacteria are selected by its production of 2 dithiothreitol, cysteine, thioglycollate, or sodium sulfide. In moles of hydrogen per mole of substrate produced. In further further or alternative embodiments, the resulting deoxygen or alternative embodiments, the various growth conditions ated biomass material is suitable for producing hydrogen 35 are selected from the group consisting of variation in tem using isolated non-heatshocked Clostridia. In further or alter perature, pressure, pH, fermentation products, exposure to native embodiments, the biomass material is further deoxy various natural gases or compounds, drug efficacy, compound genated using an agent selected from the group consisting of toxicity or survivability. oxygen Scavengers, oxygen scavenging microorganisms, cel In further or alternative embodiments, the isolated non lular membrane preparation of oxygen Scavenging microor 40 heatshock anaerobic bacteria are taken from the sealed cham ganisms, and combinations thereof. In further or alternative ber and further selected in cultivation systems after 1-7 days. embodiments, the deoxygenated water is added to the con In further or alternative embodiments, the isolated non-heat centrated, sterilized, deoxygenated, detoxified, and/or pre shock anaerobic bacteria are taken from the sealed chamber digested biomass material to facilitate growth of the isolated and further selected in cultivation systems after 1-20 weeks. non-heatshocked Clostridia. 45 In another aspect are apparatuses for selecting bacteria for In another aspect are methods for producing a nutrient use in anaerobic hydrogen production which comprise sealed suitable for non-heatshocked anaerobic bacteria to produce chambers capable for providing anaerobic environments and hydrogen comprising removing Substantially all of the oxy multiplicities of growth conditions. In an embodiment of this gen from a biomass by a method selected from pressurized aspect, the bacteria are collected from bovine rumen, Soil steam, autoclaving, addition of a reducing agent, or combi 50 samples, sludge, anaerobic bacteria cultures, aerobic bacteria nations thereof. cultures, anaerobic sediments from fresh or brackish waters, In another aspect are nutrients Suitable for non-heat sewage, animals, animal feces, insect digestive tract, dental shocked anaerobic bacteria to produce hydrogen comprising samples, hydrothermal soils, hydrothermal pools or deep prepared by removing Substantially all of the oxygen from a water hydrothermal vents. In further or alternative embodi biomass by a method selected from pressurized steam, auto 55 ments, the apparatuses further contain Substantially deoxy claving, addition of a reducing agent, or combinations genated water. thereof. In an embodiment of this aspect, the nutrients are In further or alternative embodiments, the multiplicities of further prepared by concentrating the biomass using centrifu growth conditions are selected from the group consisting of gation, reverse osmosis or a combination thereof, and steril variation in temperature, pressure, pH, fermentation prod izing the biomass by means of pressurized steam or autoclav 60 ucts, exposure to various natural gases or compounds, drug ing. efficacy, compound toxicity or survivability. In further or Isolation/Enrichment System alternative embodiments, the sealed chambers have shapes In another aspectare methods for selecting bacteria for use selected from the group consisting of circular, cylindrical, in anaerobic hydrogen production comprising isolating spherical, square, or rectangular forms. In further or alterna anaerobic bacterial strains for their ability to use biomass as 65 tive embodiments, the sealed chambers further have shapes nutrients and measuring collected hydrogen gas produced, having volumes of 100 cm to 50,000 cm. In further or wherein the anaerobic bacteria do not require heatshocking in alternative embodiments, the apparatuses further comprise at US 8,501,463 B2 27 28 least one bacterial strain that produces hydrogen and other products. In further or alternative embodiments, the chemical chemical products anaerobically. In further or alternative products are selected from the group consisting of gases, embodiments, the apparatuses further contain particular bio Solids, solvents, Volatile organic acids, salts of volatile mass samples the bacteria use as nutrients for producing organic acids, and combinations thereof. In further or alter hydrogen gas and other chemical products. native embodiments, the chemical product is a gas selected Infurther or alternative embodiments, the bacteria selected from the group consisting of carbon dioxide, carbon monox from the sealed chambers are removed without substantially ide, hydrogen Sulfide, ammonia, nitrogen, and combinations disturbing the anaerobic environment. In further or alterna thereof. In further or alternative embodiments, the chemical tive embodiments, the bacteria are selected from the sealed product is a solid that comprises sulfur. In further or alterna chambers by use of needles or by means of a vacuum. In 10 further or alternative embodiments, the apparatus comprises tive embodiments, the solid is elemental sulfur. In further or at least two Substantially parallel glass plates. In further or alternative embodiments, the solvents are selected from the alternative embodiments, the apparatus further comprises a group consisting of acetone, butanol, ethanol, propanol, iso seal on at least one side that is penetrable by needles. propanol. 1.2-propanediol, and combinations thereof. In fur Use of Bacteria and Bacterial Cultures to Produce Hydrogen 15 ther or alternative embodiments, the Volatile organic acids are and Other Chemical Products selected from the group consisting of formic acid, acetic acid, In another aspect are bacterial cultures which comprise propionic acid, butyric acid, Valeric acid and combinations purified bacterial strains, in which the bacterial strains have thereof. In further or alternative embodiments, the salts of not undergone a heatshocking process and they produce Volatile organic acids comprise anions selected from the hydrogen by anaerobically fermenting biomass. In further or group consisting of formate, acetate, propionate, butyrate, alternative embodiments, the biomass has been deoxygen Valerate, and combinations thereof. In further or alternative ated. In further or alternative embodiments, are the biomass embodiments, the salts of Volatile organic acid comprise cat has been sterilized. Further or alternative embodiments are ions selected from the group consisting of alkali metal ions, the biomass has been concentrated. In further or alternative alkaline earth ions, ammonium ions, and combinations embodiments, the biomass has been deoxygenated and con 25 thereof. In further or alternative embodiments, the cations are centrated. In further or alternative embodiments, the biomass Na', K", Ca, Mg", NH, and combinations thereof. In has been sterilized, deoxygenated, concentrated, detoxified, further or alternative embodiments, the bacteria cultures and/or pre-digested. In further or alternative embodiments, comprise Substantially purified single bacterial strains. In the bacterial strains have been selected by means of a Knowl further or alternative embodiments, the substantially purified edge Management System. In further or alternative embodi 30 single bacterial strains are at least 95% purified. In further or ments, the bacterial strains have been selected by means of alternative embodiments, the Substantially purified single isolation/enrichment systems. In further or alternative bacterial strains are at least 99% purified. In further or alter embodiments, the bacterial strains have been isolated by native embodiments, the Substantially purified single bacte means of isolation/enrichment systems. In further or alterna rial strains are at least 99.5% purified. tive embodiments, the bacterial strains have been isolated by 35 In further or alternative embodiments, the Knowledge means of isolation/enrichment systems and have been Management System comprises a program product for use in selected by means of a Knowledge Management System. In a computer that executes program instructions recorded in a further or alternative embodiments, the biomass comprises computer-readable media to obtain candidate bacterial spe material from at least one member of the Capsicum. In cies for use in the anaerobic production of hydrogen from a still further or alternative embodiments, the member of the 40 particular biomass. In further or alternative embodiments, the genus Capsicum is selected from the group consisting of program product comprises a recordable medium and a plu Capsicum anuum, Capsicum baccatum, Capsicum chinense, rality of computer-readable program instructions on the Capsicum gemnifolium, Capsicum frutescens, Capsicum recordable media that are executable by the computer to pubescens, and combinations thereof. In further or alternative perform a method comprising: a) determining a bacterial embodiments, the biomass comprises material from at least 45 species of interest; b) determining the appropriate informa one member of the genus Allium. In still further or alternative tion for the bacterial species of interest; c) repeating steps a-b embodiments, the member of the genus Allium is selected for other bacterial species; and d) comparing appropriate from the group consisting of Allium sativum, Allium cepa, information collected from step c to assess optimal candidate Allium Schoenoprasum, Allium tuberosum, Allium ampelo bacterial species for use in the anaerobic production of hydro prasum, and combinations thereof. In further or alternative 50 gen from a particular biomass. In further or alternative embodiments, the biomass comprises Sugar products. In fur embodiments, the appropriate information is collected by ther or alternative embodiments, the Sugar products include conducting cultivation systems to identify various bacterial material from at least one member of the genus Saccharum. In species. In further or alternative embodiments, the cultivation still further or alternative embodiments, the member of the systems comprise growing various bacterial species on par genus Saccharum is a species selected from the group con 55 ticular substrates, under various growth conditions to opti sisting of Saccharum spontaneum, Saccharum robustum, mize the bacterial hydrogen production. Saccharum officinarum, Saccharum barberi, Saccharum sin Infurther or alternative embodiments, the isolation/enrich ense, Saccharum edule, and combinations thereof. In further ment system is a sealed chamber containing Substantially or alternative embodiments, the biomass comprises cellulose deoxygenated water and at least one biomass sample used to products. In still further or alternative embodiments, the cel 60 anaerobically ferment the at least one biomass. In further or lulose products includes wood pulp. In further or alternative alternative embodiments, the isolation/enrichment system embodiments, the biomass comprises material from at least ultimately hosts an anaerobic environment. The isolation/ one species selected from the group consisting of Solanum enrichment system can provide a multiplicity of growth con Esculentum, Solarium melongena, Solarium tuberosum, ditions. In further or alternative embodiments, the isolation/ Lycopersicon esculentum, Beta vulgaris, and combinations 65 enrichment system has a shape selected from the group thereof. In further or alternative embodiments, the bacteria consisting of a circular, cylindrical, spherical, square, or rect cultures are used to produce hydrogen and other chemical angular form. US 8,501,463 B2 29 30 In further or alternative embodiments, the bacterial strains FIG. 13 presents one potential procedure to concentrate are used to produce hydrogen by anaerobically fermenting biomass. biomass in anaerobic fermentation apparatuses. In further or FIG. 14 presents one potential Schematic showing the multi alternative embodiments, the anaerobic fermentation appara fermentation scheme used for optimal hydrogen production. tuses comprises: Sterilization and deoxygenation systems; 5 FIG. 15 presents one potential schematic of the showing anaerobic digesters containing a population of Substantially the various uses for the anaerobic fermentation products. purified anaerobic bacteria and equipped with an anaerobic FIG. 16 presents one potential schematic of the machinery digester control systems; plurality of pipelines and pumps for components used for hydrogen production. introducing and re-circulating biomass, and removal pipe FIG. 17 presents one potential schematic demonstrating lines connected to the anaerobic digesters for removing 10 the use of a turbine system to generate low pressure steam for chemical products from the anaerobic digesters. In further or sterilization and deoxygenation of a biomass alternative embodiments, the anaerobic digester control sys FIG. 18 presents one potential schematic of one embodi tems are used to optimally operate the anaerobic digesters and ment of the anaerobic digester control system which comprise: process control tools; metrology tools to 15 acquire metrology data relating to anaerobic fermentation DETAILED DESCRIPTION OF THE INVENTION parameters; process controllers operatively coupled to the process control tools and the metrology data, wherein the Glossary of Certain Terms process controllers comprise decision making units, input/ output boards, and database units to store the metrology data. The term “anaerobic' or “anoxic', as used herein, refers to Other objects, features and advantages of the methods and an environment in which the oxygen has been Substantially compositions described herein will become apparent from the removed. By way of example only, the oxygen concentration following detailed description. It should be understood, how may be less than 10000 ppm, or the oxygen concentration ever, that the detailed description and the specific examples, may be less than 1000 ppm, or the oxygen concentration may while indicating specific embodiments, are given by way of 25 be less than 100 ppm, or the oxygen concentration may be less illustration only, since various changes and modifications than 50ppm, or the oxygen concentration may be less than 20 within the spirit and scope of the invention will become ppm apparent to those skilled in the art from this detailed descrip The term “anaerobic fermentation', as used herein, refers tion. to an anaerobic process in which biomass is degraded to 30 chemical products such as, by way of example only, hydro BRIEF DESCRIPTION OF THE DRAWINGS gen, carbon dioxide, Volatile organic acids, hydrogen Sulfide, methane, sulfur, and carbon monoxide. FIG. 1 presents one potential Schematic showing the vari The term “anaerobic respiration', as used herein, refers to ous aspects of the present invention. a process in which organic Substrates are degraded to CO. FIG.2 presents one potential schematic illustrating the use 35 but using a substance other than oxygen as the terminal elec of the Knowledge Management System for utilization of a tronacceptor. Somebacteria respire by using nitrate or Sulfate particular biomass. ions as the terminal electron acceptor during respiration. FIG. 3 presents one potential screen shot of a display page The term “assemblage' as used herein, refers to combina from the Knowledge Management System that lists bacterial tion of components interconnected to achieve a desired func species and traits. 40 tion. The assemblage may be a standalone system or a system FIG. 4 presents one potential screen shot of a display page incorporated into another assemblage. The function of the from the Knowledge Management System showing a pre assemblage includes, but is not limited to, the production of ferred test that displays specific traits the bacteria are tested chemical products by anaerobically fermenting biomass, the for a response. monitoring and control of anaerobic fermentation systems, FIG. 5 presents one potential screen shot of a display page 45 and generation power and low pressure steam. from the Knowledge Management System detailing appro The terms “biomass', as used herein, refers to any animal priate information collected on the bacteria Bilophila wad or plant derived material that contains one or more compo sworthia. nents that can be converted, bioconverted or biodegraded into FIG. 6 presents one potential screen shot of a display page a useful material by anaerobic fermentation. from the Knowledge Management System detailing a 50 The term “bacteria culture', as used herein, refers to grow description of a catalase indicator test. ing of bacteria in a specially prepared nutrient medium. FIG. 7 presents one potential bock diagram of a system in The term “energy crops, as used herein, refers to plants the Knowledge Management System that performs sample which are grown for use as a source of biomass for fuel. processing and analysis in a biological workstation. The term “feedstock', as used herein, refers to a source of FIG. 8 presents one potential block diagram of a computer 55 chemical products used industrially to generate new chemical in the Knowledge Management System, illustrating the hard products and compositions, and it also refers to biomass ware components included in a computer that can provide the SOUCS. functionality of workstations and computers. The term "fermentation', as used herein, refers to a bio FIG. 9 presents one potential diagram of one embodiment logical process in which organic compounds are partially of the isolation/enrichment system used to isolate anaerobic 60 degraded; for example, yeasts ferment Sugars to alcohol. bacteria. The terms "heatshocking' and “heatshocking process', as FIG. 10 presents one potential process flow diagram of a used herein, refers to the process of heating a microbial com preferred embodiment of the anaerobic digester system. munity, such as an inoculum or a sample containing multiple FIG. 11 presents one potential procedure to prepare biom bacterial strains, to eliminate undesired strains, such as non ass material. 65 spore forming (vegetative) bacteria, e.g. methanogens, with FIG. 12 presents one potential procedure to deoxygenate desired strains, such as spore forming bacteria, Surviving the biomass. procedure for future growth in better conditions. A non-lim US 8,501,463 B2 31 32 iting example of a heatshocking process is to heat up the product processing, food waste, Solids isolated from fermen mixture of bacteria to 105°C. for up to 2 hours. tation cultures, municipal sewer waste, animal manure, ani The terms "heatshocking” and “heatshocking process', as mal urine, animal parts, fish parts, and combinations thereof. used herein, may also refer to the induction of specific chemi The future energy economy may have an important role for cal pathways and/or a redirection of protein synthesis that 5 hydrogen as a clean, carbon dioxide neutral energy source for involves diversion from induction and synthesis of other pro use in, by way of example only, fuel cell vehicles and for teins. decentralized electricity generation in Stationary fuel cell sys The term “heatshocked’, as used herein, refers to having tems. In fuel cells, hydrogen can be converted to electricity undergone a heatshocking process. very efficiently, producing only water as a waste product, thus The term “metrology tool, as used herein, refers to as 10 drastically reducing carbon dioxide, nitric oxide, particulate measurement tool, with the information obtained referred to and other emissions that accompany the use of fossil fuels. herein as “metrology data. Although the use of hydrogen produced from fossil sources The term “substantially deoxygenated, as used herein, can lead to a Substantial reduction of emissions, the energy refers to a biomass or bacteria culture media in which there efficiency of the production-to-end-use chain (from natural has been a reduction of the oxygen concentration. By way of 15 gas to hydrogen to electricity) is limited. This is due to energy example only, the oxygen concentration may be less than losses in the hydrogen production phase with concomitant 10000 ppm, or the oxygen concentration may be less than carbon dioxide capture. In the long run, hydrogen would 1000 ppm, or the oxygen concentration may be less than 100 ideally be produced from renewable sources such as the elec ppm, or the oxygen concentration may be less than 50 ppm, or trolysis of water with renewable electricity, or by means of the oxygen concentration may be less than 20 ppm. In biomass gasification, or biological fermentation or photobio embodiments when “substantially deoxygenated’ refers spe logical hydrogen production. With large-scale implementa cifically to a reduction of the concentration of oxygen in an tion of renewable energy production, hydrogen can be a clean aqueous solution, then the term 'substantially deoxygenated carrier of energy for storage and transport. means less than 5 ppm of oxygen in the aqueous solution, Hydrogen can be produced from a variety of feedstocks including less than 4 ppm of oxygen, less than 3 ppm of 25 using a variety of process technologies. Feedstock options oxygen, less than 2 ppm of oxygen, and less than 1 ppm of include fossil resources such as coal, natural gas, and petro OXygen. leum, and renewable resources Such as biomass, Sunlight and The term “substantially parallel as used herein, refers to wind. Process technologies include thermochemical, biologi the distance between walls of the isolation/enrichment sys cal, electrolytic and photolytic. Associated with each poten tem which may differ by a certain amount along the length of 30 tial feedstock or energy source are unique challenges and the isolation/enrichment system. By way of example only, the benefit trade-offs. Fossil feedstocks, for example, are widely distance between walls of the isolation/enrichment system available and relatively well understood and accepted, but may differ up to 5% along the length of the isolation/enrich until carbon sequestration becomes feasible, greenhouse gas ment system, or the distance between walls of the isolation/ emissions make fossil feedstocks environmentally difficult. enrichment system may differ up to 1% along the length of the 35 Biomass, on the other hand, has very small or even negative isolation/enrichment system, or the distance between walls of net carbon emissions, but has presented challenges with bio the isolation/enrichment system may differ up to 0.5% along mass collection, transportation, availability, and storage. the length of the isolation/enrichment system. Perfectly par Hydrogen produced from biomass fermentation has not been allel walls would have no difference in width along the length cost competitive with gasoline because biomass and capital of the isolation/enrichment system. 