JPH08294396A - Production of hydrogen gas - Google Patents
Production of hydrogen gasInfo
- Publication number
- JPH08294396A JPH08294396A JP12456295A JP12456295A JPH08294396A JP H08294396 A JPH08294396 A JP H08294396A JP 12456295 A JP12456295 A JP 12456295A JP 12456295 A JP12456295 A JP 12456295A JP H08294396 A JPH08294396 A JP H08294396A
- Authority
- JP
- Japan
- Prior art keywords
- hydrogen
- genus
- anaerobic
- producing
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 title claims abstract description 169
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 86
- 239000001257 hydrogen Substances 0.000 claims abstract description 166
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 166
- 241000894006 Bacteria Species 0.000 claims abstract description 80
- 230000000243 photosynthetic effect Effects 0.000 claims abstract description 57
- 241000193403 Clostridium Species 0.000 claims abstract description 52
- 241000190831 Chromatium Species 0.000 claims abstract description 48
- 244000005700 microbiome Species 0.000 claims abstract description 41
- 239000010815 organic waste Substances 0.000 claims abstract description 26
- 229910052717 sulfur Inorganic materials 0.000 claims abstract description 14
- 239000011593 sulfur Substances 0.000 claims abstract description 14
- 241000192497 Oscillatoria Species 0.000 claims description 26
- 241000192700 Cyanobacteria Species 0.000 claims description 17
- 238000012258 culturing Methods 0.000 claims description 9
- 230000000813 microbial effect Effects 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 abstract description 46
- 230000004151 fermentation Effects 0.000 abstract description 45
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 abstract description 10
- 150000002431 hydrogen Chemical class 0.000 abstract description 9
- 238000002156 mixing Methods 0.000 abstract description 6
- 238000010170 biological method Methods 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 41
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 35
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 35
- 238000000034 method Methods 0.000 description 21
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 19
- 239000008103 glucose Substances 0.000 description 19
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 18
- 230000001580 bacterial effect Effects 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 235000014113 dietary fatty acids Nutrition 0.000 description 16
- 229930195729 fatty acid Natural products 0.000 description 16
- 239000000194 fatty acid Substances 0.000 description 16
- 150000004665 fatty acids Chemical class 0.000 description 16
- 239000000758 substrate Substances 0.000 description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 15
- 239000007789 gas Substances 0.000 description 15
- 239000005416 organic matter Substances 0.000 description 15
- 239000000126 substance Substances 0.000 description 14
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 13
- 239000013505 freshwater Substances 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 230000002407 ATP formation Effects 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 9
- 230000034659 glycolysis Effects 0.000 description 9
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 9
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 9
- 239000002028 Biomass Substances 0.000 description 8
- 108010020056 Hydrogenase Proteins 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 8
- 241000193171 Clostridium butyricum Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 230000007613 environmental effect Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 241000894007 species Species 0.000 description 7
- 108010020943 Nitrogenase Proteins 0.000 description 6
- -1 and in particular Chemical compound 0.000 description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 6
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000002351 wastewater Substances 0.000 description 6
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 5
- 241000191023 Rhodobacter capsulatus Species 0.000 description 5
- 241000190932 Rhodopseudomonas Species 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000001569 carbon dioxide Substances 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 230000001079 digestive effect Effects 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 235000000346 sugar Nutrition 0.000 description 5
- 150000004763 sulfides Chemical class 0.000 description 5
- 241000195628 Chlorophyta Species 0.000 description 4
- 241001464430 Cyanobacterium Species 0.000 description 4
- 150000001720 carbohydrates Chemical class 0.000 description 4
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 4
- 230000027756 respiratory electron transport chain Effects 0.000 description 4
- 239000010802 sludge Substances 0.000 description 4
- 241001474374 Blennius Species 0.000 description 3
- 102000018832 Cytochromes Human genes 0.000 description 3
- 108010052832 Cytochromes Proteins 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 229910052804 chromium Inorganic materials 0.000 description 3
- 239000011651 chromium Substances 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 229910001873 dinitrogen Inorganic materials 0.000 description 3
- 239000002803 fossil fuel Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000029553 photosynthesis Effects 0.000 description 3
- 238000010672 photosynthesis Methods 0.000 description 3
- 102220201851 rs143406017 Human genes 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 241000190857 Allochromatium vinosum Species 0.000 description 2
- 241000192542 Anabaena Species 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000192656 Nostoc Species 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 241000195663 Scenedesmus Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000004065 wastewater treatment Methods 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000195585 Chlamydomonas Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 241000192520 Oscillatoria sp. Species 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 241000191025 Rhodobacter Species 0.000 description 1
- 241000191043 Rhodobacter sphaeroides Species 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 241000192707 Synechococcus Species 0.000 description 1
- 241000190988 Thermochromatium tepidum Species 0.000 description 1
- 241000191001 Thiocapsa Species 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 230000001925 catabolic effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- ZCDOYSPFYFSLEW-UHFFFAOYSA-N chromate(2-) Chemical compound [O-][Cr]([O-])(=O)=O ZCDOYSPFYFSLEW-UHFFFAOYSA-N 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000009569 heterotrophic growth Effects 0.000 description 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
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- 238000001179 sorption measurement Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 238000009423 ventilation Methods 0.000 description 1
Classifications
-
- Y02W10/12—
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、水素ガス生産方法に係
り、特に、有機性廃棄物を基質として、嫌気性の従属栄
養性水素生産菌群と光合成微生物の混合共生培養により
水素ガスを生産する方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing hydrogen gas, and in particular, hydrogen gas is produced by mixed symbiotic culture of anaerobic heterotrophic hydrogen-producing bacteria with photosynthetic microorganisms using organic waste as a substrate. On how to do.
【０００２】[0002]
【従来の技術】化石燃料に代替するエネルギー源とし
て、クリーンな水素エネルギーが注目され、安価で、大
量生産を目的とした水素生産方法が研究されているが、
その主流は化学的及び／又は電気化学的方法による技術
が中心となっており、結局、化石燃料によるエネルギー
供給が不可欠であるために、環境汚染を招いている。こ
のような観点から、化石燃料に依存せず、かつ、環境汚
染を惹起しない微生物学的な水素ガス生産技術の研究開
発が精力的に行なわれてきている。水素ガスを生産する
微生物は、光合成微生物と非光合成微生物とに大別され
る。水素ガスを生産する光合成微生物としては、光エネ
ルギーの照射下で水を分解し、酸素を発生する光合成微
生物（微細藻類）で次のような属種があげられる。 ＊緑藻類 ・クラミドモナス（Chlamidomonas)属 ・クロレラ（Chlorella)のある属種 ・セネデスムス（Scenedesmus)属 ＊藍藻類 ・オッシラトリア（Oscillatoria) 属 ・シネココッカス（Synechococcus)2. Description of the Related Art Clean hydrogen energy has been attracting attention as an alternative energy source to fossil fuels, and an inexpensive hydrogen production method for mass production has been studied.