40 costs are high. Improved biomass/agriculture technology The term “substantially purified as used herein, refers to (higher yields per acre, etc.); lower cost biomass collection, a bacterial culture in which there has been a reduction of some transportation, and storage options; and improved biomass biological component in the culture media Such as, by way of preparation are needed. In addition, biomass Sources are often example only, viruses, pathogens, toxins and bacteria Strains, seasonal in nature, therefore biomass-flexible processes and/ other than the bacterial strain of interest. 45 or cost effective biomass storage are needed for year round Hydrogen Production operation. One approach to hydrogen production that may prove eco All renewable energy sources are ultimately based on Solar nomically viable uses biological conversion of energy crops energy which is made available through photovoltaic cells, and industrial and agricultural wastes and residues into wind energy or stored as chemical energy in biomass. Thus, hydrogen and other chemical products. Hydrogen gas is an 50 biomass essentially stores Solar energy in chemical form, environmentally friendly energy source, as it may be used to wherein the potential energy is releases in the form of hydro generate electricity without producing greenhouse gas emis gen during anaerobic fermentation. In addition, hydrogen sions. Currently, most hydrogen is produced by 'steam production may also result from direct photobiological pro reforming of methane, which predominantly relies on natu cesses. Biomass sources available for conversion into hydro ral gas as a source of methane and energy. In this process, 55 gen include, but are not limited to, dedicated bioenergy crops natural gas and a chemical catalyst are mixed with steam at and/or less expensive residues, such as organic waste from high temperatures and pressure to form hydrogen and carbon regular agricultural farming and wood processing (biomass dioxide. In this process a non-renewable energy source is residues). In contrast, direct photobiological hydrogen pro used to generate hydrogen, thus; it would be ideal to replace duction, does not require biomass, as wateris directly cleaved the fossil fuels with renewable energy sources, such as hydro 60 by photosynthetic microorganisms. Regardless, hydrogen gen production via biomass fermentation. Potential hydrogen production by these biological means is attractive due to the production may be derived from renewable energy sources renewable energy nature of Solar energy. Such as, but not limited to, energy crops, Surplus agricultural A distinction can be made between the use of dry biomass products, waste from Sugar production and processing facili (such as wood) and the use of wet biomass sources such as the ties, animal waste from Zoos, waste from fruit processing 65 organic fraction of domestic waste, agro-industrial wastes industries, waste from pulp and paper mills, silvaculture resi and slurries, and wastewater. Dry biomass is used for thermal dues, waste from wood processing, waste from agricultural conversion processes that require minimal water content Such US 8,501,463 B2 33 34 as green electricity generation (via combustion or gasifica order to maintain electrical neutrality. In aerobic environ tion) or the production of renewable diesel fuel through gas ments, oxygen is reduced and water is the product, however, ification, followed by Fischer-Tropsch synthesis. Wet biom in anaerobic environments no oxygen is present and therefore asS and residues are typically less Suitable for thermal other compounds act as electronacceptors. For instance, pro conversion because transport and drying require a consider tons accept electrons and are thereby reduced to molecular able amount of energy, which leads to a limited or even hydrogen (H). In “dark fermentation a wide variety of bac negative overall carbon dioxide reduction. The available teria use the reduction of protons to dispose of reducing amount of wet biomass and residues is however considerable, equivalents which results from primary metabolism. Other Such that their use as sources for renewable energy production examples of alternative electron acceptors in anaerobic envi is desirable. Biological conversion processes are particularly 10 ronments are nitrate with nitrogen gas (N2) as the product or useful for this application because they are catalyzed by sulfate with dihydrogensulfide (H2S) as the reduced product. microorganisms in an aqueous environment at low tempera Even organic compounds can act as electron acceptors. For ture and pressure. Furthermore these techniques are well instance, the microbial production ofbutanol occurs through Suited for decentralized energy production in Small-scale the reduction of butyric acid. The capacity to reduce other installations in locations where biomass or wastes are avail 15 electron acceptors than oxygen requires the presence of a able, thus avoiding energy expenditure and costs for trans specific enzyme system in the micro-organisms: hydrogen port. The general expectation is that biological processes may producing bacteria possess hydrogenase enzymes; nitrate play a substantial role in the production of renewable gaseous reducing bacteria possess an elaborate set of enzymes cata and liquid biofuels including methane, hydrogen, bioethanol lyzing the stepwise reduction of nitrate to nitrogen etc. and ABE (Acetone-Butanol-Ethanol). Anaerobic microbiological decomposition is a process in Hydrogen production from biomass has been thought to be which micro-organisms derive energy and grow by metabo an expensive process. This is reflected when comparing the lizing organic material in an oxygen-free environment result higher costs for biomass, distribution, and fixed capital costs ing in the production of various end-products. The anaerobic relative to other production methods such as natural gas digestion process can be subdivided into four phases, each reforming. In addition, due to the heterogeneity of biomass, 25 requiring its own characteristic group of micro-organisms: the localized production of biomass, and the relatively high hydrolysis (conversion of non-soluble biopolymers to soluble costs of gathering and transporting biomass Sources, digester organic compounds), acidogenesis (conversion of soluble complexes dedicated for biomass gasification are limited to organic compounds to Volatile organic acids, also referred to midsize-scale operations. Additionally, factors not associated as Volatile fatty acids, and carbon dioxide), acetogenesis in the economic analysis are fertilizer costs, the environmen 30 (conversion of carbohydrates or volatile organic acids to tal impact associated with the production, harvest, and trans acetate and hydrogen), and methanogenesis (conversion of port of biomass, or any potential degradation in land quality organic acids or acetate and carbon dioxide plus hydrogen to associated with intensive bioenergy crop farming. Increased methane gas). The acidogenic bacteria excrete enzymes for efficiency of continuous hydrogen production may be hydrolysis and convert Soluble organics to Volatile organic obtained by improvements in Systems, methods and compo 35 acids and alcohols. Volatile organic acids and alcohols are sitions used for anaerobic bacterial fermentation. The ability then converted by acetogenic bacteria into acetic acid or to effectively utilize a variety of biomass types, and the devel hydrogen and carbon dioxide. Methanogenic bacteria then opment of localized energy production from decentralized use acetic acid, hydrogen and carbon dioxide to produce fuel cell power stations, may ultimately decrease biomass methane. harvest and transportation issues. 40 Bio-methane production through anaerobic digestion of Production of Hydrogen and Other Chemical Products from wastes and wastewater using mixtures of bacteria species is Biomass an established technology. The final biogas product is a mix The production of hydrogen is a ubiquitous, natural phe ture of methane (55-75 Vol.%) and carbon dioxide (25-45 vol. nomenon under anaerobic conditions. A wide variety of bac %) which can be used for heating, upgrading to natural gas teria, from Swamps, sewage, hot springs, and the rumen of 45 quality or co-generation of electricity and heat. The biogas cattle are able to convert organic matter to hydrogen, carbon yield varies with the type and concentration of the biomass dioxide and other chemical products including, but not lim and process conditions. However, there are many disadvan ited to, acetic acid, butryric acid, lactate, and ethanol. In tages of conventional anaerobic treatment for methane pro general, these bacteria live in the close proximity to other duction, including a high sensitivity of methanogenic bacteria bacteria which consume the chemical products, including 50 to a large number of chemical compounds, the first start-up of hydrogen, producing their own endproducts like methane and an installation without the presence of proper seed sludge can carbon dioxide. In this way, a stable ecosystem is formed be time-consuming due to the low growth yield of anaerobic wherein hydrogen consumers remove hydrogen, and allow bacteria, and the anaerobic treatment can be accompanied by continued growth of hydrogen producers, which would oth odor due to the formation of sulfide when treating wastewater erwise be inhibited by high concentrations of hydrogen. 55 containing Sulfurous compounds. During this methane pro In anaerobic environments, hydrogen is commonly pro duction through anaerobic digestion of wastewater and resi duced during microbial breakdown of organic compounds. dues (including sewage sludge, manure and the organic frac When organic compounds are the sole carbon and energy tion of municipal waste) hydrogenis an intermediary product, Source providing metabolic energy under anaerobic condi which is not available because it is rapidly taken up and tions, the process is termed "dark hydrogen fermentation’, or 60 converted into methane by methane-producing microorgan “dark fermentation'. When light is required to provide addi isms. Also present are organisms that compete for nutrients tional energy, the process belongs to the category of photo and create undesirable end products. The final gas produced biological processes and is termed "photo-fermentation'. in this method is also contaminated with hydrogen Sulfide, When bacteria grow on organic Substrates (heterotrophic ammonia, and siloxanes which damage energy producing growth), these substrates are degraded by oxidation to pro 65 equipment. This method can also be time consuming and vide building blocks and metabolic energy for growth. This resource intensive due to extended hydraulic retention times. oxidation generates electrons which need to be disposed of in Thus, to achieve efficient hydrogen production from anaero US 8,501,463 B2 35 36 bic fermentation, as achieved using the methods disclosed identify an optimal bacterial species for use in producing herein, methanogenic bacteria mixed with hydrogen produc hydrogen from a particular biomass, rather than trying to ing bacteria, thereby eliminating the possibility of hydrogen genetically engineer the bacteria to ferment the biomass. consumption. The methods described herein decouple hydro However, bacteria may be obtained by genetic modification gen formation and consumption Such that hydrogen is avail of known bacterial strains and evaluated in the isolation/ able as the final product. enrichment system. The genetic modification of the bacterila Anaerobic Production of Hydrogen and Other Chemical strains may results from transformation procedures, bacterial Products from Biomass conjugation, transduction, interaction of bactrial strains with The present approach takes advantage of emulating a natu mutagens and combinations thereof. The mutagens used may ral environment for anaerobic bacterial growth in order to 10 include, but are not limited to, chemicals, ultraviolet light, achieve optimal hydrogen production. This is contrary to and radioactive element. previous less efficient attempts at hydrogen production using By way of example volatile fatty acids produced include anaerobic fermentation by genetically modifying bacteria or formic, acetic, propionic, isobutyric, butyric, isovaleric, by forcing bacteria to exist in unnatural environments and Valeric, isocaproic, caproic, and heptanoic acids. By way of coaxing them to produce hydrogen. FIG. 1 depicts one 15 example alcohol products from fermentation include ethanol, approach of the methods described herein, wherein hydrogen ethanol, propanol, isobutanol, butanol, isopentanol, pentanol, is produced under anaerobic conditions, using anaerobic bac acetone, propanol, isopropanol, and 1, 2-propanediol. By teria to anaerobically ferment different types of biomass. way of example non-volatile products from fermentation Central to the approach is the Anaerobic Digester, in which include pyruvic, lactic, oxalacetic, oxalic, methyl malonic, the natural environment for anaerobic bacterial growth is malonic, fumaric, succinic. emulated to achieve optimal hydrogen production. The Knowledge Management System digester operating parameters (such as, by way of example One aspect of the methods described herein is the creation only, pH, pressure and temperature) are monitored and of a database by compilation of information regarding the adjusted accordingly in order to control and maintain the hydrogen producing characteristics of various isolated bacte appropriate environment. Similarly, the concentrations of the 25 ria genera and species, along with optimal biomass used by fermentation products are monitored and the products the respective bacteria and optimal conditions. This database removed from the digester in order to maintain optimal is incorporated into a Knowledge Management System, anaerobic fermentation conditions in the digester, with opti which is an effective tool for exploitation of anaerobic hydro mal hydrogen production. By way of example only, Such gen production as an alternative energy source. The type of products removed may include organic acids/solvents/solids, 30 biomass available for hydrogen production via anaerobic fer bacterial biomass, carbon dioxide and hydrogen. mentation may vary. As shown in FIG. 2, the Knowledge One aspect of the approach, shown in FIG. 1, is the selec Management System allows for the identification of bacteria, tive isolation of anaerobic bacteria, which use particular types with known hydrogen producing characteristics for a particu of biomass, and are abundant hydrogen producers. This selec lar biomass, and the optimal digesteroperating parameters for tion, modification and maximization is achieved by utilizing 35 anaerobic fermentation of the specific biomass available. an anaerobic bacteria isolation/enrichment system in which a This system therefore can minimize the time required for the variety of food sources are arranged, and the system is deoxy discovery, research, and development process, and thereby genated and inoculated with a mixture of bacteria obtained increase the efficiency of an energy plant based on anaerobic from different sources. The bacteria are allowed to grow in the fermentation. isolation/enrichment system, and those that thrive on a par 40 The Knowledge Management System software includes ticular biomass are removed and evaluated for specific Sub user friendly options that aid the user in navigating the sys strate utilization and end products generated. These bacteria tem. Such options include creating new identifications, open are placed in a separate cultivation system for continued ing identifications, saving an identification, opening a ses growth using the optimal biomass indicated from the isola Sion, saving a session, importing data, finding an organism, tion/enrichment system. The cultivation systems allow for the 45 finding an indicator test, finding a reference, finding a photo, isolation of bacteria on particular Substrates under various comparing organisms, showing related organisms, finding growth conditions to optimize hydrogen production. Various common traits, adding organisms, adding traits, adding the growth conditions may include variation in temperature, pH, Source, user login, user logout, checkout of organisms, check fermentation products, exposure to various natural gases or ing of organisms, testing the server, reports on identifiability, compounds, drug efficacy, compound toxicity or Survivabil 50 column Sorting, preferred tests, sorting organisms, starting ity. The cultivation system aids in determining the primary over, and switch to relatedness mod. The Knowledge Man food source for particular bacteria strains and the optimal agement System houses data that can be quickly accessed and growing conditions. The isolated bacteria are then harvested manipulated into a user friendly form Such as a graph or from the cultivation system and stored for later use as inocu spreadsheet. In particular, the Knowledge Management Sys lum for hydrogen production in anaerobic digesters. Of 55 tem can narrow down which bacteria uses particular nutrients importance to the Successful operation of the anaerobic for producing a specific amount of hydrogen. digester is the selection of nutrients for a particular bacteria FIG.3 is an example of a screen shot from the Knowledge species, and the preparation, detoxification, sterilization and/ Management System that displays various traits that were or pre-digestion of nutrients prior to introduction into the determined by indicator tests listed along the top row of digester. The selection of nutrients is known from the enrich 60 columns and the various organisms listed in the first left hand ment/incubation process, in which the bacteria species was column. FIG. 4 is an example of a screen shot from the originally isolated. Finally, the information obtained for a Knowledge Management System that displays a Preferred particular species of bacteria, Such as, by way of example Test which groups only a particular subset of traits. FIG. 5 is only, optimal biomass, optimal growth pH, optimal growth an example of a screen shot from the Knowledge Manage