The mainstream of the technology is mainly based on chemical and / or electrochemical methods, and eventually energy supply by fossil fuels is indispensable, resulting in environmental pollution. From this point of view, research and development of microbiological hydrogen gas production technology that does not depend on fossil fuel and does not cause environmental pollution has been vigorously carried out. Microorganisms that produce hydrogen gas are roughly classified into photosynthetic microorganisms and non-photosynthetic microorganisms. As photosynthetic microorganisms that produce hydrogen gas, photosynthetic microorganisms (microalgae) that decompose water to generate oxygen under irradiation of light energy include the following genus species. ＊ Green algae ・ Chlamidomonas genus ・ A genus with Chlorella ・ Scenedesmus genus ＊ Cyanobacteria ・ Oscillatoria genus ・ Synechococcus
【０００３】また、他の一つは、有機物を資化する過程
で余剰の水素をガスとして放出する光合成細菌であり、
その主たるものとして次の属種があげられる。 ＊非紅色硫黄細菌 ・ロドスピリラム（Rhodospirillu
m) 属 ・ロドシュードモナス（Rhodopseudomonas) ＊紅色硫黄細菌 ・クロマチウム（Chromatium) 属 ・チオカプサ（Thiocapsa)属 光合成微生物による水素生産及び／又は有機性排水処理
に関する研究は、地球環境問題が顕在化する以前の１９
７５年以来精力的に行なっており、評価すべき研究成果
を上げている。海洋性光合成細菌であるクロマチウム
（Chromatium) 、或いは海洋性藍藻に属するオッシラト
リア（Oscillatoria) のある種の菌株を用いて、光合成
微生物の高分子ゲルによる固定化技術による安定した連
続水素生産、蜜柑ジュース製造排水（濃厚排水の処理は
不能）の処理と水素生産、或いは明暗同調培養による水
素生産と窒素の除去技術などが業績として上げられ、こ
れらの研究成果は「光合成微生物による水素生産」（鈴
木周一編；バイオマス・エネルギー変換、講談社・サイ
エンティフィック、ｐｐ．１９４〜２２８〔１９８
３〕）に集大成されている。The other is a photosynthetic bacterium that releases excess hydrogen as a gas in the process of assimilating organic matter,
The main genera include the following genera and species. * Non-red sulfur bacterium-Rhodospirillu
m) Genus ・ Rhodopseudomonas ＊ Red-colored sulfur bacterium ・ Chromatium genus ・ Thiocapsa genus Studies on hydrogen production and / or organic wastewater treatment by photosynthetic microorganisms have been conducted before the emergence of global environmental problems. 19
Since 1975, he has been energetically conducting research results that should be evaluated. Stable continuous hydrogen production by polymer gel immobilization technology of photosynthetic microorganisms using a certain strain of marine photosynthetic bacterium Chromatium or marine cyanobacterium Oscillatoria The achievements were the treatment of wastewater (concentration of concentrated wastewater is not possible) and hydrogen production, or the technology of hydrogen production and nitrogen removal by synchronized dark and light culture, and the results of these researches were "Hydrogen production by photosynthetic microorganisms" (edited by Shuichi Suzuki). Biomass / Energy Conversion, Kodansha Scientific, pp. 194-228 [198]
3]).
【０００４】然し、この膨大で、貴重な研究成果が過去
から現在にいたる過程で得られているにも拘らず、現時
点で実用化規模で稼働している水素生産施設、或いは排
水処理施設は存在しない。 その主な理由は次の通りであると思考される。 光合成微生物の増殖には、太陽エネルギーの連続的
な照射が不可欠である。 光合成細菌の培養には、通常、広大な面積を必要と
する。 水素還元酵素であるニトロゲナーゼの失活、ヒドロ
ゲナーゼの安定化のための固定化技術、その他が必要で
あり、技術的な困難が伴う。 必要面積を縮少するための濃厚培養技術、及び培養
装置が必要である。（コストアップ要因となる） 太陽光の集光、培養槽内への光の均等分散（エネル
ギー利用効率の向上）。However, despite the fact that this enormous and valuable research result has been obtained in the process from the past to the present, there is a hydrogen production facility or a wastewater treatment facility which is currently operating on a practical scale. do not do. It is thought that the main reasons are as follows. Continuous irradiation of solar energy is essential for the growth of photosynthetic microorganisms. Cultivation of photosynthetic bacteria usually requires a vast area. Deactivation of nitrogenase, which is a hydrogen reductase, immobilization technology for stabilizing hydrogenase, etc. are necessary, and technical difficulties are involved. A dense culture technique and a culture device to reduce the required area are required. (Because of cost increase) Concentration of sunlight, even distribution of light in the culture tank (enhancement of energy use efficiency).
【０００５】この光合成細菌による水素ガス生産に対し
て、非光合成細菌による水素生産の研究も、広範に行な
われている。この種の水素生産菌は偏性、或いは通性嫌
気性菌に属し、その代表的な細菌は前記の通りである
が、従属栄養的な増殖の過程で、有機物を分解し、この
過程で生ずる余剰の電子をヒドロゲナーゼで還元する事
により水素を生産し、エネルギー生産に伴う還元力の調
整を水素生産により自動的に行なっている。この嫌気性
の従属栄養性水素生産菌は、有機物を分解し、結果とし
て水素を生産できるので、高濃度の有機性廃棄物、その
他のバイオマスをエネルギーとしての再資源化できると
同時に処理・処分が可能であり、将来性のある環境保全
技術になり得るものである。然し、現段階では、この従
属栄養性水素生産菌による水素生産にも下記のような欠
点がある。[0005] In contrast to the hydrogen gas production by the photosynthetic bacteria, research on hydrogen production by non-photosynthetic bacteria has been extensively conducted. This kind of hydrogen-producing bacterium belongs to obligate or facultative anaerobic bacterium, and its representative bacterium is as described above, but it decomposes organic matter in the process of heterotrophic growth and occurs in this process. Hydrogen is produced by reducing excess electrons with hydrogenase, and the reducing power associated with energy production is automatically adjusted by hydrogen production. This anaerobic heterotrophic hydrogen-producing bacterium is capable of decomposing organic matter and producing hydrogen as a result, so that high-concentration organic waste and other biomass can be recycled as energy and treated and disposed of at the same time. It is a viable and promising environmental conservation technology. However, at this stage, hydrogen production by this heterotrophic hydrogen-producing bacterium has the following drawbacks.