temperature, and fermentation products, are compiled into a 65 ment System that displays appropriate information for the database. A Knowledge Management System, which bacteria Bilophila wadsworthia. Such information may includes the database of information, can be later utilized to include habitat, growth rate, identification, where it is nor US 8,501,463 B2 37 38 mally associated or found, information from in vitro, in Vivo, Also provided are systems that automate the optimal bio and molecular studies, subgroups or strains, growth condi mass detection process for particular bacterial genera and tions, diameter, form, elevation, margin, color, density, gen species using a computer programmed for identifying posi eral information, colony morphology, cellular morphology, tive and negative results based upon the methods provided references, photos of gram stain, photos of blood plates, and herein. The methods herein can be implemented, for example, photos of BBE/LKV plates. by use of the following computer systems and using the The Knowledge Management System houses appropriate following calculations, systems and methods. An exemplary information for each bacterial species. Appropriate informa automated testing system includes various identification tion may be collected from Scientific literature and may con workstation that includes analytical instruments, such as a 10 CCD camera or a microscope or other instruments for cap sist of information regarding bacteria growth on various Sub turing the image of a bacterial sample or instruments that can strates, bacteria sensitivity to a condition and/or bacteria measure bacterial physiological changes, and a computer for production of metabolites. Such traits may also be assayed for data analysis which is capable of communicating with the by indicator tests. By way of example, Such traits may include analytical instrument. growth, reactions or production from: acetic acid major meta 15 In an exemplary embodiment, the computer is a desktop bolic product, acetic acid minor metabolic product, ADH, computer system, Such as a computer that operates under ALP, alpha-fucosidase, alpha-galactosidase, alpha-glucosi control of the “Microsoft Windows: operation system of dase, arabinose, ArgA, bacillus, beta-galactosidase, beta-glu Microsoft Corporation or the “Macintosh' operating system cosidase, beta-glucuronidase, beta-NAG, beta-xylosidase, of Apple Computer, Inc., that communicates with the instru box car shape, butyric acid major metabolic product, butyric ment using a known communication standard Such as a USB, acid minor metabolic product, CAMP, caproic acid major parallel or serial interface. metabolic product, catalase, cellobiose, chartreuse fluores For example, systems for analysis of bacterial samples are cence, chymotrypsin, CO. growth, coccus, desulfoviridin, provided. The system includes a processing station that per double Zone beta-hemolysis, esculin hydrolysis, F/F forms the various indicator tests. For example, the system required, fructose, gelatin hydrolysis, glucose, glycogen, 25 may include a processing station that performs the nitrate gram reaction, growth in bile, His A, I-arabinose, indole, indicator test and a catalase indicator test. The nitrate indica isobutyric acid major metabolic product, isobutyric acid tor test determines an organism’s ability to reduce nitrate to minor metabolic product, isocapronic acid major metabolic nitrite. A nitrate disk is placed on the plate at the time of product, isocapronic acid minor metabolic product, isovaleric inoculation. After 24-48 hours, reagents A (Sulfanilic acid acid major metabolic product, isovaleric acid minor meta 30 dissolved in glacial acetic acid) and B (1.6-Cleve’s acid dis bolic product, lactate converted to propionate, lactic acid Solved in glacial acetic acid) are added and a positive test is major metabolic product, lactic acid minor metabolic prod indicated by a bright magenta color. If no color is seen within uct, lactose, leithinase, LeuA, lipase, maltose, mannitol, man a few minutes, Zinc dust is added to make Sure the nitrate has nose, melezitose, melibiose, milk clot formed, milk digested, not reduced beyond nitrite. Zinc dust, too, reduces nitrate to motile, N-Acetyl-beta-gulcosaminidase, nitrate, ONPG 35 nitrite. Magenta coloration at this point is interpreted as a (Beta-galactosidase), oxygen tolerance, Phea, phenylaceric negative result because it indicates that the nitrate was not acid minor metabolic product, pigment, pitting of agar, ProA, previously reduced by the organism. No color change after propionic acid major metabolic product, Pyra, raffinose, red the addition of Zinc is a positive result, because the organism fluorescence, reverse CAMP test, rhamnose, ribose, salicin, reduced the nitrate beyond nitrite. The catalase indicator test, sensitive to colistin, sensitive to kanamycin, sensitive to SPS, 40 an example of which is shown in FIG. 6, is when the presence sensitive to Vancomycin, Sorbitol, spore former, starch of catalase is determined by the addition of 15 percent hydro hydrolysis, strictly anaerobic, Subterminal spore location, gen peroxide to a loop of cells placed on a glass slide. The Succinic acid major metabolic product, Succinic acid minor formation of gas bubbles indicates a positive reaction. Thered metabolic product, Sucrose, terminal spore location, threo blood cells in brucella and other blood agar plates contain nine converted to propionate, trehalose, trypsin, Tyra, urease, 45 catalase and scraping the media may give a false-positive Valeric acid major metabolic product, Valeric acid minor reaction. metabolic product, Xylan, and Xylose. Bacterial response may The system may also include a spectral analysis system include positive, negative, or no response. Bacterial response that analyzes the bacterial color changes from a CCD camera may also be measured by Smell, color, growth, non-growth, which processes results from a nitrate indicator test or a gas death, symbiosis and non-symbiosis. 50 formation analysis system that analyzes formation of gas by The Knowledge Management System software also bacteria from a CCD camera which processes results from a includes statistical modeling programs which aid in identify catalase indicator test; and a complete data analysis system, ing an optimal biomass for anaerobic bacterial hydrogen pro Such as a computer programmed to identify which biomass is duction. Such methods include Hidden Markov Models, phy optimal for a particular bacteria to produce hydrogen anaero logenetic inferences which include clustering methods such 55 bically. The system can also include a control system that as (UPGMA) Unweighted Pair Group Methods using Arith determines when processing at each station is complete and, metic averages, distance and parsimony methods, maximum in response, moves the sample to the next test station, and likelihood estimation and Chomsky hierarchy. For example, continuously processes samples one after another until the given a set of growth conditions by which indicator tests have control system receives a stop instruction. been performed to identify traits of bacteria, use of probabi 60 FIG. 7 is an example of a block diagram of a system that listic modeling may infer whether using different growth performs sample processing and biological workstation 702 conditions deter or optimize production of hydrogen. Also and an analysis computer 740. At the biological workstation, use of phylogenetic trees may aid to infer relationships one or more samples 706 are received and prepared for analy between bacterial species and assist in determining for sis at a sample processing station 708, where the above example the likelihood of whether certain unperformed indi 65 described indicator tests can take place. The samples are then cator tests will result in either similar or dissimilar results moved to an analysis station 712, where analysis of the bac compared to that of a related bacteria species. teria Such as spectral or gas formation analysis can be pro US 8,501,463 B2 39 40 cessed and recorded. The samples are moved from the pro computers 708, 712, and 704. Those skilled in the art will cessing station 708 to the analysis station 712 by either a appreciate that the stations and computers illustrated in FIG. computer-controlled robotic device 710 or by manual pro 7 can all have a similar computer construction, or can have cessing, not shown. alternative constructions consistent with the capabilities and The robotic device can include subsystems that ensure respective functions described herein. The FIG. 8 construc movement between all of the stations that preserve the integ tion is especially suited for the data analysis computer 704 rity of the samples 706 and ensure valid test results. The illustrated in FIG. 7. Subsystems can include, for example, a mechanical lifting FIG. 8 displays an exemplary computer 800 such as might device or arm that can pick up a sample from the sample comprise a computer that controls the operation of any of the processing station 708 and then move to and deposit the 10 stations and analysis computers 708, 712, and 704. Each processed sample for analysis at the analysis station 712. computer 800 operates under control of a central processor The analysis station 712 produces data that identifies and unit (CPU) 802, such as a “Pentium microprocessor and quantifies the positive and negative results of the indicator associated integrated circuit chips, available from Intel Cor tests of the sample 706 being measured. Those skilled in the poration of Santa Clara, Calif., USA. A computer user can art will be familiar with the biological monitoring systems, 15 input commands and data from a keyboard and computer Such as microscopes, that can be used to identify positive or mouse 804, and can view inputs and computer outputs at a negative bacterial reactions or a CCD camera, which can be display 806. The display is typically a video monitor or flat used to image the results. The data is provided from the panel display. The computer 800 also includes a direct access analysis station 712 to the analysis computer 704, either by storage device (DASD) 808, such as a hard disk drive. The manual entry of measurements results into the analysis com computer includes a memory 810 that typically comprises puter or by communication between the analysis station and Volatile semiconductor random access memory (RAM). Each the analysis computer. For example, the analysis station 712 computer includes a program product reader 812 that accepts and the analysis computer 704 can be interconnected over a a program product storage device 814, from which the pro network 714 such that the data produced by the analysis gram product reader can read data (and to which it can option station can be obtained by the analysis computer. The net 25 ally write data). The program product reader can comprise, work 714 can comprise a local area network (LAN), or a for example, a disk drive, and the program product storage wireless communication channel, or any other communica device can comprise removable storage media Such as a mag tions channel that is suitable for computer-to-computer data netic floppy disk, a CD-R disc, a CD-RW disc, or DVD disc. exchange. Each computer 800 can communicate with the other FIG.7 The processing function of the analysis computer 704 and 30 systems over a computer network 820 (Such as, for example, the control function of the biology workstation 702 can be the local network 714 or the Internet or an intranet) through a incorporated into a single computer device, if desired. In that network interface 818 that enables communication over a configuration, for example, a single general purpose com connection 822 between the network 820 and the computer. puter can be used to control the robotic device, not shown, and The network interface 818 typically comprises, for example, to perform the data processing of the data analysis computer 35 a Network Interface Card (NIC) that permits communication 704. Similarly, the operations of the analysis station 712, the over a variety of networks, along with associated network sample processing operations of the sample processing sta access Subsystems, such as a modem. tion 708, or the other additional stations, not shown, can be The CPU 802 operates under control of programming performed under the control of a single computer. instructions that are temporarily stored in the memory 810 of Thus, the processing and analysis functions of the stations 40 the computer 800. When the programming instructions are and computer 708, 712, and 704 can be performed by a executed, the computer performs its functions. Thus, the pro variety of computing devices, if the computing devices have gramming instructions implement the functionality of the a Suitable interface to any appropriate Subsystems (such as a respective workstation or processor. The programming mechanical arm of the robotic device, not shown) and have instructions can be received from the DASD 808, through the Suitable processing power to control the systems and perform 45 program product storage device 814, or through the network the data processing. connection 822. The program product storage drive 812 can The data analysis computer 704 can be part of the analyti receive a program product 814, read programming instruc cal instrument or another system component or it can be at a tions recorded thereon, and transfer the programming instruc remote location. The computer system can communicate with tions into the memory 810 for execution by the CPU 802. As the instrument, for example, through a wide area network or 50 notes above, the program product storage device can com local area communication network or other Suitable commu prise any one of multiple removable media having recorded nication network. The system with the computer is pro computer-readable instructions, including magnetic floppy grammed to automatically carry out steps of the methods disks and CD-ROM storage discs. Other suitable program herein and the requisite calculations. For embodiments that product storage devices can include magnetic tape and semi use color changing patterns (for a reference) based on the 55 conductor memory chips. In this way, the processing instruc indicator test used in the processing station, a user enters the tions necessary for operation in accordance with the methods spectral identification for the bacteria sample. These data can and disclosure herein can be embodied on a program product. be directly entered by the user from a keyboard of from other Alternatively, the program instructions can be received into computers or computer systems linked by network connec the operating memory 810 over the network 820. In the net tion, or on removable storage medium such as a data CD, 60 work method, the computer 800 receives data including pro minidisk (MD), DVD, floppy disk, jump disk or other suitable gram instructions into the memory 810 through the network storage medium. Next, the user initiates execution Software interface 818 after network communication has been estab that operates the system in which the spectral identification is lished over the network connection 822 by well-known meth constructed for the bacteria imaged. ods that will be understood by those skilled in the art without FIG. 8 is a block diagram of a computer in the system 800 65 further explanation. The program instructions are then of FIG. 7, illustrating the hardware components included in a executed by the CPU 802 thereby comprising a computer computer that can provide the functionality of the stations and process. US 8,501,463 B2 41 42 It should be understood that all the stations and computers the tube is filled with water from the lake or river. The tube is of the system 700 illustrated in FIG. 7 can have a construction then capped (not sealed), allowing air flow to the top of the similar to that shown in FIG. 8, so that details described with water column, and placed near a window with Supplementary respect to the FIG. 8 computer 800 will be understood to strip lights. All the organisms are present initially in low apply to all computers of the system 700. It should be appre numbers, but after incubation for 2 to 3 weeks the different ciated that any of the communicating stations and computers types of microorganisms proliferate and occupy distinct can have an alternative construction, so long as they can Zones where the environmental conditions favor their specific communicate with the other communicating stations and activities. The large amount of cellulose added initially pro computers illustrated in FIG. 7 and can support the function motes rapid microbial growth which Soon depletes the oxy ality described herein. For example, ifa workstation is unable 10 gen in the sediment and in the water column. Only the very to receive program instructions from a program product top of the column remains aerated because oxygen diffuses device, then it is not necessary for that workstation to include very slowly through water from the capped tube opening. The that capability, and that workstation will not have the ele only organisms that can grow in the anaerobic conditions are ments depicted in FIG. 8 that are associated with that capa those that ferment organic matter and those that perform bility. 15 anaerobic respiration. For example, Clostridium species are Bacteria Selection, Modification and Maximization strictly anaerobic and start to grow when the oxygen is The various species of bacteria and various forms of bio depleted in the sediment. Cellulose-degrading Clostridium mass described herein may be used in all aspects of anaerobic species degrade the cellulose to glucose and then ferment the production of hydrogen and other chemical products pre glucose producing a range of simple organic compounds (e.g. sented herein. For example, the various species of bacteria acetic acid), carbon dioxide, and hydrogen as the fermenta may be used in the anaerobic fermentation apparatuses tion end products. Sulphur-reducing bacteria, such as Des described herein, using any form of biomass presented as a ulfo vibrio, utilize these fermentation products by anaerobic food source. respiration, using either Sulphate or other partly oxidised In contrast to known anaerobic methods, which generate forms of Sulphur (e.g. thiosulphate) as the terminal electron hydrogen from mixed bacteria cultures obtained from soil or 25 acceptor, generating large amounts of HS by this process. other samples, the methods described herein produces hydro The HS can react with any iron in the sediment, producing gen using specific isolated anaerobic bacteria Strains, chosen blackferrous sulphide, and some of the HS diffuses upwards for the ability of particular strains to utilize specific biomass/ into the water column, where it is utilized by other organisms. nutrients as food sources. The approach is to isolate and The diffusion of HS from the sediment into the water column culture specific bacterial strains which are high hydrogen 30 enables anaerobic photosynthetic bacteria to grow, resulting producers for particular biomass. By way of example only, a in a Zone of green Sulphur bacteria immediately above the particular bacteria strain may yield large quantities of hydro sediment, followed by a Zone of purple sulphur bacteria. The gen from cellulosic material, whereas a different isolated green and purple Sulphur bacteria gain energy from light strain may yield large quantities of hydrogen from sewage. reactions and produce their cellular materials from CO in Thus, selection and isolation of specific bacterial strains with 35 much the same way as plants do. However, HS is used as the enhanced fermentation properties for specific biomass allows reductant instead of water and rather than generate oxygen for optimization of hydrogen production as the type of Sub during elemental Sulphur is formed. The strate available varies. purple Sulphur bacteria (e.g. Thiocapsa) typically have large The Winogradsky column can be used to demonstrate the cells and they deposit Sulphur granules inside the cells, metabolic diversity of , and how the activities of 40 whereas green Sulphur bacteria have Smaller cells and typi different microorganisms enable other organisms to grow in cally deposit sulphur externally. Note that the sulphur (or an interdependent or symbiotic manner. In addition, the col sulphate formed from it) produced by these photosynthetic umn demonstrates how elements, such as Sulphur, nitrogen, bacteria returns to the sediment where it can be recycled by carbon and other elements are cycled in natural environments. Desulfovibrio as part of the sulphur cycle in natural waters. These columns are complete, self-contained recycling sys 45 Most of the water column above the photosynthetic bacteria is tems, driven only by energy from light. All life on earth can be coloured bright red by a large population of purple non categorized in terms of the organisms carbon and energy Sulphur bacteria. These include species of Rhodopseudomo Source. Energy can be obtained from light reactions (pho nas, Rhodospirillum and Rhodomicrobium. These bacteria totrophs) or from chemical oxidations of organic or inorganic are photoheterotrophs, as they grow in anaerobic conditions, Substances (chemotrophs); the carbon for cellular synthesis 50 gaining their energy from light reactions but using Volatile can be obtained from CO (autotrophs) or from preformed organic acids (e.g. acetic acid) as their carbon Source for organic compounds (heterotrophs). Combining these catego cellular synthesis. The Volatile organic acids that they use are ries, we get the four basic life strategies: photoautotrophs the fermentation products of other anaerobic bacteria (e.g. (e.g. plants), chemoheterotrophs (e.g. animals, fungi), photo Clostridium species), but the purple non-Sulphur bacteria are heterotrophs and chemoautotrophs. Only in the bacteria do 55 intolerant of high HS concentrations, so they occur above the we find all four basic life strategies. The prokaryotic bacteria Zone where the green and purple Sulphur bacteria are found. and exhibit an astonishing metabolic diversity, which Nearer the top of the water column the purple non-sulphur far exceeds that of animals, plants, fungi and other higher bacteria disappear due to the oxygenated water. A variety of organisms, and Winogradsky columns also demonstrate how microorganisms can grow in the oxygenated Zone at the top of microorganisms occupy highly specific microsites according 60 the water column; in particular Sulphur-oxidising bacteria, to their environmental tolerances and their carbon and energy and . Any HS that diffuses into the aerobic requirements. Zone can be oxidized to Sulphate by Sulphur-oxidising bacte A typical Winogradsky column is usually a glass or plastic ria. These bacteria are chemosynthetic or chemoautotrophic tube, about 30 cm tall and 5 cm diameter. Mud from the organisms, since they gain energy from oxidation of H2S, and bottom of a lake or river, Supplemented with cellulose (e.g. 65 they synthesize their own organic matter from CO. Similar newspaper), Sodium Sulphate and calcium carbonate, is then types of organism occur in Soils, gaining energy from the added to the lower one-third of the tube, and the remainder of oxidation of ammonium to nitrate, which then leaches from US 8,501,463 B2 43 44 the Soil and can accumulate in water Supplies. The photosyn bacteria. The bacteria in the isolation/enrichment system may thetic cyanobacteria also grow in the aerobic Zones, and are thrive on a particular food source as they continue to grow the only bacteria that have oxygen-evolving photosynthesis under anaerobic conditions. The bacteria isolated in this sys like that of plants. Once the cyanobacteria start to grow they tem may include, by way of example only, species such as oxygenate the water, which forces the anaerobic bacteria 5 Abiotrophia defectiva, Acidaminococcus fermentans, Actino toward the bottom, thereby allowing the cyanobacteria and baculum Schalii, Actinomyces europaeus, Actinomyces other aerobes to occupy a larger portion of the Winogradsky finkei, Actinomyces georgiae, Actinomyces gerensceriae, column. Actinomyces graevenitzii, Actinomyces israelii, Actinomyces The Winogradsky column is essentially a miniature eco meyeri, Actinomyces naeslundi, Actinomyces neuii species system, wherein the metabolic diversity of bacteria allows for 10 anitratus, Actinomyces neuii species neuii, Actinomyces Ond interdependent relationships to exist throughout the column. Ontolyticus, Actinomyces radicidentis, Actinomyces radin The fermentation products of one species are consumed by gae, Actinomyces turicensis, Actinomyces urogenitalis, Acti another, thereby cycling various elements, such as Sulphur, nomyces viscosis, Anaerorhabdus fircosa, Arcanobacterium nitrogen, and carbon, and maintaining the life cycle. Without bernardiae, Arcanobacterium hemolyticum, Arcanobacte manipulating the growth of one species over another, the 15 rium pyogenes, Atopobium minutum, Atopobium parvulum, Winogradsky column demonstrates how microorganisms Atopobium rimae, Atopobium vaginae, Bacteroides caccae, occupy highly specific locations according to their environ Bacteroides capillosus, Bacteroides coagulans, Bacteroides mental tolerances and their carbon and energy requirements. distasonis, Bacteroides eggerthii, Bacteroides forsythus, The initial food source is common throughout the initial , Bacteroides merdae, Bacteroides ova sediment, and the variety of species required for the develop tus, Bacteroides putredenis, Bacteroides pyogenes, Bacteroi ment of the “ecosystem” is initially present. des splanchnicus, Bacteroides Stercoris, Bacteroides tectus, The apparatus, methods and processes for bacterial isola Bacteroides thetaiotaomicron, Bacteroides uniformis, tion and enrichment described herein include anaerobic bac Bacteroides ureolyticus, Bacteroides vulgatus, Bifidobacte teria isolation/enrichment systems designed to be ultimately rium adolescentis, Bifidobacterium angulatum, Bifidobacte anaerobic and to optimize growth of bacteria species depend 25 rium bifidum, Bifidobacterium breve, Bifidobacterium ing on the food source provided. In this manner bacteria with catenulatum, Bifidobacterium denticolens, Bifidobacterium optimal fermentation properties are selected out for maxi dentium, Bifidobacterium infantis, Bifidobacterium inopina mum hydrogen production. In addition, the apparatus, meth turn, Bifidobacterium longum, Bifidobacterium ods and processes for bacterial isolation and enrichment pseudocatenulatum, Bilophila wadsworthia, Bulleidia ext described herein may also allow for the growth and isolation 30 cructa, Campylobacter gracilis, Campylobacter hominis, of anaerobic bacteria which utilize fermentation products of Campylobacter rectus, Campylobacter Showae, Capnocy other species as food sources, thereby increasing hydrogen tophaga granulosa, Capnocytophaga haemolytica, production. These features illustrate the difference between Clostridium argentinense, Clostridium baratii, Clostridium the apparatus, methods and processes for bacterial isolation bifermentans, Clostridium botulinum types A B F. and enrichment described herein, and a Winogradsky col 35 Clostridium botulinum types B E F. Clostridium botulinum l, types C D. Clostridium butyricum, Clostridium cadaveris, The isolation/enrichment system can take any shape, Clostridium carnis, Clostridium clostridioforme, including, but not limited to, circular, cylindrical, spherical, Clostridium difficle, Clostridium glycolicurn, Clostridium square, or rectangular form, and may have a Volume ranging hastiforme, Clostridium histolyticum, Clostridium indolis, from 100 cm to 50,000 cm. One embodiment of the anaero 40 Clostridium innocuum, Clostridium limosum, Clostridium bic bacteria isolation/enrichment system described herein is malenominaturn, Clostridium novyi type A, Clostridium shown in FIG. 9. Here, the isolation/enrichment system paraputrificum, Clostridium perfingens, Clostridium pili resembles an ant farm or an aquarium, wherein two parallel forme, Clostridium putrificurn, Clostridium rarnosurn, glass plates with a spacer are sealed on three sides to create an Clostridium septicum, Clostridium sordellii, Clostridium open trough. The top of the trough may eventually be closed 45 sphenoides, Clostridium sporogenes, Clostridium subtermi and sealed to ensure anaerobic conditions remain within the male, Clostridium symbiosum, Clostridium tertium, sealed chamber, although, venting systems may be used to Clostridium tetani, Collinsella aerofaciens, Corynebacte prevent pressure build up due to gas evolution during fermen rium matriuchotii, Cryptobacterium curturn, Desulfomonas tation. pirgra, Dialister pneumosintes, Eggerthella lenta, Eikenella Preparation of the anaerobe bacteria isolation/enrichment 50 corrodens, Eubacteria sulci, Eubacteria tenue, Eubacterium system involves obtaining various potential food sources, combesii, Eubacterium contortum, Eubacterium cylindroi Such as, by way of example only, vegetable materials, garlic des, Eubacterium infirmum, Eubacterium limosum, Eubacte material, shredded hay, grass clippings, shredded newspaper, rium minutum, Eubacterium moniliforme, Eubacterium sawdust, corn starch, oatmeal, and arranging these food modatum, Eubacterium rectale, Eubacterium saburreum, Sources at various locations throughout the isolation/enrich 55 Eubacterium saphenurn, Eubacterium WALI, Eubacterium ment. In addition, any of the biomass known in the art may yuri, Filifactor alocis, Filifactor villosus, Finegoldia magna, also be used. The anaerobe bacteria isolation/enrichment sys Fusobacterium gonidiaformans, Fusobacterium mortiferum, tem is filled with deoxygenated water, inoculated with a mix Fusobacterium naviforme, Fusobacterium necrophorum, ture of bacteria species, and the top is then sealed. The head Fusobacterium nucleatum, Fusobacterium russii, Fusobac space above the deoxygenated water is purged to remove any 60 terium ulcerans, Fusobacterium varium, Gemella morbil oxygen present. The mixture of bacteria species used in the lorum, Granulicatella adiacens, Granulicatella elegans, anaerobe bacteria isolation/enrichment system may be Holdemaniafiliformis, Lactobacillus acidophilus, Lactoba obtained from cattle rumen, a sample of soil, sludge, an cillus brevis, Lactobacillus canteforme, Lactobacillus casei, anaerobic bacteria culture, an aerobic bacteria culture, Lactobacillus crispatus, Lactobacillus fermentum, Lactoba anaerobic sediments from fresh or brackish waters, sewage, 65 cillus gasseri, Lactobacillus iners, Lactobacillus jensenii, animal feces, hydrothermal soils and pools, deep water Lactobacillus leichmannii, Lactobacillus Oris, Lactobacillus hydrothermal vents or other potential source of anaerobic paracasei subspecies paracasei, Lactobacillus paraplan US 8,501,463 B2 45 46 tarum, Lactobacillus plantarum, Lactobacillus rhamnosus, may be used in the anaerobic fermentation apparatuses Lactobacillus salivarius, Lactobacillus uli, Lactobacillus described herein, using any form of biomass presented as a vaginalis, Leptotrichia buccalis, Leptotrichia sanguinegens, food source. Megasphaera elsdenii, Micromonas micros, Mitsuokella The methods described herein allows for the preparation of multiacida, Mobiluncus curtisii species curtisii, Mobiluncus 5 the biomass, which includes, but is not limited to, harvesting curtisii species holmesii, Mogibacterium pumilum, Mogibac or protecting the biomass quickly before spoilage, steriliza terium timidum, Mogibacterium vescum, Peptococcus niger; tion (i.e. pasteurizing and/or acidifying), neutralization (i.e. Peptostreptococcus anaerobius, Peptostreptococcus asac ammonium hydroxide), removal of oxygen (deoxygenation), charolyticus, Peptosfeptococcus harei, Peptostreptococcus and concentration adjustments, detoxification, and/or pre hydrogenalis, Peptostreptococcus indolyticus, Peptostrepto 10 digestion, prior to fermentation by isolated anaerobic bacte ria. coccus ivorii, Peptostreptococcus lacrimalis, Peptostrepto Harvesting or protecting the biomass before spoilage or a coccus lactolyticus, Peptostreptococcus Octavius, Pep state whereby the biomass is useless is a pre-condition to to streptococcus previotii, Peptostreptococcus previotii/ optimize preparation of the biomass for bacterial consump tetradius, Peptostreptococcus tetradius, Peptostreptococcus 15 tion. This is to ensure optimal hydrogen production from a trisimilis, Peptostreptococcus vaginallis, Porphyromonas biomass. An example of harvesting or protecting biomass assacharolytica, Porphyromonas Cangingivalis, Porphy before spoilage is when biomass is harvested and used within romonas canoris, Porphyromonas can sulci, Porphyromonas a 24 hour period. An another example may include refrigera cantoniae, Porphyromonas circumdentaria, Porphyromonas tion of the biomass to prevent spoilage. crevioricanis, Porphyromonas endodontalis, Porphyromonas Pre-digestion includes the pre-treatment of biomass to get gingivalis, Porphyromonas gingivicanis, Porphyromonas it into a condition for the bacteria to feed upon. By way of guilae, Porphyromonas levi, Porphyromonas macacae, Pre example only, pre-digestion may include addition of other votella albensis, PrevOtella bivia, Prevotella brevis, Prevo insects, animals, enzymes, and/or solutions to modify the tella bryantii, PrevOtella buccae, Prevotella buccalis, Prevo biomass prior to introducing it to the bacteria. For example, tella corporis, Prevotella dentalis, Prevotella denticola, 25 the breakdown of cellulose into glucose may be a pre-treat Prevotella disiens, Prevotella enoeca, Prevotella hep ment/pre-digestion. Another example is the pre-treatment of arinolytica, Prevotella intermedia, Prevotella loescheii, Pre manure into an anaerobic substrate feedstock for bacteria votella melanogenica, PrevOtella nigrescens, PrevOtella Ora through use of an organism that breakdown bile/urea within lis, Prevotella Oris, Prevotella Oulorum, Prevotella pallens, the manure. Prevotella ruminicola, Prevotella tannerae, PrevOtella ver 30 The steps of Sterilization, deoxygenation, concentration oralis, PrevOtella zoogleoformans, Propionibacterium acnes, adjustments, detoxification, and/or pre-digestion may occur Propionibacterium avidum, Propionibacterium granulosum, in a variety of orders such as deoxygenation of biomass first Propionibacterium proionicus, Pseudoramibacter alactolyti followed by concentration adjustments, sterilization, detoxi cus, Rothia dentocariosa, Ruminococcus hansenii, Rumino fication, and/or pre-digestion second or concentration adjust coccus productus, Slakia exigua, Slakia heliontrinireducens, 35 ments, sterilization, detoxification, and/or pre-digestion first Staphylococcus saccharolytics, Streptococcus anginosus, followed by deoxygenation of biomass second or all steps Streptococcus contellatus, Streptococcus pleomorphus, simultaneously or other combinations thereof. An example of Streptococcus intermedius, Sutterella wadsworthensis, Tis a process flow diagram is shown in FIG. 10: double arrows sierella praeacuta, and Veillonella species, or genera contain display the order of deoxygenation of biomass before con ing Such species. 40 centration adjustments, sterilization, detoxification, and/or The anaerobic bacteria isolation/enrichment system may pre-digestion and the singe arrow displays the order of con be large, allowing for increased available space for different centration adjustments, sterilization, detoxification, and/or bacteria species which may utilize the same food source, and pre-digestion before deoxygenation of biomass. therefore minimizes competition between species. Thus, it After the concentration step, it may be necessary to adjust may be possible to isolate various high Volume hydrogen 45 the concentration of the biomass depending on what is used. producing strains grown on a common food source and then For example, fats or meats which have high protein content further differentiate them by subdividing the food source into would be diluted to an approximate 5% Sugar concentration different categories. For instance, by way of example only, equivalent before use. In addition, managing the proper rate at the isolation of high hydrogen producers grown on agricul which the biomass is fed to the bacteria needs to be accounted. tural waste, and then Subdividing the agriculture waste into 50 The dynamic feed rate should be balanced with the exponen specific crops, such as potatos, tomatos, garlic, cranberries, tial growth rate of the bacteria as well as the rate of utilization. and the like. Once it is evident that a bacterial colony is Oilier factors that should be monitored may include hydrogen specific for a particular biomass type, the bacteria are production, pH levels and acid production. removed from the isolation/enrichment system, transferred Examples of biomass available for hydrogen production for more controlled culture, grown, and the isolated bacteria 55 via “dark fermentation' include, but are not limited to, mash species is harvested and stored for later use to inoculate and of starched materials derived from corn, wheat, oats, and ferment a specific biomass in the digester. Removal of bacte other grain materials, beet molasses, blackstrap molasses, ria from the isolation/enrichment system may be done at any citrus molasses, invert Sugar, Sucrose, fructose, glucose, time period. By way of example only, bacteria may be wood Sugar, Xylose, animal or fish tissue or parts, plant parts, removed after 1-7 days, or earlier, or after 1-20 weeks, or 60 fruits, Sorghum, cheese whey, vegetables, plant processing longer. Bacteria may be removed from the system by any waste, animal manure or urine, Solids isolated from fermen means, for example, by use of a needle, Syringe or vacuum. tation cultures, bovine manure, poultry manure, equine Nutrient Selection and Preparation manure, porcine manure, bovine urine, poultry urine, equine The various species of bacteria and various forms of bio urine, porcine urine, wood shavings or chips, slops, shredded mass described herein may be used in all aspects of anaerobic 65 paper, cotton burrs, grain, chaff, seed shells, hay, alfalfa, production of hydrogen and other, chemical products pre grass, leaves, sea shells, seed pods, corn shucks, weeds, sented herein. For example, the various species of bacteria aquatic plants, algae and fungus and combinations thereof. US 8,501,463 B2 47 48 FIG. 11 illustrates an example of the preparation of biom hydrogen, carbon dioxide, and a variety of organic com ass for hydrogen production that may include but is not lim pounds. For instance, during fermentation of Sugars, hydro ited to the removal of non-biomass material, an optional gen, carbon dioxide and volatile organic acids, such as filtering or sifting of biomass material step, an optional pre butyrate and acetate, are produced, whereas, during the fer conditioning of biomass material step or combinations mentation of amino acids and fatty acids, a variety of foul thereof. Biomass material or feed stock is selectively chosen Smelling compounds are formed. Clostridia also produce for optimal hydrogen production depending on the specific extracellular enzymes to degrade large biological molecules anaerobic bacteria used. Grit, Such as dirt, sand, Soil, stones, in the environment into fermentable components. Hence, pebbles, rocks, feathers, hair and other such materials, may be Clostridia play an important role in nature in biodegradation removed prior to addition of the feed stock to the anaerobic 10 and many other nutrient cycles. digester; however, grit can be removed at any point along the Members of genus Clostridia are ubiquitous in nature and process. Equipment such as clarifiers, settling tanks, mul may be found in soil and in the gastrointestinal tracts of tiphase tanks, and/or filters can be used to remove the grit. animals and humans. Clostridia are Gram-positive, spore Deoxygenation of the biomass prior to introduction to a forming, mostly motile, rod shaped anaerobic bacteria. A digester, either as batch or continuous processes, ensures 15 Gram-stain is a good method for differentiating Clostridium viability of the anaerobic bacteria which may die on exposure from other genera, as the cell incorporates the dye while the to oxygen. Oxygen exposure also inactivates the enzyme spore remains unstained. Most Clostridium species are complex required for hydrogen production. Also, for continu strictly anaerobic, as their vegetative cells are killed by expo ous fermentation systems it rminimizes oxygen introduction Sure to oxygen. However, they can Survive as spores in aero and limits the need to continuously purge the digester. FIG. 12 bic conditions, and therefore are able to wait for anaerobic shows an example of a process flow diagram of the deoxy conditions required for active anaerobic bacteria growth. genation of biomass material. Deoxygenation of biomass Clostridium butyricum, Clostridium acetobutylicum, material