【０００６】嫌気性菌による有機物（グルコースで代表
させる）からの水素生産の発酵反応式は通常式１で表さ
れる。 Ｃ6 Ｈ12Ｏ6 ＋２Ｈ2 Ｏ＝２ＣＨ3 ＣＯＯＨ＋２ＣＯ2 ＋４Ｈ2 ・・・・・式１ （標準自由エネルギー＝＋１８４ｋｊ／ｍｏｌ） 式１から容易に理解できるように、グルコース１モルか
ら生産される水素は４モルに過ぎず、光合成細菌の水素
生産菌１２モル（式２）に対して極めて少量である。 Ｃ6 Ｈ12Ｏ6 ＋６Ｈ2 Ｏ＝６ＣＯ2 ＋１２Ｈ2 ・・・・・・・・・ 式２ この水素生産量格差の最大の原因は、光合成細菌による
水素生産では、この反応の推進力となるアデノシン三リ
ン酸（ＡＴＰ）が、少なくとも太陽エネルギーが供給さ
れている限り、理論的には無限大であるのに対して、嫌
気性細菌による有機物からの水素生産は、有機物を最終
的に水と炭酸ガスまで分解するのに１８４ｋｊ／ｍｏｌ
のエネルギーが不足する。従って、嫌気性細菌による水
素発酵は完全な吸エルゴン反応であり、不足ＡＴＰ量に
対応する分だけ発酵液中に各種の低級脂肪酸が蓄積し、
そのために水素生産量が少ないだけでなく、発酵消化液
の２次処理を必要とする。A fermentation reaction formula for hydrogen production from an organic substance (represented by glucose) by an anaerobic bacterium is usually represented by Formula 1. C 6 H 12 O 6 + 2H 2 O = 2CH 3 COOH + 2CO 2 + 4H 2 Formula 1 (standard free energy = + 184 kj / mol) As can be easily understood from Formula 1, hydrogen produced from 1 mol of glucose. Is only 4 moles, which is an extremely small amount relative to 12 moles of hydrogen-producing bacteria of photosynthetic bacteria (formula 2). C 6 H 12 O 6 + 6H 2 O = 6CO 2 + 12H 2 ········· formula 2 biggest cause of the hydrogen production disparities, the hydrogen production by photosynthetic bacteria, the driving force of the reaction Adenosine triphosphate (ATP) is theoretically infinite as long as at least solar energy is supplied, whereas anaerobic bacteria produce hydrogen from organic matter by converting it to water. 184 kj / mol to decompose carbon dioxide
Run out of energy. Therefore, hydrogen fermentation by anaerobic bacteria is a complete absorption ergon reaction, and various lower fatty acids accumulate in the fermentation broth in an amount corresponding to the insufficient ATP amount,
Therefore, not only the hydrogen production amount is small, but also the secondary treatment of the fermentation digestion liquid is required.
【０００７】 不足ＡＴＰ量＝１８４ｋｊ／ｍｏｌ÷３０．５ｋｊ／ｍｏｌ・ＡＴＰ ≒６．０ｍｏｌ・ＡＴＰ／ｍｏｌ・Ｃ6 Ｈ12Ｏ6 ・・・・・式３ 嫌気性細菌（水素生産菌）によるＡＴＰ生産量は、理想
的条件でグルコース１モル当り解糖系で４モル、酸発酵
工程で３モルの計７モルであり、何らかの手段・方法で
ＡＴＰを補填・補完しない限り、水素発酵はこれ以上正
方向に進行せず、低級脂肪酸が蓄積した状態で動的平衡
状態に達する。この問題を解決するために、次のような
研究が知られている（水素発酵；バイオマスから水素生
産、微生物、Ｖｏｌ．３，Ｎｏ．６，ｐｐ．４２〜４
９，１９８７）。即ち、嫌気性細菌は光照射が不要であ
り、強力な有機物分解能を持っており、バイオマスから
水素生産ができる。また、通気も必要なく、大型のタン
クで培養できるという利点がある。然し、有機物の完全
な分解ができず、有機酸を蓄積することが欠点であり、
水素発生率も低い。Insufficient ATP amount = 184 kj / mol / 30.5 kj / mol.ATP.apprxeq.6.0 mol.ATP / mol.C 6 H 12 O 6 ... Formula 3 ATP by anaerobic bacteria (hydrogen-producing bacteria) The amount of production is 7 mol, which is 4 mol per mol of glucose under the ideal conditions in the glycolysis system and 3 mol in the acid fermentation process, and unless the ATP is supplemented or supplemented by some means or method, the hydrogen fermentation is more than this. It does not proceed in the positive direction, and reaches a dynamic equilibrium state when lower fatty acids are accumulated. In order to solve this problem, the following studies are known (hydrogen fermentation; hydrogen production from biomass, microorganisms, Vol. 3, No. 6, pp. 42-4).
9, 1987). That is, anaerobic bacteria do not need light irradiation, have a strong ability to decompose organic substances, and can produce hydrogen from biomass. Further, there is an advantage that the culture can be performed in a large tank without the need for ventilation. However, the drawback is that organic substances cannot be completely decomposed and organic acids are accumulated,
The hydrogen generation rate is also low.
【０００８】これに対して光合成細菌は一般に有機物の
資化性能は低いが、光エネルギーを利用できるため、有
機物を完全に水と炭酸ガスにまで分解できる。また、有
機酸等嫌気性発酵の生産物を好んで利用する。このよう
に、嫌気性細菌と光合成細菌は基質の代謝機能において
相補的関係にあるので、これらの２種類の細菌を混合し
て用いれば基質の完全分解が可能と考えられる。このよ
うな発想により、クロストリジウム ブチリカム（Clos
tridium butyricum)とロドシュードモナス スファエロ
イデス（Rhodopseudomonas sphaeroides) を混合培養す
ると、グルコース１モルから７モルの水素が回収できた
と報告している。一方、混合培養ではなく、クロストリ
ジウム ブチリカム（Clostridium butyricum)の発酵廃
液を光合成細菌に与えた場合は、合計でも４モル以下の
水素しか得られなかったことから、混合培養において
は、互いの細菌の水素発生効率を高める相乗効果も生じ
ていると考えられると報告している。然し、この発想に
よる混合培養での水素生産量は最高でも７モルであり、
光合成細菌の１２モルに比較すると、まだ可成りの格差
がある。On the other hand, photosynthetic bacteria generally have low assimilation performance for organic substances, but they can utilize light energy to completely decompose organic substances into water and carbon dioxide. Also, products of anaerobic fermentation such as organic acids are preferably used. As described above, since anaerobic bacteria and photosynthetic bacteria have a complementary relationship in the metabolic function of the substrate, it is considered that the substrate can be completely decomposed by using these two types of bacteria in combination. Based on this idea, Clostridium butyricum (Clos
It has been reported that, when tridium butyricum) and Rhodopseudomonas sphaeroides were mixed and cultured, 1 to 7 mol of glucose could be recovered. On the other hand, when the fermentation effluent of Clostridium butyricum was fed to the photosynthetic bacteria instead of the mixed culture, only 4 mol or less of hydrogen was obtained in total. It is reported that a synergistic effect that increases the generation efficiency is also considered to occur. However, the hydrogen production amount in the mixed culture based on this idea is at most 7 mol,
There is still a considerable difference when compared to 12 moles of photosynthetic bacteria.
【０００９】また、それ以前にも嫌気性細菌と光合成細
菌の混合培養による水素生産の実験を行い、次のような
結果を得ている。即ち、基質としてグルコース（＋各種
必要成分を加えた人工培地）を用い、嫌気性水素生産菌
としてクロストリジウム ブチリカム（Clostridium bu
tyricum)ＩＦＯ１３９４９、光合成細菌として新しく発
見した非硫黄光合成細菌ロドシュードモナス（Rhodopse
udomonas）ＳＰ・ＲＶ（Rhodopseudomonas capsulata・
・・・ＦＥＲＭＰ−７２５４として寄託）を混合し、培
養温度３０℃、さらに嫌気性条件下で１０ｋｌｕｘの光
照射（嫌気明条件下）を行いながら混合培養し、計算
上、１モルのグルコースから７モル〜９モルの水素を得
ている（J.Ferment.Technol.,Vol. ６２，No. ６，ｐ．
５３１〜５３５，１９８４、特公昭６３−４９９９４号
公報）。何れにしても、有機性廃棄物などのバイオマス
から光合成微生物を介在せしめて水素生産を行なう場合
には、人工的な光エネルギー使用は水素生産コストの面
から実用性は期待できず、さらに、光エネルギーを太陽
光に依存する場合には、恒久的に安定した光エネルギー
を供給することは現実的に不可能である。Further, before that, an experiment of hydrogen production by mixed culture of anaerobic bacteria and photosynthetic bacteria was conducted, and the following results were obtained. In other words, glucose (+ artificial medium containing various necessary components) was used as a substrate, and Clostridium butyricum was used as an anaerobic hydrogen-producing bacterium.
tyricum) IFO 13949, a newly discovered non-sulfur photosynthetic bacterium Rhodopse
udomonas) SP ・ RV (Rhodopseudomonas capsulata ・
... (deposited as FERMP-7254) is mixed, and the mixture is cultured at a culture temperature of 30 ° C. under light irradiation of 10 klux (anaerobic condition) under anaerobic conditions. Mol. To 9 mol of hydrogen is obtained (J. Ferment. Technol., Vol. 62, No. 6, p.