can include but are not limited to a single deoxygen Clostridium pasteurianum, and Clostridium barati produce ation step or optional multiple deoxygenation steps of biom hydrogen by anaerobic fermentation of glucose, with ass material. Deoxygenation of biomass material can include 25 Clostridium butyricum being the most effective H producer. but are not limited to using pressurized steam, oxygen free gas These bacteria may be utilized to obtain hydrogen for use as Sparging, autoclaving, a reducing agent (Such as by way of fuel or as a chemical agent in the chemical industry. In addi example only, dithiothreitol, cysteine, thioglycolate, or tion, Clostridium species may be referred to as solventogenic sodium sulfide), or combinations thereof. The use of pressur as they also are able to produce solvents as the result of ized steam also serves to thoroughly sterilize the media and 30 continued fermentation of volatile organic acids (VOA) at the simultaneously convert any complex carbohydrates into a expense of hydrogen production, such as, by way of example form more easily acted upon by the bacteria. only, butyrate to butanol and/or acetate to acetone. The biomass concentration may have an effect on the bac Provided complete conversion occurs dark fermentation of teria metabolic pathways, which may affect the yield of biomass comprising Sucrose produces eight moles of hydro hydrogen. However, concentrating the biomass prior to intro 35 gen per mole of Sucrose, or alternatively four moles of hydro duction to a digester, either as batch or continuous processes gen per mole of glucose. The other products of the dark in combination with efficient removal of fermentation prod fermentation process are carbon dioxide and acetic acid, a ucts, such as hydrogen, carbon dioxide and Volatile organic precursor to acetone. If all of the substrate were converted to acids, and control of the fermentation media pH increases the butyric acid, then two moles of hydrogen may be produced yield of hydrogen per unit volume of the digester. FIG. 13 is 40 per mole of glucose. an example of a process flow diagram of the concentration of FIG.14 is a schematic showing the basic elements involved biomass material. Concentration of biomass material or in the conversion of complex substrates, through intermediate optional steps of concentration of biomass material can compounds, to hydrogen and carbon dioxide. First, complex include, but are not limited to, centrifugation, reverse osmo organic compounds are hydrolyzed by extracellular enzymes sis, filtering, boiling, removal of liquids, lyophilization, other 45 to generate free sugars which are further fermented to hydro methods known in the art of condensing material into a solid gen, carbon dioxide, and Smaller organic products, primarily or semi-solid form or combinations thereof. VOAS, such as, but not limited to, acetate, formate, butyrate Fermentation Products and propionate. Continued fermentation of the VOAS can Some anaerobic fermentation end products are beneficial produce the corresponding solvent, such as acetone from to humans and are the basis of a number of industries, such as 50 acetate. Although acidogenic fermentation produces acetate, alternative energy, brewing industry, and the dairy industry. other VOAS are formed, which can be further metabolized by Examples of microorganisms and fermentation end products acetic acid producing (acetogenic) bacteria to yield hydro include, but are not limited to: Streptococcus, which produce gen, carbon dioxide and acetate. The acetate formed may be lactate, formate, ethanol, and acetate when grown under Subsequently converted into hydrogen and carbon dioxide by anaerobic conditions: Propionibacterium, produce propionic 55 photofermentation, or be isolated and purified for alternative acid acetic acid, and CO by fermentation of glucose: uses Such as industrial chemical applications. Escherichia coli species can anaerobically produce acetic Although hydrogen may be produced by acidogenesis and acid, lactic acid, Succinic acid, ethanol, CO, and H. Entero acetogenesis, the production of VOAS or solvents may be bacter species can anerobically produce formic acid, ethanol, changed or manipulated depending on the composition of the 2,3-butanediol, lactic acid, CO, and H2, and Clostridium 60 fermentation medium and the Clostridia species utilized. For species can anerobically produce butyric acid, butanol, instance, by way of example only, Some cellulose-degrading acetate, acetone, isopropyl alcohol, CO, and H2. Clostridia species degrade cellulose to glucose and then fer To obtain alternative energy sources such as hydrogen from ment the glucose, producing hydrogen, carbon dioxide, and a anaerobic fermentation, it is evident that various bacteria, range of VOAS, Such as formate, acetate, propionate, such as, Escherichia coli, Enterobacter and Clostridia, are 65 butyrate, and Succinate, as fermentation end products. By active hydrogen producers under anaerobic conditions. way of example, Clostridium thermoaceticum, Clostridium Clostridia are able to ferment various types of biomass into thermoautotrophicum, and Clostridium magnum, convert US 8,501,463 B2 49 50 glucose or Sucrose to acetate, whereas; Clostridium acetobu Monitoring H, pH, Pressure and Temperature tylicum, which is a saccharolytic bacterium, produces acetate, While direct and indirect photolysis systems produce pure butyrate, carbon dioxide, hydrogen, and some lactate when H, dark-fermentation processes produce a mixed biogas con grown in glucose-limited conditions at a neutral pH. In com taining primarily H and carbon dioxide (CO), but which parison, other nonpathogenic species grown in glucose-lim may also contain lesser amounts of methane (CH), carbon ited media, such as Clostridium barati, produce large monoxide (CO), and/or hydrogen sulfide (H2S). The gas com amounts of lactate in addition to acetate and butyrate, while position presents technical challenges as fuel Sources, as it Clostridium butyricum produces large amounts of formate in may require Scrubbing and purification before use in some addition to acetate and butyrate. In contrast, Clostridium pro fuel cells. pionicum fermentation of lactate produces propionate, 10 Anaerobic bacteria known to produce hydrogen during acetate, hydrogen and carbon dioxide. Regardless of the Sol dark-fermentation of carbohydrate-rich substrates include, vents or VOAS produced, hydrogen and carbon dioxide may but are not limited to, species of Ruminococci, Archaea, and be obtained from anaerobic fermentation using any of these Clostridia. Carbohydrates are a good Substrate for hydrogen species with a variety of food sources. producing fermentations. Glucose, isomers of hexoses, or The generation of large quantities of hydrogen is one 15 polymers in the form of starch or cellulose, yield different aspect of the methods described herein. The approach used to quantities of H. per mole of glucose, depending on the fer achieve efficient hydrogen production is analogous to some mentation pathway and end-product(s). For instance, the wild ecosystems comprising various anaerobic bacteria spe highest theoretical yield of H is associated with acetate as the cies of the same genus and/or bacteria of different genera: fermentation end-product, whereina theoretical maximum of wherein the fermentation waste (products) of one bacteria 4 mole H per mole of glucose is obtained. Alternatively, colony is used as a food source for a different bacteria colony. when butyrate is the end-product, a theoretical maximum of 2 If waste is allowed to accumulate near the colony, the colony moles H. per mole of glucose is obtained. In practice, how may die or its metabolic pathways alter to metabolize the ever, high H yields may be associated with a mixture of waste. This latter step may allow the colony to survive, how acetate and butyrate fermentation products, and low H yields everit may not flourish. The diffusion of waste away from one 25 are associated with propionate and reduced end-products, colony to a different colony, where it is consumed, creates a Such as alcohols (i.e. methanol, ethanol, propanol, isobutanol, concentration gradient which continually bleeds away waste. butanol, isopentanol, pentanol, acetone, propanol, isopro This symbiotic type relationship allows both colonies to panol, and 1,2-propanediol), solvents and lactic acid. Note flourish, without any changes in metabolic pathways. Alter that potential hydrogen molecules remain contained in Vola natively the different bacteria species or genera may cohabi 30 tile organic acid products including, but not limited to acetate, tate as one colony, with bacteria feeding on the waste of butyrate, and the like. Thus, fermentation products produced neighboring bacteria. by a bacterium depend on the environmental conditions in Mimicking natural symbiotic type ecosystems for the which it grows. Reduced fermentation products like ethanol, anaerobic bacterial production of hydrogen is one aspect of butanol, and lactate, contain hydrogen that has not been lib the methods described herein. The initial bioprocess for 35 erated as gas. For instance, the metabolism of Clostridium anaerobic production of hydrogen from carbohydrate based pasteurianum, which is a high Volume H and Volatile organic biomass is the heterotrophic fermentation (dark fermenta acid (VOA) producer, can be shifted toward solvent produc tion) of carbohydrates to hydrogen, carbon dioxide and Vola tion by having high glucose concentrations. To maximize the tile organic acids (solvent precursors). The accumulation of yield of H, the metabolism of the bacterium may be directed these fermentation products decreases the pH, which can kill 40 away from Solvents (ethanol, acetone and butanol) and the bacteria, or it can initiate changes in the bacteria metabo reduced acids (lactate), and directed more towards volatile lism wherein continued fermentation (solventogenesis) of the organic acids. Volatile organic acids to solvents occurs at the expense of The concentration of hydrogen, carbon dioxide, carbon hydrogen yield. In addition, the accumulation of hydrogen monoxide, and/or hydrogen Sulfide, both in Solution and in and carbon dioxide may affect hydrogen yield and bacteria 45 the head space above the dark fermentation media, increase as viability, respectively. A high hydrogen partial pressure may dark fermentation proceeds. The yield of hydrogen during limit the rate of fermentation or may cause a metabolic shift, dark fermentation is dependent on process conditions such as thereby increasing the conversion of solvent precursors to pH, temperature, buffer capacity, hydraulic retention time solvent, and severely affect the yield of hydrogen produced. (HRT), and gas partial pressure; each of which may affect The presence of a high concentration of carbon dioxide and 50 metabolic balance. To ensure optimal hydrogen production Volatile organic acids may also decrease the pH of the fer occurs during anaerobic dark fermentation, these process mentation media, resulting in bacteria death. In addition, parameters, as well as the concentration offermentation prod solvent formation from reduction of volatile organic acids by ucts, such as Volatile organic acids, hydrogen, carbon dioxide, Solventogenesis may also results in a decrease in pH, and carbon monoxide and hydrogen sulfide, need to be monitored potential bacteria mortality. Similarly, anaerobic fermenta 55 either continuously or at regular intervals. tion of biomass containing Sulfur compounds, such as, by way Temperature is an important parameter in establishing the of example only, proteins and hydrogen Sulfide (H2S) which fermentation rate and determining the stability of the fermen is converted into elemental Sulfur by purple and green pho tation process. Digesters fermentation reactions can be oper tosynthetic bacteria. High concentrations of H2S are toxic to ated at mesophilic (25-40 C), thermophilic (40-65 C), bacteria and result in bacteria death. Therefore, in order to 60 extreme thermophilic (65-80 C), or hyperthermophilic (>80 maintain optimal hydrogen production it is desirable to C) temperatures. Higher gas yields may be obtained using remove the dark fermentation products, such as hydrogen, thermophilic conditions; however this requires energy to heat carbon dioxide, and volatile organic acids (VOAs) in order to the digester. A 5 C to 10 C fluctuation in temperature may avoid these issues. Dark fermentation and removal of the result in an imbalance in fermentation and lead to digester heterotrophic fermentation products can proceed as either a 65 instability for either temperature range, however thermo continuous process, or batch process, or a combination of philic digestion is more sensitive to Such fluctuations. There both continuous and batch processes. fore, to ensure optimal anaerobic fermentation it is necessary US 8,501,463 B2 51 52 to monitor and control the digester temperature. The digester and bacteria. On-line monitoring methods do not require temperature can be monitored using thermal sensors, such as, sterilization, provided no recirculation of the media occurs, by way of example only, thermometers, thermopiles, and plus it allows for sample clean up by filtering of bacteria and thermocouples, which are placed either inside the digester or particulates from the test stream. Examples of on-line pH on the outside surface of the digester. Similarly, infra-red 5 monitoring instrumentation are, but not limited to, glass thermal sensors may be used as non-invasive thermal moni membrane type pH electrodes, solid state type pH electrodes toring methods by measuring the radiative heat from the (e.g. silicon nitride based and metal oxide based), optodes digester. However, using infra-red sensors, or monitoring the (optical type pH electrodes using fluorescence or absorbance inside temperature by measuring the outside Surface tempera measurements of pH sensitive dyes), and flow injection ture, requires calibration to correlate the outside temperature 10 analysis (FIA). The same methodologies used for on-line with the inside temperature of the digester. Regardless of the monitoring may be used for periodic sampling. The pH infor method used to measure the digester temperature, the infor mation obtained may be used for feedback in a control loop to mation can be used in a control loop as feedback to the system the system used to adjust the pH and thereby maintain the used to heat the digester, Such as, by way of example only, hot optimal pH for optimal growth. Examples of methods to plates, or heating mantles. The system used to heat the 15 adjust the pH include, but are not limited to, addition of digester is adjusted manually, or may be automated by com deoxygenated solutions of Sodium hydroxide to increase the puter control, to maintain the optimal temperature needed. pH and addition of deoxygenated solutions of hydrochloric The heat required by these processes can be provided by the acid to decrease the pH. This control of the pH adjustment waste exhaustheat from power generating equipment. A non system may be achieved manually, or may be automated by limiting example of such a process is the use of low pressure computer control. steam exhausted from a back pressure turbine system. The partial pressure of H (pH2), is a parameter which may The fermentation process and products may be affected by be monitored during continuous H. Synthesis as it reflects the the pH of the fermentation media. During dark fermentation concentration of H. Hydrogen synthesis pathways may be at pH values greater than about 5.6 (depending on the bacteria sensitive to H concentrations and which can then be subject strain), the exponential growth of bacteria, Such as, by way of 25 to end-product inhibition. As H concentrations increase. He example only, Clostridium acetobutylicum, generates large synthesis decreases and metabolic pathways shift to produc quantities of fermentation products. Such as Volatile organic tion of more reduced substrates Such as lactate, ethanol, acids (e.g. formic acid, acetic acid, propionic acid, butyric acetone, butanol, or alanine. In addition, temperature also acid, and Valeric acid), carbon dioxide and hydrogen. The affects the pH2 such that, by way of example only, for optimal formation of carbonic acid (from carbon dioxide) and volatile 30 H synthesis, pH,<50 kPa at 60° C.; pH-20 kPa at 70° C.: organic acids causes the pH to decrease, and as the acids pH2 and pH.<2 kPa at 98°C. The increase in temperature is accumulate, particularly in batch cultures, growth becomes aimed at better performance of the hyperthermophiles hydro linear and gradually stops. In addition, the bacteria may die if genases, because the affinity for hydrogen decreases and the the pH decreases significantly. Also, when the pH drops to thermodynamic equilibrium of hydrogen formation from 4.5-5.0, a shift infermentation processes may occur, resulting 35 acetate, or other Volatile organic acids, is favored at higher in solventformation (solventogenesis). The shift to solvento temperatures. In addition, at large hydrogen and carbon diox genesis is induced by high intracellular concentrations of ide concentrations there is potential for the onset of acetoge acids, low pH, a growth limiting factor (Such as phosphate or nesis, resulting in the formation of acetate from hydrogen and Sulfate depletion) and high concentrations of glucose and carbon dioxide. Although this conserves the hydrogen in a nitrogen compounds. Solvent formation, allows for further 40 different chemical form, it decreases the potential dark fer metabolism by preventing excessive acidification, but this mentation yield of hydrogen. In addition, the rate of fermen may be at the expense of hydrogen yield. Solventogenesis tative hydrogen production may be inhibited by high partial requires the induction of a new set of enzymes catalysing the pressures (i.e. concentration) of hydrogen. formation of acetone, butanol and ethanol from glucose and The measurement of hydrogen is achieved using tech assimilated acids. Also, solventogenic Clostridia, depending 45 niques known in the art, and may be carried out in the head on the Strain and fermentation conditions, can produce other space inside the digester or externally in the pipe/tube system alternative solvents such as propanol, isopropanol and 1.2- used remove the biogas from the digester. Described below propanediol. are examples of hydrogen gas measurement systems, how Optimal hydrogen production from dark fermentation may ever, any hydrogen sensing or measurement system known in be maintained and solventogenesis may be minimized by 50 the art may be used. As with pH monitoring, any of the monitoring of the fermentation media pH, either continu hydrogen measurements may be used either continuously or ously or by periodic sampling. In-line and/or on-line moni by periodic sampling, and may used as in-line and/or on-line toring methods may be used for continuous pH monitoring. monitoring methods. Hydrogen gas measurement may be In-line monitoring methods use instrumentation which can be achieved using fiber optic sensors based on the chemochro directly inserted into the digester, whereas, on-line monitor 55 mic reaction of certain transition metal oxides, such as tung ing involves diverting a small stream of media to a location sten oxide or W03 with hydrogen in air, wherein, the reaction where it is either continually sampled and analyzed by fixed is catalyzed by palladium or platinum, and the color change of instrumentation, or manually sampled and tested elsewhere. the film in the presence of hydrogen is detected by reflectance The stream of media is then either re-circulating back into the spectroscopy. Another method is to use thermal conductivity digester, after appropriate sterilization, or taken off as waste. 60 detectors (TCD), in which the thermal conductivity of the Examples of in-line pH monitoring instrumentation are, but digester gas is compared to that of a reference gas, and the not limited to, glass membrane type pH electrodes, Solid State difference in conductivity can then be calibrated to give a type pH electrodes (e.g. silicon nitride based and metal oxide hydrogen concentration value. Thermal conductivity detec based), and optodes (optical type pH electrodes using fluo tion may also be used in conjunction with gas chromatogra rescence or absorbance measurements of pH sensitive dyes). 