531 to 535, 1984, JP-B-63-49994). In any case, when hydrogen is produced from biomass such as organic waste by interposing photosynthetic microorganisms, artificial light energy use cannot be expected from the viewpoint of hydrogen production cost. When relying on sunlight for energy, it is practically impossible to provide permanently stable light energy.
【００１０】[0010]
【発明が解決しようとする課題】以上詳述したように、
有機性廃棄物などのバイオマスを処理すると共に、水素
生産をも達成できる水処理技術、有機性廃棄物の再資源
化技術の出現が強く要望されているが、現在のところ実
用的な技術は開発されていない。本発明は、上記従来技
術の問題点を解消し、安定して大量の水素を生産できる
全く新規な発想による革新的な生物学的水素生産方法を
提供することを課題とする。DISCLOSURE OF THE INVENTION As described in detail above,
There is a strong demand for the emergence of water treatment technology and organic waste recycling technology that can achieve hydrogen production as well as the processing of biomass such as organic waste, but currently practical technology is being developed. It has not been. An object of the present invention is to solve the above-mentioned problems of the prior art and to provide an innovative biological hydrogen production method based on a completely new idea that can stably produce a large amount of hydrogen.
【００１１】[0011]
【課題を解決するための手段】上記課題を解決するため
に、本発明では、各種の有機性廃棄物から水素ガスを生
産する方法において、該有機性廃棄物中で、偏性嫌気性
の従属栄養性水素生産菌であるクロストリジウム（Clos
tridium)属に属する微生物群と、水素生産性光合成微生
物である紅色硫黄細菌クロマチウム（Chromatium) 属及
び／又は藍藻であるオッシラトリア（Oscillatoria) 属
に属する微生物とを、嫌気暗条件下で共生せしめ、混合
培養することを特徴とする水素ガス生産方法としたもの
である。前記方法において、混合培養は減圧発酵槽ある
いは微嫌気減圧発酵槽で行なうことができる。In order to solve the above-mentioned problems, the present invention provides a method for producing hydrogen gas from various kinds of organic wastes, in which the obligate anaerobic subordination is contained in the organic wastes. Clostridium, a nutrient-producing hydrogen bacterium
A microorganism group belonging to the genus tridium and a microorganism belonging to the genus Oromatella (Oscillatoria), which is a hydrogen-producing photosynthetic microorganism, and the red sulfur bacterium Chromatium and / or cyanobacteria, are allowed to coexist under anaerobic dark conditions and mixed. This is a hydrogen gas production method characterized by culturing. In the above method, the mixed culture can be carried out in a vacuum fermentation tank or a slightly anaerobic vacuum fermentation tank.
【００１２】このように、本発明は、各種の有機性廃棄
物を基質として嫌気性の従属栄養性水素生産菌により、
解糖系とＴＣＡサイクル系で水素を生産し、同時に生成
される低級脂肪酸を光合成細菌であるクロマチウム（Ch
romatium) 属に属する細菌群及び／又は藍藻の一種であ
る水素生産性藻類オッシラトリア（Oscillatoria) 属に
属する藍藻群を嫌気暗条件、即ち、光エネルギーを供給
しない環境条件下で混合共生培養し、この嫌気的環境下
で生産されるＡＴＰにより前記の低級脂肪酸を資化さ
せ、水素に転換し、有機性廃棄物から光エネルギーなし
で、安定して大量の水素を生産するシステムである。As described above, according to the present invention, various kinds of organic wastes are used as substrates for anaerobic heterotrophic hydrogen-producing bacteria.
The lower fatty acids that produce hydrogen at the same time as glycolysis and TCA cycle and are simultaneously produced are photosynthetic bacteria chromateum (Ch
romatium) and / or hydrogen-producing algae belonging to the genus Oscillatoria, which is a type of cyanobacteria, are mixed and co-cultivated under anaerobic dark conditions, that is, under environmental conditions that do not supply light energy. It is a system in which the above lower fatty acid is assimilated by ATP produced in an anaerobic environment and converted into hydrogen, and a large amount of hydrogen is stably produced from organic waste without light energy.
【００１３】本発明方法においては、通常の土壌、各種
の有機性廃水及び／又は各種汚泥など有機物を濃厚に含
む有機性廃棄物に、野性的に生息している偏性嫌気性の
従属栄養性水素生産菌の存在に着目し、該菌の機能を有
効に利用して、有機性廃棄物の主要構成成分である繊維
質、炭水化物、脂肪、蛋白質及び／又は低級脂肪酸など
の有機物を解糖系、ＴＣＡサイクル系で分解、低分子化
する事を第一の特徴としている。この分解過程で菌体内
に生じた余剰電子はヒドロゲナーゼによって水素になっ
て水と共に菌体外に運びだされ（水素生産）、エネルギ
ー生産に伴う還元力、還元雰囲気の調整をＮＡＤの還元
型（ＮＡＤＨ）を生成する事によって調整している。In the method of the present invention, an obligately anaerobic heterotrophic habitat that wildly inhabits organic wastes rich in organic matter such as ordinary soil, various organic wastewaters and / or various sludges. Focusing on the existence of hydrogen-producing bacteria, effectively utilizing the functions of the bacteria, glycolysis system for organic substances such as fibers, carbohydrates, fats, proteins and / or lower fatty acids, which are the main constituents of organic waste. The first feature is that it decomposes in the TCA cycle system and becomes low molecular weight. The surplus electrons generated in the cells during this decomposition process are converted into hydrogen by hydrogenase and carried out of the cells along with water (hydrogen production), and the reducing power and reducing atmosphere associated with energy production are adjusted to the reduced form of NAD (NADH). ) Is adjusted by generating
【００１４】通常の微生物であれば、ミトコンドリアの
内部での電流反応によって、即ち、チトクロームによる
電子伝達によって電子は酸素に付与されて水を生ずると
共に、それに共役して微生物活動、機能の推進力となる
ＡＴＰが適量生成されるが、本発明の主役を演ずる偏性
嫌気性の従属栄養性水素生産菌クロストリジウム（Clos
tridium)属は、電子伝達系としてＮＡＤ→ＦＡＤ→ＵＱ
は備えているが、チトクローム電子伝達系が欠落してい
る。従って、クロストリジウム（Clostridium)属の細菌
群は解糖系、ＴＣＡサイクル系におけるＡＴＰ生産量が
基本的に少なく、そのために、水素発酵反応が吸エルゴ
ン反応となり、完全反応を達成するにはエネルギーが不
足する。そのため発酵消化液中に低級脂肪酸が蓄積し、
水素生産量が光合成微生物に対して３０％程度に過ぎな
い。In the case of ordinary microorganisms, electrons are added to oxygen by the current reaction inside the mitochondria, that is, by electron transfer by cytochromes to generate water, and at the same time, they are coupled with oxygen to drive microbial activity and function. ATP is produced in an appropriate amount, but the obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium (Clos) that plays the main role of the present invention.