65 phy (GC) as a method to identify the various components of In-line methods require sterilization of the measuring system the fermentation gas mixture and obtain their respective con and may have issues with electrode fouling for particulates centrations. The application of GC with TCD detection for US 8,501,463 B2 53 54 gas analysis is know to one skilled in the art. Sensing is based thermal based detectors, or hot wire anemometers. The sys on resistance changes upon hydrogen adsorption to platinum tem used for gas removal may be manually controlled, or may and/or palladium, field effect transistors based on carbon be automated using computer control. Methods for removal nanotubes are other approaches used to monitoring hydrogen of fermentation gases from digesters will be known to those concentrations in digesters. The hydrogen concentration skilled in the art, however, one possible method is to control information obtained may be used for feedback in a control a vacuum system attached to the digester. loop to the system used to control the removal offermentation Gas partial pressure may affect the metabolic balance and gases from the digester. In addition, the gas flow rate may be therefore affects the hydrogen yield obtained from dark fer monitored to assist in controlling the removal of gases from mentation. The digester pressure may be monitored using the digester. Typical methods for measuring flow rate are 10 methods and techniques known to one skilled in the art. thermal based detectors, or hot wire anemometers. The sys However, examples of pressure sensing methods used to tem used for gas removal may be manually controlled, or may monitor the digester pressure are solid state pressure sensors be automated using computer control. Methods for removal or manometers, either attached to the digester or incorporated of fermentation gases from digesters will be known to those with the outlet pipe/tube. The pressure information obtained skilled in the art, however, one possible method is to control 15 may be used for feedback in a control loop to the system used a vacuum system attached to the digester. to control the removal of fermentation gases from the Similarly, carbon dioxide in dark fermentation biogas mix digester. In addition, the gas flow rate may be monitored to tures may be monitored using measurement systems know to assist in controlling the removal of gases from the digester. one skilled in the art. However, Some examples for detecting Typical methods for measuring flow rate are thermal based carbon dioxide are fiber optic sensors based fluorescence and detectors, or hot wire anemometers. The system used for gas absorbance changes with pH sensitive dyes, non-dispersive removal may be manually controlled, or may be automated infra-red detection, field effect transistors based on carbon using computer control. Methods for removal offermentation nanotubes, pyroelectric detectors, thermal conductivity gases from digesters will be known to those skilled in the art, detectors, and thermal conductivity detectors coupled with however, one possible method is to control a vacuum system gas chromatography. The carbon dioxide concentration infor 25 attached to the digester. mation obtained may be used for feedback in a control loop to In dark-fermentation processes, the gas produced is a mix the system used to control the removal of fermentation gases ture of primarily H and CO, but may also contain other from the digester. In addition, the gas flow rate may be moni gases such as CO. methane, and HS, depending on the bio tored to assist in controlling the removal of gases from the mass and bacteria present. If the digester becomes contami digester. Typical methods for measuring flow rate are thermal 30 nated with undesirable bacteria it is likely that other fermen based detectors, or hot wire anemometers. The system used tation gases, such as methane (CH) from methanogenesis for gas removal may be manually controlled, or may be auto and ammonia (NH) from nitrate or nitrite reduction may be mated using computer control. Methods for removal offer present. Early detection of these contamination gases may mentation gases from digesters will be known to those skilled allow for quick containment and remedy of the contamina in the art, however, one possible method is to control a 35 tion. One method for detection and quantification of contami vacuum system attached to the digester. nation gases is, but not limited to, thermal conductivity detec Carbon monoxide in dark fermentation biogas mixtures tion in conjunction with gas chromatography (GC). This may be monitored using measurement systems know to one method allows identification of the various components of the skilled in the art. However, some examples for detecting fermentation gas mixture and obtains their respective concen carbon monoxide are electrolytic sensors, colorimetric sen 40 trations. sor, MOS detectors (Metal Oxide Semiconductor Sensor), Optimal hydrogen production from dark fermentation may thermal conductivity detectors, and thermal conductivity be maintained and solventogenesis may be minimized by detectors coupled with gas chromatography. The carbon monitoring, either continuously or by periodic sampling, the monoxide concentration information obtained may be used nutrient concentration in the fermentation media. In-line and/ for feedback in a control loop to the system used to control the 45 or on-line monitoring methods may be used for nutrient removal of fermentation gases from the digester. In addition, monitoring. In-line monitoring methods use instrumentation the gas flow rate may be monitored to assist in controlling the which can be directly inserted into the digester, whereas, removal of gases from the digester. Typical methods for mea on-line monitoring involves diverting a small stream of media suring flow rate are thermal based detectors, or hot wire to a location where it is either continually sampled and ana anemometers. The system used for gas removal may be 50 lyzed by fixed instrumentation, or manually sampled and manually controlled, or may be automated using computer tested elsewhere. The stream of media is then either re-circu control. Methods for removal of fermentation gases from lating back into the digester, after appropriate sterilization, or digesters will be known to those skilled in the art, however, taken off as waste. Examples of in-line nutrient monitoring one possible method is to control a vacuum system attached to instrumentation are, but not limited to, glucose sensors (opti the digester. 55 cal or amperometric). In-line methods require Sterilization of Hydrogen Sulfide in dark fermentation biogas mixtures the measuring system and may have issues with electrode may be monitored using measurement systems know to one fouling from particulates and bacteria. On-line monitoring skilled in the art. However, some examples for detecting HS methods do not require sterilization, provided no recircula are conductiometric sensors (CuO SnO2) based on resis tion of the media occurs, plus it allows for sample cleanup by tance changes upon exposure to H2S, thermal conductivity 60 filtering of bacteria and particulates from the test stream. detectors, and thermal conductivity detectors coupled with Examples of on-line nutrient monitoring instrumentation are, gas chromatography. The hydrogen Sulfide concentration but not limited to, glucose sensors (optical or amperometric), information obtained may be used for feedback in a control flow injection analysis (FIA) methods, high performance liq loop to the system used to control the removal offermentation uid chromatography (HPLC), high performance liquid chro gases from the digester. In addition, the gas flow rate may be 65 matography coupled with Mass Spectrometry (HPLC-MS), monitored to assist in controlling the removal of gases from and capillary electrophoresis (CE). The same methodologies the digester. Typical methods for measuring flow rate are used for on-line monitoring may be used for periodic Sam US 8,501,463 B2 55 56 pling. The nutrient concentration information may be used for microbe present within the anaerobic digester. Rapid removal feedback in a control loop to the system used to add nutrients and purification of the H and removal of diluting (CO, CH) into the digester, and thereby maintain the optimal nutrient and/or contaminating (CO, H2S) gases allows for maintaining concentration for optimal growth. The control of nutrient continuous H. Synthesis. addition may involve adjusting a valve, either manually or 5 As discussed above, the pH2 is a parameter which may be computer controlled, to increase or decrease addition of monitored during continuous H. Synthesis, since with deoxygenated nutrient solutions into the digester. increasing H concentrations, H. Synthesis decreases and In addition, monitoring of the Volatile organic acid fermen metabolic activity shifts to pathways that synthesize more tation products, either continuously or by periodic sampling, reduced Substrates. Thus, efficient gas removal bypasses is needed for optimal hydrogen production from dark fermen 10 these pathways, thereby increasing H2 production. The con tation. In-line and/or on-line monitoring methods may be centration of CO, also affects the rate of synthesis and final used for Volatile organic acid monitoring. Examples of on yield of H due to Succinate and formate synthesis using CO, line Volatile organic acid monitoring instrumentation are, but pyruvate and nicotinamide adenine dinucleotide (NADFf) via not limited to, Fluorescence Immunoassay (FIA) methods, the hexose monophosphate pathway. This pathway competes high performance liquid chromatography (HPLC), high per 15 with reactions in which H is synthesized by NADH-depen formance liquid chromatography coupled with Mass Spec dent hydrogenases (which oxidize NADH to NAD+). Thus, trometry (HPLC-MS), capillary electrophoresis (CE), and efficient removal of CO from the fermentation system ion chromatography. The same methodologies used for on reduces competition for NADH, and results in increased H. line monitoring may be used for periodic sampling. The Vola synthesis. tile organic acid concentration information may be used for Methods of removal offermentation gases include, but are feedback in a control loop to the system used to remove the not limited to, sparging with N or argon (Ar) gas, applying a Volatile organic acid from the digester, and thereby maintain vacuum to the digester head space, or using membrane tech the minimal volatile organic acid concentration necessary for nologies, such as, by way of example only, hollow fiber/ optimal growth. Volatile organic acids may be removed by silicone rubber membranes and non-porous, synthetic poly cross flow dialysis, with the rate of removal dependent on the 25 vinyltrimethylsilane (PVTMS) membranes. flow rate of the media on the opposite side of the dialysis The hydrogen and carbon dioxide produced by dark fer membrane. The control of flow rate may involve adjusting a mentation in the anaerobic digester may be cleaned or puri pump, either manually or computer controlled, to increase or fied by a scrubber to remove moisture, vapor, droplets, Sus decrease the flow rate of the deoxygenated media used to pended Solids or other Such contaminants. The Scrubber can remove the Volatile organic acid from the digester as they 30 comprise one or more of a filter, desiccant, Zeolite, activated diffuse across the dialysis membrane. carbon, fiber, countercurrent wash solution, mixer, homog Changes in the fermentation buffer capacity may be enizer, or other such components typically used in association obtained with knowledge of the digester pH and the concen with or comprised within gas scrubbers. Such components are trations of the volatile organic acids. If the buffer capacity is well known to those of ordinary skill in the art of gas process too low, such that a slight increase in fermentation rate may 35 ing. In general, hydrogen sulfide (HS) is an undesired by cause a rapid decrease in pH, then fresh deoxygenated media product or off-gas, which is removed from the desired hydro may be added. The buffer capacity information may be used gen gas; however it may be used as feed stock for bacteria for feedback in a control loop to the system used to add Such as, by way of example only, , to deoxygenated media into the digester, and thereby maintain obtain more hydrogen and elemental Sulfur. the optimal buffer capacity. The control of media addition 40 The gases which exit the anaerobic digester or the scrubber may involve adjusting a valve, either manually or computer are then optionally separated into their individual compo controlled, to increase or decrease addition of deoxygenated nents using conventional gas separation equipment, which is media into the digester. known to those of ordinary skill in the art for separating gas Removal of the dark fermentation products, such as hydro mixtures. By way of example only, HS and CO can be gen, carbon dioxide, hydrogen Sulfide, carbon monoxide and 45 removed from the gas product stream with the use of mem the Volatile organic acids during fermentation creates condi branes which are permeable to HS or CO and not permeable tions for use of concentrated nutrients and the ability to main to hydrogen. Alternatively, palladium-silver alloy? ceramic tain optimal hydrogen production. Without this removal pro composite membranes with high selectivity and flux rate for cess hydrogen production would be diminished due to hydrogen may be used for separating hydrogen from CO and initiation of alternate metabolic pathways at high concentra 50 HS. Thus, CO and H2S may be selectively stripped from the tion of fermentation products. Therefore, separation of gas product stream yielding purified H. In addition, the, CO microbial growth and product formation improves hydrogen may be selectively stripped from HS using a variety of meth production yields, however optimizing the process condi ods for H2S removal. Such as, by way of example only, a tions, such as pH, and temperature also improve hydrogen monoethanolamine Girbotol type process. The CO may then yield. 55 be isolated and purified for later use. Alternatively, the CO Removal of Hydrogen, Carbon Dioxide and Hydrogen Sul and HS mixture may be used as feed stock for purple sulfur fide bacteria, wherein the HS is converted into hydrogen and Dark-fermentation systems have great potential as practi elemental sulfur, and the CO is subsequently removed from cal biohydrogen systems by incorporating rapid gas removal the H to yield purified CO and H. and separation, rapid Volatile organic acid removal and sepa 60 Alternatively, the gases which exit the anaerobic digester ration, and bioreactor design. In dark-fermentation processes, are separated and purified using a differential compression the gas produced in the anaerobic digester is a mixture of H system. The differential compression process involves ini and CO, but may also contain other gases such as CO, CH, tially drying the mixture of gaseous anaerobic fermentation HS, or ammonia (NH), which is be present in fermentation products, such as, but not limited to, hydrogen, and carbon media and headspace above the reaction solution. The content 65 dioxide, then compressing and cooling the mixture to trans of each gas in the headspace and the reaction solution varies form at least one of the gases into a liquid form. The remain according to the conditions, feed stock and/or anaerobic ing gaseous products may then be compressed and cooled at US 8,501,463 B2 57 58 a different location using different conditions, thus making the Volatile organic acids from the fermentation culture, and the process a differential compression process. The condi generally, dead-end filtration, cross-flow filtration and ultra tions used for compressing and cooling vary depending on the filtration use positive pressure to force fluid through the filter gas mixture. By way of example only, the removal and puri element. Alternatively; methods without the use of positive fication of hydrogen from carbon dioxide may use a pressure pressure to force fluid through a filter element, such as reverse of 58 bars at a temperature of 15° C. to liquefy the carbon osmosis, dialysis or electro-dialysis, may be used for the dioxide allowing for easy separation of the gaseous hydrogen. removal of the volatile organic acids from the dark fermen Once separated, the hydrogen may be optionally processed tation culture. with one or more dehydration stages, then compressed and The use of dialysis for the removal of volatile organic acids alternatively stored in pressurized storage vessels or tanks, or 10 from the bacteria used for dark fermentation relies on diffu used directly as a fuel to generate electricity via fuel cells or sion of the Volatile organic acids across a semi-permeable combustion/turbine type systems. The generated electricity membrane and leaving the bacteria behind. A concentration may then be used to operate the anaerobic digester and asso gradient is needed for the process to occur and this can be ciated facility, or can be added to the external power system achieved continuously or in a batch mode. In the batch mode via connection to the external grid. If the purified hydrogenis 15 the dark fermentation culture is poured into a dialysis bag stored, it can be used later as a fuel source, or used as a (constructed from semi-permeable membrane material) and chemical component in an industrial process. Similarly, the the filled bag is placed into a container of fresh fermentation purified CO can be directly used as feed stock for alternate media or water. A concentration gradient is present between fermentation processes, it can be stored and used later as feed the inside of the bag and the outside, and diffusion of soluble stock, it can be used as a greenhouse Supplement to enhance ions and molecules across the semi-permeable membrane. greenhouse plant, fruit and vegetable production, it can be Large molecules, particles and bacteria which are unable to used as a refrigerant in the anaerobic fermentation facility, it pass through the semi-permeable membrane remain inside can be used as a fire-extinguishing material, or it can be used the bag. This process continues until equilibrium is reached, as chemical component in an industrial process, such as Soda after which a fresh container of fresh fermentation media or ash production. In addition, collected carbon dioxide may be 25 water is used for thorough removal of Volatile organic acids. used commercially such as, but are not limited to, in the In a continuous process a semi-permeable membrane may be production of carbonated beverages, in water softening, in the used to separate the dark fermentation digester from a system manufacture of aspirin, used as a refrigerating agent, used in in which fresh fermentation media or water is flowing past, the Solvay process for the preparation of sodium carbonate, thereby creating a concentration gradient and continuously used to provide an inert atmosphere for packaging and storage 30 removing volatile organic acids as they diffuse into the flow of foods, and used as pressurizing medium and propellant in ing stream. aerosol cans of food, fire extinguishers, target pistols, and for Electro-dialysis is a membrane process, during which ions inflating life rafts. are transported through semi permeable membrane, under the Substantial gains in H production can also be achieved influence of an electric potential. The membranes are cation through optimization of bioreactor designs. By way of 35 or anion-selective, allowing either positive ions or negative example only, fixed-bed bioreactors enhance H production ions to flow through. Cation-selective membranes may be by using activated carbon as a Support matrix, plus a mem negatively charged polyelectrolytes, which rejects negatively brane filter system for removal of the biogas. The support charged ions and allows positively charged ions to flow matrix allows for retention of the H producing bacteria through. Cation-selective membranes may be Sulphonated within the bioreactor, and the membrane system maintains 40 polystyrene, while anion-selective membranes may be poly low gas partial pressures. styrene with quaternary ammonia functional groups. Par Removal of Volatile Organic Acids ticles and bacteria are too large to pass through the membrane The Volatile organic acids generated during dark fermen and are not removed. Electro-dialysis can also be used in a tation are removed as their accumulation may have an effect continuous or batch process as described above for dialysis. on the anaerobic bacteria’s ability to produce hydrogen. In 45 The filtrate obtained from the above methods may be fur particular, as fermentation proceeds the pH decreases, and ther purified with the use of reverse osmosis. Reverse osmo continued fermentation may yield pH-neutral components sis, also known as hyperfiltration, process uses a membrane Such as acetone and butanol at the expense of hydrogen pro that is semi-permeable, allowing the fluid to pass through it, duction. Removal of these products can be either continu while rejecting ions from crossing. This in effect is a method ously or in batch mode, wherein both continuous and batch 50 to concentrate and purify the Volatile organic acids obtained removal methods include, but are not limited to, dead-end from dark fermentation. The process of reverse osmosis filtration, cross-flow filtration, ultrafiltration, pervaporation, requires a driving force to push the fluid through the mem and dialysis. Dead-end filtration involves the use of a filter brane, and the most common force is pressure from a pump. element through which the media is passed while leaving the Most reverse osmosis technology uses a process known as bacteria and other solid particulates behind. The use of a 55 cross flow to allow the membrane to continually clean itself, dead-end filter can be continuous, with fresh media being analogous to cross flow filtration. As some of the fluid passes introduced to replenish the quantity filtered, alternatively through the membrane the rest continues downstream, dead-end filtration can be a batch process. One issue encoun Sweeping the rejected species away from the membrane. tered with dead-end filtration is clogging and potential block Reverse osmosis is capable of rejecting bacteria, salts, Sugars, age of the filter element, which can limit the through put of a 60 proteins, particles, dyes, and other constituents that have a continuous system. To overcome clogging issues a cross flow molecular weight of greater than 150-250 daltons (Da). The filtration system can be used, wherein tangential flow of separation of ions with reverse osmosis is aided by charged media, bacteria and other particulates occurs across the filter membranes. Thus, dissolved ions that carry a charge, such as element minimizing the potential for clogging. Cross-flow salts, are more likely to be rejected by the membrane than filtration can also be used in a batch mode approach. Ultra 65 those that are not charged. Such as organics. In this manner, filtration can be used for bacteria not filterable using other any solvents produced, such as, by way of example only, pore sizes. The presence offilter systems effectively removes acetone, may be removed while maintaining the Volatile US 8,501,463 B2 59 60 organic acids in the flow stream. The larger the charge and the teria may be used to obtain hydrogen and elemental Sulfur larger the particle, the more likely it will be rejected. from HS in an anaerobic lithotrophic process. The volatile organic acids may be removed from the are all gram negative, but otherwise repre anaerobic fermentation system and then purified by osmosis sent a diverse range of organisms such as the purple pho as a salt. The Volatile organic acids anions, including, but not totrophic, nitrifying bacteria and enteric bacteria, as well as limited to, formate, acetate, propionate, butyrate, and Valer the bacteria responsible for animal bioluminescence. They ate, may be combined with alkali metal cations, alkaline earth can be divided into five sections (depending on their RNA), cation, ammonium ion, and combinations thereof to form a referred to by the Greek letters alpha, beta, gamma, delta and salt. By way of example only, the cations may be Na', K", epsilon. Photosynthetic protobacteria (anoxygenic photoau Ca", Mg", NH, or combinations thereof. 10 totrophy) are found in alpha, beta, and gamma groups, in Convert Volatile Organic Acids to H/CO particular purple Sulfur bacteria are generally beta or gamma The filtrate obtained from dead-end filtration, cross-flow proteobacteria, and purple non-Sulfur bacteria are primarily filtration, ultrafiltration, or dialysis of the dark fermentation alpha proteobacteria. Alpha, beta, and gamma groups also stage may contain other Volatile organic acids than acetic include chemolithotrophs and chemoorganotrophs. In con acid. Therefore, the filtrate may be fed into separate anaerobic 15 trast, the delta and epsilon proteobacteria are not photosyn digesters and further metabolized by acetic acid producing thetic and are all chemoorganotrophs. (acetogenic) bacteria to yield hydrogen, carbon dioxide and Most purple bacteria are strict anaerobes and live in the acetate. A close monitoring of hydrogen concentrations is sediment of ponds and lakes. Purple phototrophic bacteria use necessary for acetogenic bacteria conversion of fatty acids energy from Sunlight in a process known as anoxygenic pho (e.g., formic, acetic, propionic, isobutyric, butyric, isovaleric, tosynthesis and can be divided into two groups depending Valeric, isocaproic, caproic, and heptanoic acids) and alco whether or not hydrogen sulfide (HS) is used as an electron hols into acetate, hydrogen, and carbon dioxide because donor for carbon dioxide reduction. If HS is used as an acetate formation may be reduced under relatively high H electron donor then they are called purple sulfur bacteria, if partial pressure. The use of acetogenic bacteria for generation HS is not used as an electron donor, then they are known as of acetate not only increases hydrogen output, but it is an 25 purple non-Sulfur bacteria. For purple non-sulfur bacteria, alternative approach to increasing the acetate concentration in hydrogen can be used as the reducing agent, although some the filtered dark fermentation media. The acetate generated purple non-sulfur bacteria may use other compounds. Purple during acetogenic bacteria fermentation may be used as feed sulfur bacteria fix CO, to live, whereas non-sulfur purple stock for other fermentation stages in separate anaerobic bacteria can grow aerobically in the dark by respiration on an digesters, such as, by way of example only, photo-fermenta 30 organic carbon Source. tion (FIG. 14). With light, and under anaerobic conditions, Purple sulfur bacteria normally are anaerobic or reduced organic compounds, like acetic acid, can be con microaerophilic, and are often found in sulfur springs or Verted to hydrogen and carbon dioxide by certain microor stagnant water. An environment they can be found in is the ganism, Such as, by way of example only, purple bacteria illuminated but anoxic Zones of these aquatic environments. (Chromatium, Rhodospirillium, Rhodomicrobium). There 35 The presence of oxygen hinders their growth. Purple sulfur fore, photo-fermentation may be added as another fermenta bacteria oxidize HS or S as an electron donor for carbon tion stage to obtain more hydrogen from the photohet dioxide reduction during the dark reaction of photosynthesis. erotrophic fermentation of acetate into hydrogen and carbon The HS is oxidized to produce granules of elemental sulfur, dioxide. The hydrogen and carbon dioxide can be separated which in turn may be oxidized to form sulfuric acid and and purified. 40 therefore acidifies the environment. Some of the sulfur oxi Alternatively, the filtrate obtained from dead-end filtration, dizers are acidophiles that are able to grow at a pH of 1 or less. cross-flow filtration, ultrafiltration, or dialysis of the dark The photosynthetic proteobacteria may be placed into two fermentation stage, or the media, concentrated by reverse families, the and the Ectothiorhodospiraceae. osmosis, may be further purified to isolate the various volatile Ectothiorhodospiraceae are gamma-proteobacteria, but are organic acids. Isolation and purification methods can include 45 distinctive because they deposit sulfur on the outside of their distillation, adsorption chromatography, ion exchange chro cells, whereas Chromatiaceae deposit sulfur inside their cells. matography, and affinity chromatography. Such purified This behavior may be utilized to obtain elemental sulfur from Volatile organic acids may then be used as either commercial oxidation of HS, which has been obtained from certain types products, or as precursors or reactants for synthesis of com of biomass during anaerobic dark fermentation. mercial products. 50 Lithoautotrophic sulfur oxidizers are found in environ Convert HS with Appropriate Bacteria to H/CO and Sulfur ments rich in H.S. Such as Volcanic hot springs and fuma Proteobacteria or “purple bacteria' may be the largest and roles, and deep-sea thermal vents. Some are found as Sym most physiologically diverse group of bacteria, which exhibit bionts and endosymbionts of higher organisms. Since they a wide variety of types of metabolism: aerobic, anaerobic, can generate energy from an inorganic compound and fix CO heterotrophic, autotrophic, phototrophic, and lithotrophic. 55 as autotrophs, they may play a fundamental role in primary The use of carbon dioxide as the carbon Source and an inor production in environments that lack Sunlight. In addition, ganic compound (water, or H2S) as the source of reducing Some are hyperthermophiles that grow at temperatures of power is reflected in the terms autotroph and (re 115°C., while some are halophilic (salt loving), such as the spectively) which are used for an organism performing this genus Halothiobacillus, which can be found in Soda lakes and reaction. With light as the ultimate source of energy, the 60 salterns. organism would also be termed a phototroph. Thus, in addi Most purple non-sulfur photosynthetic bacteria are able to tion to protobacteria being anoxygenic photoheterotrophs, grow as photoheterotrophs, photoautotrophs or chemohet proteobacteria include chemolithotrophs and chemoorgan erotrophs. A few species are capable of anaerobic growth otrophs, wherein are able to fix carbon, while though most species are aerobic. The mode of growth is organotrophs require fixed carbon as a nutrient. Thus, purple 65 determined by the available conditions, such as, availability bacteria may be used for H and CO production from acetate of light (needed for phototrophic growth), the degree of by a photoheterotrophic process, or alternatively, purple bac anaerobiosis (oxygen level), the availability of CO as a car US 8,501,463 B2 61 62 bon source for autotrophic growth, and the availability of ited to, carbon dioxide). By way of example only, acetic acid organic compounds (such as simple Sugars, Volatile organic obtained from the dark fermentation and acetogenic conver acids, and aromatic compounds) for heterotrophic growth. sion can be purified, and optionally concentrated into glacial Therefore, photo-fermentation may be used to obtain more acetic acid (99.5% pure acetic acid), and used as feedstock for hydrogen from the photoheterotrophic fermentation of 5 the chemical industry. Non-limiting examples of the use of acetate into hydrogen and carbon dioxide by purple non acetic acid include the production of fibers and resins such as Sulfur. Typical purple non-sulfur bacteria genera are Rho cellulose acetate used in making acetate rayon, the production dospirillum and Rhodopseudomonas and Rhodobacter: of pharmaceuticals, bleaches, preservatives and photographic Chemotrophic growth for the purple non-sulfur bacteria is chemicals, fertilizers, plastics, nonflammable motion-picture achieved by respiration, although there are some exceptional 10 strains and species which can obtain energy by fermentation film, photographic film, lacquers, and paint Solvents. In addi or anaerobic respiration. In addition, it was thought that tion, the acetic acid obtained by anaerobically fermenting a purple non-sulfur bacteria could not use hydrogen Sulfide as biomass, as described herein, may be used to make acetate an electron donor for the reduction of carbon dioxide when esters which may be used as solvents for various resins in growing photoautotrophically, hence the use of “non-sulfur 15 protective coatings and for formulating inks. Such acetate in their group name. Sulfide can be used if present in a low esters include, but are not limited to, amyl, butyl, ethyl, concentration, however, higher concentrations of H2S (in methyl, and propyl acetates which may be used as solvents in which the purple sulfur bacteria and green sulfur bacteria can quick-drying lacquers, cements and adhesives. thrive) are toxic. The volatile organic acids obtained from anaerobic fer Use of Fermentation Products mentation, as described herein, may be used in the synthesis FIG. 15 is a schematic showing one approach toward pro of other chemical products. By way of example only, a Vola ducing and processing various fermentation products for their tile organic acid and a mineral may be admixed and reacted to use. The fermentation products, also referred to as chemical form a composition. This is demonstrated by an exemplary products, may include, but are not limited to, gases, Volatile embodiment in which acetic acid, obtained from anaerobic organic acids, Solids and solvents. These chemical products 25 fermentation of a biomass, is admixed and reacted with dolo may be obtained from dark fermentation, acetogenesis, mite (calcium magnesium carbonate) to form calcium mag photo-fermentation, Solventogenesis, fermentation with pro nesium acetate (reaction Scheme 1). teolytic bacteria, or combinations thereof. The gases pro duced include, but are not limited to, hydrogen, carbon diox ide, carbon monoxide, hydrogen Sulfide, methane, ammonia, 30 Scheme 1 and nitrogen; which are removed from the various digesters, separated and purified for use as feedstock for chemical O industry, as energy sources for power production, or as prod CaCO3/MgCO3 + ch--on Her ucts themselves. The Volatile organic acids include, but are not limited to, formic acid, acetic acid, propionic acid, butyric 35 Dolomite Acetic acid acid, and Valeric acid; which are removed from the various O digesters, separated and purified for use as feedstock for Ca(Ac)2Mg(Ac)2 wherein Ac is ch--o chemical industry, feedstock for other fermentation pro cesses, or as products themselves. In addition, the Volatile Calcium Magnesium Acetate organic acids may be removed from the digester and con 40 verted to the respective salts of the volatile organic acids, which comprise at least one cation. The anions of Such salts The calcium magnesium acetate formed in this manner include, but are not limited to, formate, acetate, propionate, may be used as an anti-freeze agent, a deicing agent, or butyrate, and Valerate, while the at least one cation includes, an anti-icing agent. In addition, glacial acetic acid may but is not limited to, alkali metal ions (such as, by way of 45 be used as described above, wherein the glacial acetic example only Na" and K"), an alkaline earth ions (such as, by acid is formed by concentrating the acetic acid obtained way of example only Ca" and Mg"), and ammonium ion from anaerobic fermentation of a biomass. (NH). The solvents produced include, but are not limited, to Similarly, Volatile organic acids may be admixed with an acetone, butanol, propanol, isopropanol. 1.2-propanediol. oxide and liquid ammonia and reacted to form a composition. and the Solid may be compounds which comprise Sulfur, or 50 This is demonstrated by an exemplary embodiment in which may be elemental sulfur. The solvents and solids are removed acetic acid, obtained from anaerobic fermentation of a biom from the various digesters, separated and purified and may be ass, is admixed and reacted with Zinc oxide to form Zinc used as feedstock for chemical industry, as energy sources for ammonium acetate (reaction Scheme 2). power production, or as products themselves. The fermentation products, such as gases, Volatile organic 55 acids, salts of Volatile organic acids, Solids and solvents may Scheme 2 be used as feedstock in other industries. Such as, but not limited to, polymer industry, industrial synthesis industry, photographic industry, coatings industry, fertilizer industry, ZnO + NH4OH + CH-C-OH -- printing industry, and combinations thereof. In addition, the 60 Zine Ammonium Acetic acid Volatile organic acids may also be used as feedstock for other oxide hydroxide fermentation processes, such as, but not limited to, acetoge nesis (to produce more acetic acid from other Volatile organic acids), photo-fermentation (to produce more hydrogen and ZnNH4(Ac)3 wherein Acis CH-C-O other fermentation gases, such as, but not limited to, carbon 65 Zinc Ammonium Acetate dioxide) and solventogenesis (to produce solvents and more hydrogen and other fermentation gases, such as, but not lim US 8,501,463 B2 63 64 The Zinc ammonium acetate formed in this manner may be ings, stove-top, ovens, and barbeques for cooking, and dryers used as a fertilizer or a seed germination enhancer. Again for drying clothes or commercial products Such as plastics, the glacial acetic acid may also be used, wherein the polymers, plywood, paper, and pharmaceutical products. glacial acetic acid is formed by concentrating the acetic Combustion of solvents obtained as fermentation products acid obtained from anaerobic fermentation of a biomass. may be used to generate steam to run turbines, used in internal Hydrogen plus catalysts are used extensively in the chemi combustion generators and turbine generators. Such solvent cal industry to hydrogenate a variety of starting materials to include, but are not limited to, acetone, butanol, ethanol, create chemical products for further chemical synthesis use. propanol, isopropanol. 1.2-propanediol and or other solvents. The hydrogen obtained from anaerobic fermentation of a There are various types of fuel cells, each with particular biomass, as described herein, may be used for hydrogenation 10 operating conditions and fuel requirements. The hydrogen processes in the chemical industry. Such processes include, produced as described herein may be used in any of them. but are not limited to, the following examples: Alkaline fuel cells (AFC) utilize hydroxyl ions (OH) as the a) Hydrogenation of unsaturated hydrocarbons and aro mobile ion (derived from potassium hydroxide, KOH), oper matics ate in the 50 to 200° C. range, and are sensitive to the presence e.g. benzene to cyclohexane using Ni /Pt Al2O3 15 of CO. Phosphoric acid fuel cells (PAFC) utilize protons catalyst, wherein the cyclohexane, is used as starting (H) as the mobile ion and operate at approximately 200° C. material for nylon production or as a solvent, PAFC systems were the first fuel cells produced commer b) Hydrogenation of ketones and aldehydes cially and are used as stationary power sources, generating up e.g. acetone to mesityl oxide to methyl isobutyl ketone to 200 kW of electricity. The high operating temperature and (MIBK) for use as a solvent corrosive nature of the electrolyte makes them unsuitable for c) Hydrogenation of nitrogen containing compounds use in mobile and transportation applications. Molten carbon e.g. nitrobenzene to aniline using a Cucatalysts such as ate fuel cells (MCFC) utilize carbonate ions (CO) as the NiS/CuS, wherein the aniline is used as a starting mobile ion, operate at approximately 650° C., and can take material for dyes, pharmaceuticals, polymers, and H, CO, CO, and/or CH as fuel, which means they can use Solvents. 25 natural gas, coal gas, or biogas as fuel Sources. Like PAFC, Hydrogen gas is an environmentally friendly energy MCFC are used as stationary power Sources, generating elec Source, as it can be used generate electricity without produc tricity in the MW range. Solid oxide fuel cells (SOFC) utilize ing greenhouse gas emissions. Thus, a hydrogen based sys oxygen radicals (O) as the mobile ion and operate between tem offers totally clean energy Supplies with no pollution. 