The genus tridium is NAD → FAD → UQ as an electron transfer system.
, But lacks the cytochrome electron transport system. Therefore, the bacterial group of the genus Clostridium basically has a small amount of ATP production in the glycolysis system and the TCA cycle system, and therefore the hydrogen fermentation reaction becomes an absorption ergon reaction, and energy is insufficient to achieve a complete reaction. To do. Therefore, lower fatty acids accumulate in the fermentation digestive juice,
Hydrogen production is only about 30% of photosynthetic microorganisms.
【００１５】このクロストリジウム（Clostridium)属と
光合成細菌がグルコースを基質とした場合の発酵反応式
は前記式１及び式２で示したが、クロストリジウム（Cl
ostridium)属による水素発酵では、その反応達成率を１
００％とするには、標準自由エネルギーとして１８４ｋ
ｊ／ｍｏｌ・グルコースが不足し、この熱量は大凡６．
０モルＡＴＰ／モル・グルコースに相当する。本発明の
主役として、自然界に広く生息している通性嫌気性従属
栄養水素生産菌の代表菌属であるエンテロバクター（En
terobacter) 属、或いはクレブシエラ（Klebsiera)属で
代替することも考えられるが、この両菌属も電子伝達系
に不備があり、前段階でのエネルギー生成反応（分解・
異化）である発エルゴン反応から得られる自由エネルギ
ーを、エネルギー消費反応である吸エルゴン反応過程に
伝達する中心的役割を果たしている高エネルギー物質Ａ
ＴＰの生産量が不足し、目的とする物質の生産、即ち水
素生産量が著しく制限される。The fermentation reaction formulas when the genus Clostridium and the photosynthetic bacterium use glucose as a substrate are shown in the above formulas 1 and 2, and the Clostridium (Cl)
In hydrogen fermentation by genus ostridium, the reaction achievement rate is 1
To achieve 100%, the standard free energy is 184k.
There is a shortage of j / mol / glucose, and this calorific value is about 6.
Equivalent to 0 mol ATP / mol glucose. As a protagonist of the present invention, Enterobacter (En), which is a representative genus of facultative anaerobic heterotrophic hydrogen-producing bacteria widely inhabiting in the natural world, is used.
terobacter) or Klebsiera genus may be substituted, but both of these genus also have deficiencies in the electron transfer system, and the energy generation reaction (decomposition
A high-energy substance A that plays a central role in transmitting free energy obtained from the catabolic ergon reaction, which is an energy-consuming reaction, to the absorbing ergon reaction process.
The production amount of TP is insufficient, and the production of the target substance, that is, the hydrogen production amount is significantly limited.
【００１６】また、通性嫌気性細菌を水素生産の目的に
使用すると、余剰電子のはけ口として機能しているハイ
ドロゲナーゼの酵素学的安定化を阻害し、さらに、酸素
混入・混在によりニトロゲナーゼの活性が不可逆的に阻
害される。また、重要な要因として考慮すべき事は、本
発明の対象とする各種の有機性廃棄物は、自然環境に放
置された状態で、本発明の一方の主役であるクロストリ
ジウム（Clostridium)属の細菌群だけが単独で生態系を
構成していることはあり得ず、各種の嫌気生細菌、好気
性細菌が共生・共存しており、所謂混合培養系を構成し
ている事である。本発明と直接関連する重要な、その存
在を無視し得ない細菌にメタン菌があり、従属栄養性水
素生産菌の培養条件によっては、混合培養系でメタン菌
が優占種となり、本発明が本来の目的とする水素生産が
阻害される恐れがある。When a facultative anaerobic bacterium is used for the purpose of hydrogen production, it inhibits the enzymatic stabilization of hydrogenase functioning as an outlet for surplus electrons, and further, by mixing oxygen and mixing nitrogenase. The activity is irreversibly inhibited. Further, what should be considered as an important factor is that various organic wastes of the present invention are bacteria of the genus Clostridium, which is one of the protagonists of the present invention, in a state of being left in a natural environment. The group alone cannot constitute an ecosystem alone, and various anaerobic bacteria and aerobic bacteria coexist and coexist, which is what constitutes a so-called mixed culture system. An important bacterium which is directly related to the present invention and whose existence cannot be ignored is methane bacterium, and depending on the culture conditions of the heterotrophic hydrogen-producing bacterium, methane bacterium becomes the dominant species in the mixed culture system, The original intended hydrogen production may be hindered.
【００１７】然し、クロストリジウム（Clostridium)属
とメタン細菌とは自然環境の中で生活し、かつ増殖する
に必要な最適条件に可成りの格差があることが実験的に
実証・確認されており、この条件格差を人為的に制御す
ることにより、本発明の目的とするクロストリジウム
（Clostridium)属を混合培養系の中で常に優占種として
増殖せしめる事ができる。その制御因子としては次の事
があげられる。 ＊クロストリジウム（Clostridium)属とメタン細菌との
間では増殖速度に可成りの格差がある。 ＊両属の間には、至適ｐＨ範囲に可成りの格差があり、
クロストリジウム（Clostridium)属はｐＨ５．５〜５．
８、メタン細菌はｐＨ７．８前後である。 ＊両属の間には、混合培養液（発酵環境）の好適酸化還
元電位に可成りの格差がある。 ・クロストリジウム（Clostridium)属 −１００〜−２００ｍＶ ・メタン細菌 −３５０〜−４５０ｍＶ ＊両属では、水素に対する感受性が異なり、メタン細菌
に対してクロストリジウム（Clostridium)属は可成り敏
感であり、水素の存在により活性阻害を受けやすい（全
ての微生物類が一般的に水素の害作用を受ける）。However, it has been experimentally verified and confirmed that the genus Clostridium and methane bacteria have a considerable difference in optimal conditions necessary for living and growing in a natural environment. By artificially controlling this condition disparity, the genus Clostridium, which is the object of the present invention, can always be proliferated as a dominant species in a mixed culture system. The control factors are as follows. * There is a considerable disparity in the growth rate between Clostridium and methane bacteria. * There is a considerable difference in the optimum pH range between the two genera,
The genus Clostridium has a pH of 5.5-5.
8. pH of methane bacteria is around 7.8. * There is a considerable disparity between the two genera in the preferred redox potential of the mixed culture solution (fermentation environment).・ Clostridium genus −100 to −200 mV ・ Methane bacterium −350 to −450 mV * Both genus have different susceptibility to hydrogen, and the genus Clostridium is considerably sensitive to methane bacteria, and the presence of hydrogen Is susceptible to activity inhibition by (all microorganisms are generally affected by hydrogen).