500° C. and 1000° C. Like MCFC, SOFC can utilize H, CO, Hydrogen may be produced by a number of processes, includ 30 and/or CH as fuel, which means they can use methane, coal ing electrolysis of water, thermocatalytic reformation of gas, or biogas as fuel sources. Carbon dioxide is not utilized hydrogen-rich organic compounds such as methane, and bio as a fuel and is discharged as a waste gas. Like other high logical processes. Biological production of hydrogen pro temperature fuel cell systems (PAFC and MCFC) SOFC sys vides a wide range of approaches to generate hydrogen, tems are used as stationary power sources, generating elec including direct biophotolysis, indirect biophotolysis, photo 35 tricity from the low kW to the MW range. Proton exchange fermentations, and dark-fermentation. The fermentation membrane fuel cells (PEMFC) utilize hydrogen protons (H) products, such as gases and solvents, produced as described as the mobile ion, operate in the 50-100° C. range, require herein, may be used as fuel and/or energy sources for power pure H, and are sensitive to the presence of CO. Of all fuel generation using power generation systems. Such energy and cell systems available, PEMFC systems are especially suit power production applications, include, but are not limited to, 40 able for mobile and transportation applications, such as powering motor vehicles, running turbines or fuel cells to engines for cars. Small PEMFCs, in the 1-10kW range, may produce electricity at centralized power stations, running tur be useful for electrical applications for homes. bines or fuel cells to produce electricity at localized decen Anaerobic Fermentation System tralized power stations, and generating heat by direct com The anaerobic fermentation system described herein is an bustion. The power generation systems include, but are not 45 assemblage of components, combined to produce chemical limited to, internal combustion generators, turbine genera products by anaerobically fermenting biomass. FIG. 16 is a tors, fuel cells, and Such systems may be part of a centralized schematic representation of a non-limiting example of the power station or a decentralized power station. In addition, components of Such an assemblage. The arrows between the electricity obtained locally from the decentralized power components are used to illustrate the transportation of mate station may be used for residential consumption or commer 50 rial from one component to another, continuous movement of cial consumption, or the electricity may be added to the grid material from one component to another, depiction of the next and then used residentially or commercially. step in the process or combinations thereof. In addition, each The gaseous fermentation products used as fuel or energy component represented may incorporate a single process, Sources include, but are not limited to hydrogen, carbon diox multi step processes, a single device, multiple devices, or ide, carbon monoxide, hydrogen Sulfide and methane, 55 combinations thereof. wherein the methane may be produced by anaerobic fermen In FIG. 16 the biomass is pretreated prior to being trans tation or may be formed by methanation using the hydrogen ported to the anaerobic fermentation digester. The pretreat and carbon oxides produced by anaerobic fermentation. ment process involves concentration adjustment, detoxifica Methane formation by methanation results by mixing hydro tion, sterilization, deoxygenation, and/or pre-digestion, not gen and carbon oxides, such as, by way of example only, 60 necessarily in this order. By way of example only, the biomass carbon dioxide and carbon monoxide, and a catalyst Such as, may be sterilized and deoxygenated prior to concentration by way of example only activated nickel catalysts and NiO/ adjustment, detoxification, and/or pre-digestion steps; the MgO. The methane formed either by methanation or by biomass may be concentrated prior to sterilization, detoxifi anaerobic fermentation may be used as a fuel or energy source cation, deoxygenation, and/or pre-digestion; the biomass to generate electricity from fuel cells or to generate heat by 65 may be sterilized, then deoxygenated, then detoxified, then combustion in a heat generation system. The heat generation concentrated, and then pre-digested; the biomass may be system includes, but is not limited to, furnaces to heat build detoxified, deoxygenated, then sterilized, and then concen US 8,501,463 B2 65 66 trated; the biomass may be concentrated, then sterilized, then effluent rates, gas production rate, carbon/nitrogen ratio of the deoxygenated, and then detoxified; or the biomass may be feed Stock, pressure, pH, and retention time in the digester. concentrated, then deoxygenated, then detoxified, and then These parameters may be monitored continuously or inter sterilized. The concentration adjustment step may be accom mittently, using pH sensors, gas sensors, GCS, HPLC's, plished using methods which separate solids from liquids, FIA's, pressure sensors, temperature sensors, optical densi Such as, but not limited to, centrifugation and filtration, or the tometers, refractometers, and gas flow sensors concentration adjustment step may be accomplished using The operation of the anaerobic fermentation systems methods which separate Solution phase chemicals from liquid described herein may be a continuous process or a batch Such as, but not limited to, reverse osmosis, dialysis and process. In either approach the anaerobic fermentation osmosis. The components used to concentrate biomass using 10 parameters. Such as, by way of example only, temperature, Such techniques are known to one skilled in the art. In the operation of the anaerobic fermentation system pH, pressure, gas flow, biomass concentration, and fermen described hereinsterilization can be accomplished using pas tation chemical product concentration, may be monitored and teurization and/or acidification. Other methods known in the the information used to optimally operate the anaerobic fer art may also be used to sterilize the biomass. 15 mentation digester system. The anaerobic digester control In the operation of the anaerobic fermentation system system may comprise at least one process control tool; at least described herein deoxygenation can be accomplished using one metrology tool to acquire at least one metrology data high pressure and low pressure steam. In general high pres relating to at least one anaerobic fermentation parameter; a Sure steam is generated with the use of a boiler, although any process controller operatively coupled to at least one process means to generate high pressure steam may be used. This high control tool and at least one metrology data, and wherein the pressure steam may be used for deoxygenation, or a pressure process controller comprises decision making units, input/ reducing valve may be used to lower the pressure, and the low output boards, and database units to store at least one metrol pressure steam may be used for deoxygenation. In another ogy data. The decision making unit is used in a feedback embodiment turbines, including, but not limited to, backpres control process to acquire metrology tool data from the input/ Sure turbine generators, are used to both create low pressure 25 output board and to determine control adjustments based on steam by reducing the pressure of high pressure steam and the metrology tool data and defined operational ranges for the generate electricity. The advantage associated with the use of desired anaerobic fermentation parameters. This maintains a turbine to generate low pressure Steam is that in addition to the anaerobic fermentation parameters within operational the low pressure steam being made available for deoxygen ranges and allows for optimal operation of the anaerobic ation, the turbine can be used to generate electricity which can 30 be used to operate the facility containing the anaerobic digest digester. The magnitude of the control adjustments, are modi ers. FIG. 17 is a schematic demonstrating the use of a turbine fied and returned to the input/output board, whereby by the system to generate low pressure steam for Sterilization and modified control adjustments are sent to process control tools deoxygenation of a biomass, in conjunction to the generation to adjust operating parameter of the anaerobic fermentation of electricity for use by the facility, or any other electricity 35 digester and thereby maintain the digester at optimal condi consumer. As seen in FIG. 17, a heat source (i.e. commeri tions as defined by the operational ranges. cally bought or from an anerobic Substrate source/reaction) The type of decision making units incorporated into the heats the boiler. High pressure steam enters the turbine which anaerobic fermentation systems described herein include, but causes the blades to turn and thereby turning a generator and are not limited to, computers, PROMs (Programmable Read creating electricity. Thus the steam turbine generator makes 40 Only Memory), EPROM's (Erasable Programmable Read electricity by converting a steam pressure drop into mechani Only Memory) and EEPROMs (Electrically Erasable Pro cal power to spin a generator. As the high-pressure steam grammable Read-Only Memory). The types of metrology enters the turbine and drives the generator, the lower pressure tool incorporated into the anaerobic fermentation systems steam exhausts from the turbine and is used to either heat the described herein include, but are not limited to, at least one facility, sterilize and deoxygenate a biomass, or combinations 45 pH sensor, at least one pressure sensor, at least one gas flow thereof. In addition, single backpressure turbine generators sensor, at least one temperature sensor, at least one GC, at may be used, or multistage backpressure turbine generators least one HPLC, at least one FIA, and combinations thereof. may be used to handle different steam paths form different In addition, the type of process control tool incorporated into high pressure Steam sources. the anaerobic fermentation systems described herein After pretreatment the biomass is transported to the 50 includes, but is not limited to, a valve, a hotplate, or a heating anaerobic fermentation digester (FIG. 16), where it is fer mantle. mented using anaerobic bacteria chosen for the particular FIG. 18 is a schematic of an embodiment of the anaerobic biomass present. The conditions inside the anaerobic digester digester control system, wherein, by example only, the may vary according to the anaerobic bacteria being used, the metrology tool is a pH sensor and the operating parameter to configuration of the anaerobic digester, the feedstock being 55 converted, the desired productivity of the anaerobic digester, be controlled is the pH of the anaerobic fermentation media. the chemical product being produced, and the form of bacte Adjustment of the pH is by addition of acid or base from ria (immobilized or free-flowing) used. Mobilized bacteria appropriate tanks by control of valves which couple the acid can be prepared using any methods known by the artisan of and base tanks to the anaerobic digester. Adjustment occurs to ordinary in the arts. In order to optimally operate the anaero 60 maintain the pH in a defined operational range. The pH sensor bic digester the conditions used to ferment the biomass are information is acquired by the I/O board which sends the monitored, and the fermentation parameter are then con information to the decision making unit. Depending on the trolled to achieve optimal production of chemical product and pH value and the resulting calculated control adjustment, the the maintenance of anaerobic bacteria viability. The fermen valve, to either the acid or base tanks, is opened and a prede tation parameters include, but are not limited to, Solids con 65 termined aliquot is added to the digester. The valve is then tent, nutrient Solution composition, temperature, gas content, 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. US 8,501,463 B2 67 68 ILLUSTRATIVE EXAMPLES pressure sensors, temperature sensors, and gas sensors are monitored using Lab VIEW NIDAQ software (National The following examples are provided for illustrative pur Instruments Corporation, Austin, Tex.), running a DAQPad poses only and not to limit the scope of the claims provided 6508 National Instruments I/O board. Any adjustments herein. 5 needed to the operating parameters are calculated using a laptop computer based on the sensor signals. Throughout the Example 1 fermentation process the anaerobic fermentation gas products are continuously removed from the digester using a vacuum. Batch Anaerobic Fermentation of Plant Material The gaseous products are separated and purified using differ 10 ential compression. The fermenting slurry is recycled through Stem, Leaf, Seed and Vegetable Parts of Bell Pepper a reverse osmosis system (GEOsmonics Corp., Kent, Wash.) Plants to remove the Volatile organic acids produced. The concen tration of the organic acids are measured using a gas chro Parts of bell pepper plants, including leaves, stems, Veg matograph (Model 8610C: SRI Instruments, Torrance, Calif.) etables and seeds, is obtained from a bell pepper farm and is 15 equipped with a flame ionization detector, using nitrogen as shredded using a mulching machine. The shredded material carrier gas and a fused silica capillary column 0.1 umx0.53 and water are placed into a vat, whereby the mixture is treated mmx15 m MXT-WAX (Restek, Bellefonte, Pa.) with low pressure steam for sterilization and deoxygenation. The steam treatment also assists in degrading cellulosic mate Example 3 rial for easier fermentation. The sterilized/deoxygenated mixture is then concentrated by removing a portion of the Utilizing the Enrichment/Isolation System and water using a centrifuge system. After centrifugation the con Knowledge Management System to Identify Optimal centrated/sterilized/deoxygenated material is pumped into an Biomass for Bacterial Strains in Producing anaerobic digester which has been deoxygenated by sparging Hydrogen Anaerobically with nitrogen. The anaerobic digester is a modified commer 25 cial digester (DCI Inc., St. Cloud, Minn.). After addition of Using two parallel glass plates approximately 10,000 cm the treated bell pepper material the anaerobic digester is in Volume, a sealed chamber is formed to house an enrich inoculated with a bacterial culture of Clostridium butyricum. ment/isolation system. In very concentrated spots nutrients During fermentation the temperature, pH, pressure, concen are placed within the sealed chamber either by direct place tration of anaerobic fermentation products and optical density 30 ment or metering in with a hose. Different types of nutrients are monitored and changes to the digester operating param are placed away from each other a distance so that different eters are adjusted accordingly. Throughout the fermentation spatial Zones of bacteria around the nutrients may form. process the anaerobic fermentation gas products are col Nutrients used are organic acids, diesel, food waste fats and lected, separated and purified. Upon completion of the fer cellulose. Bacteria used in the sealed chamber are harvested mentation process the digester is emptied and the slurry is 35 from decomposing bell peppers. The bacteria are introduced filtered to obtain the liquid which is further processed to into sealed chamber so that they use the nutrients as a food obtain the Volatile organic acid produced. source. After 3-4 weeks bacterial samples around the nutri ents are taken out using a syringe or tool which is non-evasive. Example 2 The bacterial samples are tested to determine whether the 40 bacteria are primary or secondary users of the food source. Continuous Anaerobic Fermentation of Plant The bacteria are isolated, put on defined medium to test what Material they use as nutrients. Two particular indicator tests that are preformed are the nitrate and catalase tests. The bacteria are Stem, Leaf, Seed and Vegetable Parts of Bell Pepper also assayed for their production of hydrogen at different Plants 45 growth conditions involving variation in temperature, pres Sure, pH, fermentation products, exposure to various natural Parts of bell pepper plants, including leaves, stems, Veg gases or compounds, drug efficacy, compound toxicity or etables and seeds, is obtained from a bell pepper farm and is survivability. shredded using a mulching machine. The shredded material Information regarding which types of bacteria species uses and water are placed into a vat, whereby the mixture is treated 50 what particular type of nutrients as a food source and optimal with low pressure steam for sterilization and deoxygenation. bacterial growth conditions for producing hydrogen are The steam treatment also assists in degrading cellulosic mate entered into the Knowledge Management System database. rial for easier fermentation. The sterilized/deoxygenated The Knowledge Management System houses data that can be mixture is then concentrated by pumping the slurry through a quickly accessed and manipulated into a user friendly form continuously fed reverse osmosis system (GE Osmonics 55 Such as a graph or spreadsheet. In particular, the Knowledge Corp., Kent, Wash.); thereby a portion of the water is Management System can narrow down which bacteria uses removed. The concentrated/sterilized/deoxygenated slurry is particular nutrients for producing a specific amount of hydro pumped into an anaerobic digester which is continuously gen. anaerobically fermenting biomass and contains a population All references cited herein, including patents, patent appli of Clostridium butyricum. The anaerobic digester is a modi 60 cations, and publications, are hereby incorporated by refer fied commercial digester (DCI Inc., St. Cloud, Minn.). Dur ence in their entirety. All of the methods disclosed and ing fermentation the temperature, pH, pressure, concentra claimed herein can be made and executed without undue tion of anaerobic fermentation products and optical density experimentation in light of the present disclosure. It will be are constantly monitored using a digester control system and apparent to those of skill in the art that variations may be changes to the digester operating parameters are adjusted 65 applied to the methods and in the steps or in the sequence of continuously by the digester control system. The digester steps of the method described herein without departing from control system is a custom built system, wherein pH sensors, the concept, spirit and scope of the invention. More specifi US 8,501,463 B2 69 70 cally, it will be apparent that certain agents that are both 3. The anaerobic fermentation apparatus of claim 1, chemically and physiologically related may be substituted for wherein the chemical product is selected from the group the agents described herein while the same or similar results consisting of a gaseous chemical product, a non-gaseous would be achieved. All such similar substitutes and modifi chemical product, and combinations thereof. cations apparent to those skilled in the art are deemed to be 4. The anaerobic fermentation apparatus of claim 1, within the spirit, scope and concept of the invention as defined wherein the anaerobic digester control system is used to opti by the appended claims. mally operate the anaerobic digester and comprises: an at least one process control tool; an at least one metrology tool What is claimed: to acquire at least one metrology data relating to at least one 1. An anaerobic fermentation apparatus using a population 10 anaerobic fermentation parameter; an a process controller of substantially purified anaerobic bacterial strain for anaero operatively coupled to the at least one process control tool and bically fermenting a biomass into a chemical product com the at least one metrology data, wherein the process controller prising: (i) a sterilization and deoxygenation system; (ii) an comprises a decision making unit, an input/output board, and anaerobic digester containing a population of Substantially a database unit to store the at least one metrology data. purified anaerobic bacteria and equipped with an anaerobic 15 5. The anaerobic fermentation apparatus of claim 1, digester control system; (iii) a plurality of pipelines and wherein the biomass comprises a material selected from the pumps for introducing and re-circulating biomass, and (iv) group consisting of energy crops, Surplus agricultural prod removal pipelines connected to the anaerobic digester for ucts, waste from Sugar production and processing facilities, removing a chemical product from the anaerobic digester, waste from fruit processing industries, waste from pulp and wherein the population of substantially purified anaerobic paper mills, Silvaculture residues, waste from wood process bacterial strain has not been Subjected to a heatshocking ing, waste from agricultural product processing, food waste, process. Solids isolated from fermentation cultures, municipal sewer 2. The anaerobic fermentation apparatus of claim 1, waste, animal manure, animal urine, animal parts, fish parts, wherein the anaerobic fermentation apparatus is a component and combinations thereof. of an assemblage for generating power. k k k k k