【００１８】以上のように、クロストリジウム（Clostr
idium)属とメタン細菌とでは、上記の作用因子に対する
感受性に相当の格差があるので、前記したように、本発
明でも、常にクロストリジウム（Clostridium)属を優占
種とし、確実に、かつ遅退なく水素発酵、水素生産を行
なうために、必要により減圧発酵、或いは微嫌気減圧発
酵を行なう事が望ましい。この事に関しては、先に出願
した特願平５−１９５３２９号に詳細に記載してある。
本発明の第二の特徴は、有機性廃棄物を基質として偏性
嫌気性の従属栄養水素生産菌クロストリジウム（Clostr
idium)から水素を生産する方法において、該菌の足らざ
る能力（ＡＴＰ生産能力）を補完するために特定の光合
成微生物を一定の比率で混合共生培養し、光合成微生物
が嫌気暗条件で生産するＡＴＰを利用して、発酵消化液
に残存している各種の低級脂肪酸を水素に転換し、総水
素生産量として光合成微生物が光エネルギー供給条件下
（嫌気明条件）で達成し得る水素生産量１２モル／モル
・グルコースに匹敵する水素生産量を確実に確保する方
法を提供する事である。As described above, Clostridium (Clostr
Since there is a considerable difference in the susceptibility to the above-mentioned agents between the genus idium) and the methane bacterium, in the present invention, as described above, the genus Clostridium is always the dominant species, and it can be reliably and delayed. In order to carry out hydrogen fermentation and hydrogen production without using it, it is desirable to carry out vacuum fermentation or slightly anaerobic vacuum fermentation as necessary. This matter is described in detail in Japanese Patent Application No. 5-195329 filed earlier.
A second feature of the present invention is that the obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium (Clostrtria) is produced using organic waste as a substrate.
In the method for producing hydrogen from (idium), ATP produced by the photosynthetic microorganisms under anaerobic dark conditions by co-cultivating specific photosynthetic microorganisms at a fixed ratio in order to complement the capacity (ATP production ability) of the bacterium. 12 mol of hydrogen production that can be achieved by photosynthetic microorganisms under light energy supply conditions (anaerobic conditions) as a total hydrogen production amount by converting various lower fatty acids remaining in the fermentation digestive liquid into hydrogen by using It is to provide a method for surely ensuring a hydrogen production amount comparable to that of mol glucose.
【００１９】本発明の特徴１と特徴２における水素発酵
の基本となる反応式は次の通りである。 ＊クロストリジウム（Clostridium)属による水素生産 Ｃ6 Ｈ12Ｏ6 ＋２Ｈ2 Ｏ→２ＣＨ3 ＣＯＯＨ＋４Ｈ2 ＋２ＣＯ2 ・・・・式４ Ｈ2 ／Ｃ6 Ｈ12Ｏ6 ＝４ｍｏｌ／ｍｏｌ ＊光合成微生物による水素生産 ２ＣＨ3 ＣＯＯＨ＋４Ｈ2 Ｏ→８Ｈ2 ＋２ＣＯ2 ・・・・・・式５ Ｈ2 ／Ｃ6 Ｈ12Ｏ6 ＝８ｍｏｌ／ｍｏｌ 式４、式５より、 Ｃ6 Ｈ12Ｏ6 ＋６Ｈ2 Ｏ→１２Ｈ2 ＋６ＣＯ2 ・・・・・ 式６ Ｈ2 ／Ｃ6 Ｈ12Ｏ6 ＝１２ｍｏｌ／ｍｏｌThe reaction formulas that are the basis of hydrogen fermentation in the features 1 and 2 of the present invention are as follows. * Clostridium (Clostridium) genus hydrogen production C 6 H 12 O 6 + 2H 2 O → 2CH 3 COOH = + 4H 2 + 2CO 2 ···· formula 4 H 2 / C 6 H 12 O 6 4mol / mol * Hydrogen production by photosynthetic microorganisms by 2CH 3 COOH + 4H 2 O → 8H 2 + 2CO 2 ... Formula 5 H 2 / C 6 H 12 O 6 = 8 mol / mol From Formula 4 and Formula 5, C 6 H 12 O 6 + 6H 2 O → 12H 2 + 6CO 2 ... Formula 6 H 2 / C 6 H 12 O 6 = 12 mol / mol
【００２０】まず第一に、本発明者が偏性嫌気性水素生
産菌クロストリジウム（Clostridium)と混合培養すべき
相手として光合成細菌に属する紅色硫黄細菌クロマチウ
ム（Chromatium) を選択した理由を説明する。前記した
通り、本発明の処理対象となる有機性廃棄物には、通
常、可成りの濃度の硫化物が含まれており、従って、こ
の種の有機性廃棄物を生物学的に処理する場合、浄化に
関与する微生物は著しく活性阻害を受るのが宿命であ
り、前記特公昭６３−４９９９７号公報で適用している
ロドシュードモナス（Rhodopseudomonas) 属に属する光
合成細菌も例外ではあり得ない。これに対して、クロマ
チウム（Chromatium) 属に属する光合成細菌（紅色硫黄
細菌）は硫黄イオン、チオ硫酸イオンなどの、所謂硫化
物自身を電子供与体として利用する事が可能であり、酵
素ニトロゲナーゼを介在して電子を水素イオンと結合す
る事により水素を生産し、エネルギー生産に伴う還元力
の増加を調整している。First, the reason why the present inventor has selected the red sulfur bacterium Chromatium belonging to the photosynthetic bacterium as a partner to be mixed with the obligate anaerobic hydrogen-producing bacterium Clostridium will be explained. As described above, the organic waste to be treated by the present invention usually contains a considerable concentration of sulfide, and therefore, when treating this type of organic waste biologically. The microorganisms involved in purification are destined to undergo remarkable activity inhibition, and the photosynthetic bacteria belonging to the genus Rhodopseudomonas applied in Japanese Patent Publication No. 63-49997 cannot be an exception. On the other hand, photosynthetic bacteria belonging to the genus Chromatium (purple sulfur bacterium) can use so-called sulfides such as sulfur ions and thiosulfate ions themselves as electron donors, and mediate the enzyme nitrogenase. Then, the electrons are combined with hydrogen ions to produce hydrogen, and the increase in reducing power accompanying energy production is adjusted.
【００２１】また、該菌は硫化物に止まらず、通常の光
合成細菌と同じようにコハク酸、フマール酸、マレイン
酸、酢酸、リンゴ酸糖の低級脂肪酸を電子供与体として
水素を発生することができる（鈴木周一編・バイオマス
・エネルギー変換；三井旭、光合成微生物による水素生
産、講談社・サイエンチフィック、ｐｐ．１９４〜２２
８、１９８３／２）。また、同文献によればクロマチウ
ム（Chromatium) 属は酢酸、ピルビン酸等を従属栄養的
に好んで資化するが、グルコース、フラクトースなどの
炭水化物からは水素を生産することが出来なかったと報
告している。有機性廃棄物を基質として水素を生産する
場合、光合成細菌が炭水化物を電子供与体として利用で
きる機能を備えていることは好ましいが、本発明のよう
に有機性廃棄物をクロストリジウム（Clostridium)属で
異化分解すると、発酵消化液に残存するのは酢酸、酪
酸、プロピオン酸、或いは蟻酸等の低級脂肪酸であり、
此等の低級脂肪酸は充分にクロマチウム（Chromatium)
属の電子供与体となり得るので、クロストリジウム（Cl
ostridium)属とクロマチウム（Chromatium) 属の混合培
養においては後者の細菌が炭水化物を電子供与体として
利用できなくても、本発明に対して致命的な要素とはな
らない。Further, the bacterium is not limited to sulfides and can generate hydrogen by using lower fatty acids such as succinic acid, fumaric acid, maleic acid, acetic acid, and malic acid sugar as electron donors, like ordinary photosynthetic bacteria. Yes (Shuichi Suzuki, Biomass / Energy Conversion; Asahi Mitsui, Hydrogen Production by Photosynthetic Microorganisms, Kodansha / Scientific, pp. 194-22
8, 1983/2). According to the same document, Chromatium genus favors assimilation of acetic acid and pyruvic acid heterotrophically, but reported that hydrogen could not be produced from carbohydrates such as glucose and fructose. There is. When hydrogen is produced using an organic waste as a substrate, it is preferable that the photosynthetic bacterium has a function of utilizing a carbohydrate as an electron donor, but as in the present invention, the organic waste is categorized as Clostridium. After catabolism, what remains in the fermentation digestive juice are lower fatty acids such as acetic acid, butyric acid, propionic acid, or formic acid.
These lower fatty acids are sufficient for Chromatium
Since it can be an electron donor of the genus, Clostridium (Cl
In the mixed culture of the genus ostridium and the genus Chromatium, even if the latter bacterium cannot utilize the carbohydrate as an electron donor, it is not a fatal element for the present invention.
【００２２】光合成細菌は、通常、太陽エネルギーの供
給によりＡＴＰが理論的に無限に生産されるので、標準
自由エネルギーが正となる吸エルゴン反応であっても完
全に進行し、例えば酢酸を基質とした場合、次に示すよ
うに反応は完結し、理論値の水素と炭酸ガスを生産す
る。 ２ＣＨ3 ＣＯＯＨ＋４Ｈ2 Ｏ→８Ｈ2 ＋４ＣＯ2 ＋（１００ｋｊ／ｍｏｌ） さらに本発明に対して有利な事にクロマチウム（Chroma
tium) 属は、嫌気暗、即ち光エネルギー照射が行なわれ
ない環境、従って菌体内での光化学系が作動しない状態
でも酢酸、ケト・グルコン酸を始め、ある種の多糖類を
資化して水素を生産する機能を具備している事である。In photosynthetic bacteria, ATP is theoretically infinitely produced normally by the supply of solar energy, and therefore, even if the absorption ergon reaction in which the standard free energy is positive is completely advanced, for example, acetic acid is used as a substrate. In that case, the reaction is completed and the theoretical values of hydrogen and carbon dioxide are produced as shown below. 2CH 3 COOH + 4H 2 O → 8H 2 + 4CO 2 + (100 kj / mol) Further, it is advantageous for the present invention that chromate (Chroma
The genus tium) anaerobically darkens, i.e., in an environment where light energy is not irradiated, that is, in a state where the photosystem in the fungus body does not operate, acetic acid, keto-gluconic acid, and other polysaccharides are assimilated to produce hydrogen. It has the function of producing.
【００２３】また、低級脂肪酸と、ある種の有機物を嫌
気暗条件で分解する過程、即ち、解糖系とＴＣＡサイク
ル系（酸発酵工程）で水素生産による還元力の増大をＮ
ＡＤＨの生成により調整し、この反応に共役する反応に
より高エネルギー物質であるＡＴＰを生産する事ができ
る。クロマチウム（Chromatium) の嫌気暗条件下でのＡ
ＴＰ生産量に関する確たるデータはないが、解糖系及び
ＴＣＡサイクル系でのＡＴＰ生産量はその代謝サイクル
から推測して３〜４ＡＴＰと考えるのが妥当である。こ
の数値を基礎にして、クロストリジウム（Clostridium)
属とクロマチウム（Chromatium) 属の混合共生培養（嫌
気暗培養）での全生物反応による理論的なＡＴＰ生産量
（基質としてグルコースを想定する）と各段階でのＡＴ
Ｐ消費量を計算したのが表１であるが、この計算結果か
らグルコースを完全分解して１２モルの水素を生産する
に必要なＡＴＰ不足量、及びクロマチウム（Chromatiu
m) 属により補完されるＡＴＰ量が明確となる。In addition, the process of decomposing lower fatty acids and certain organic substances under anaerobic dark conditions, that is, the increase in reducing power by hydrogen production in the glycolysis system and the TCA cycle system (acid fermentation process) is
ATP, which is a high energy substance, can be produced by a reaction that is adjusted by the production of ADH and is coupled to this reaction. Chromatium A under anaerobic dark conditions
Although there is no definite data on the TP production amount, it is appropriate to assume that the ATP production amount in the glycolysis system and the TCA cycle system is 3 to 4 ATP inferred from the metabolic cycle. Based on this figure, Clostridium
Theoretical ATP production (assuming glucose as substrate) and AT at each stage in all biological reactions in mixed co-cultivation of genus and Chromatium (anaerobic dark culture)
Table 1 shows the calculation of P consumption. From the calculation results, the ATP deficiency necessary for completely decomposing glucose to produce 12 mol of hydrogen and the amount of Chromatium (Chromatiu)
m) The amount of ATP complemented by the genus becomes clear.
【００２４】[0024]
【表１】 [Table 1]
【００２５】表１から容易に理解できるように、クロス
トリジウム（Clostridium)属の反応系でのＡＴＰ生産量
は７ｍｏｌ・ＡＴＰ／ｍｏｌ・Ｃ6 Ｈ12Ｏ6 、クロマチ
ウム（Chromatium) 属の反応系でのＡＴＰ生産量は３〜
４ＡＴＰ、合計１０〜１１ＡＴＰであるのに対して、水
素生産の全反応に要するＡＴＰは１０．２ＡＴＰであ
り、従って、有機性廃棄物から水素生産する場合、偏性
嫌気性水素生産菌と水素生産性光合成細菌クロマチウム
（Chromatium属) を混合培養すれば、有機物の完全分解
と理論量の水素を生産する事ができる。本発明者は、淡
水性のクロマチウム（Chromatium) 属を長野県下の硫黄
泉の湧出水から容易に発見し、また、海産性のクロマチ
ウム（Chromatium) 属を八重山列島の石垣島海岸に漂着
している大型海藻（Macroalgae) の葉面に大量に付着増
殖している菌群として採取した。これら自然界に広く分
布しており、従って本発明は特定の菌株の性状に依存す
るものではない。As can be easily understood from Table 1, the ATP production amount in the reaction system of the genus Clostridium is 7 mol.ATP / mol.C 6 H 12 O 6 and the reaction system of the genus Chromatium. ATP production is 3 ~
4ATP, 10-11 ATP in total, 10.2 ATP is required for the whole reaction of hydrogen production. Therefore, when hydrogen is produced from organic waste, obligate anaerobic hydrogen-producing bacteria and hydrogen production are required. By mixing and cultivating the sex photosynthetic bacterium Chromatium (Chromatium spp.), It is possible to completely decompose organic matter and produce a theoretical amount of hydrogen. The present inventor easily found a freshwater Chromatium genus from the spring water of a sulfur spring in Nagano Prefecture, and also drifted a marine Chromatium genus on the Ishigaki coast of the Yaeyama Archipelago. A large group of macroalgae (Macroalgae) was collected as a bacterial group that adhered to and grew on the leaf surface. It is widely distributed in these natural environments, and thus the present invention does not depend on the properties of a particular strain.
【００２６】本発明者が発見したこの淡水性、海産性の
クロマチウム（Chromatium) 属については細菌群として
の水素生産能を測定したが、嫌気明条件では２〜３μｍ
ｏｌ／ｍｇ−ｃｅｌｌ／ｈｒの高い水素生産能力を示し
た。然し、嫌気暗条件では微量の水素しか生産しなかっ
た。淡水性クロマチウム（Chromatium) 属の嫌気暗条件
下での集積培養はＡＴＣＣのＭＥＤＩＡ ＨＡＮＤＢＯ
ＯＫの液体培地から選択し、基本的にはＭｅｄｉａＦｏ
ｒｍｕｌａｔｉｏｎｓ ＡＴＣＣ３７を使用し、有機物
成分として酢酸塩３，０００ｇ／リットル、酵母抽出液
１，５００ｇ／リットルを加えた。また、海産性クロマ
チウム（Chromatium) 属の嫌気暗培養液としては、湘南
海岸の清浄な海水を用い、ＡＴＣＣ３７を基礎とする各
種成分を前記海水に溶解して滅菌し、培養した。クロマ
チウム（Chromatium) 属の菌体集積物は、前記の温泉水
及び大型海藻の葉面に付着した菌体を物理的に剥離し、
遠心分離器（３，０００Ｇ、１０分間）で濃縮し、それ
ぞれを前記の基礎培養液に接種し、２リットルの嫌気性
暗培養槽に培養液１リットルを張込み、気相部の空気を
窒素ガスで置換し、３０℃で培養した。With respect to this freshwater, marine Chromatium genus discovered by the present inventor, the hydrogen-producing ability as a bacterial group was measured.
It showed a high hydrogen production capacity of ol / mg-cell / hr. However, it produced only a small amount of hydrogen under anaerobic and dark conditions. The enrichment culture of freshwater Chromatium genus under anaerobic dark conditions is ATCC MEDIA HANDBO.
Select from OK liquid medium, basically MediaFo
Rumulations ATCC 37 was used, and 3,000 g / liter of acetate and 1,500 g / liter of yeast extract were added as organic components. As the anaerobic dark culture liquid of the marine Chromatium genus, clean seawater from Shonan coast was used, and various components based on ATCC37 were dissolved in the seawater and sterilized. Chromatium (Chromatium) genus of bacterial cells is physically detached from the hot spring water and the large seaweed leaf surfaces,
Concentrate with a centrifuge (3,000 G, 10 minutes), inoculate each of them into the above-mentioned basal culture solution, add 1 liter of the culture solution to a 2 liter anaerobic dark culture tank, and replace the air in the gas phase with nitrogen. It was replaced with gas and incubated at 30 ° C.
【００２７】培養当初から成分不明のガスが直ちに発生
したが、約１０日後からガス発生が激しくなり、ガスク
ロマトグラフで測定した結果、微量の水素と可成りの炭
酸ガス、及び微量の窒素であることが確認された。以上
の半定量的所見は、淡水性クロマチウム（Chromatium)
属、海産性クロマチウム（Chromatium) 属の細菌群につ
いてのものであり、純粋に分離した単一菌に関しての所
見ではない。然し、実際の濃厚廃水、各種汚泥などの有
機性廃棄物を対象としてクロストリジウム（Clostridiu
m)属とクロマチウム（Chromatium) 属の混合培養系から
水素生産を行なう場合には、純粋菌同士の混合培養を行
なうよりも、それぞれ、各種の菌種が混合した、所謂細
菌群集を混合培養する方が自由度の大きい、また幅のあ
る生態系の共生関係を構築することができ、外部からの
環境変動に対して順応性、適応性、抵抗力があり、それ
ぞれの細菌の機能の補完、機能の増幅が期待される。従
って、本発明では特定の純粋菌の混合培養を避け、各種
細菌群の混合培養法を意識的に選択したものである。A gas of unknown composition was immediately generated from the beginning of the culture, but the gas generation became violent after about 10 days. As a result of measurement with a gas chromatograph, a trace amount of hydrogen, a considerable amount of carbon dioxide gas, and a trace amount of nitrogen were found. Was confirmed. The above semi-quantitative findings are based on freshwater Chromatium.
Genus, marine Chromatium genus, not purely isolated single fungus. However, for actual wastewater, organic waste such as various sludges, Clostridium (Clostridiu)
When hydrogen is produced from a mixed culture system of the genus m) and the genus Chromatium, a so-called bacterial community in which various bacterial species are mixed is mixed and cultivated, rather than a mixed culture of pure bacteria. Is more flexible and has a wider range of symbiotic relationships of ecosystems, is more adaptable, adaptable, and resistant to external environmental changes, and complements the functions of each bacterium. Amplification of function is expected. Therefore, in the present invention, the mixed culture method of various bacterial groups is intentionally selected while avoiding the mixed culture of specific pure bacteria.
【００２８】因みに、前記の淡水性クロマチウム（Chro
matium) 属、及び海産性クロマチウム（Chromatium) 属
を、それぞれの培養液で培養した結果、次に示すクロマ
チウム（Chromatium) 属の菌種の類縁菌（近縁細菌）が
検出された。 ＊淡水性クロマチウム（Chromatium) 属 ・クロマチウム テピダム（Chromatium tepidum) (ATCC 43061) ・クロマチウム ビノサム（Chromatium vinosum) (ATCC 17899) ＊海産性クロマチウム（Chromatium) 属 ・クロマチウム ブデリ（Chromatium buderi) (ATCC 25588) ・クロマチウム ビノサム（Chromatium vinosum) (ATCC 35206) 以上、本発明は、偏性嫌気性水素生産菌クロストリジウ
ム（Clostridium)属と水素生産性光合成細菌クロマチウ
ム（Chromatium) 属のそれぞれの細菌群集を混合培養
し、クロストリジウム（Clostridium)属の単独培養では
不足するＡＴＰ生産量を、嫌気暗条件下で光合成細菌ク
ロマチウム（Chromatium) 属と混合培養し、該菌が生産
するＡＴＰによって補完することにより、さらに各細菌
群集を混合培養することにより、安定してほぼ理論量の
水素を生産することができる。Incidentally, the above-mentioned fresh water chromatium (Chro
As a result of culturing the matium) genus and the marine genus Chromatium in their respective culture solutions, the following related strains (related bacteria) of the genus Chromatium were detected. ＊ Freshwater Chromatium genus ・ Chromatium tepidum (ATCC 43061) ・ Chromatium vinosum (ATCC 17899) ＊ Marine Chromatium genus ・ Chromatium 25588 ・ AT Chromatium vinosum (ATCC 35206) As described above, according to the present invention, bacterial communities of the anaerobic anaerobic hydrogen-producing bacterium Clostridium and the hydrogen-producing photosynthetic bacterium Chromatium are mixed and cultured, and (Clostridium) single culture deficiency in ATP production is mixed with the photosynthetic bacterium Chromatium genus under anaerobic dark conditions and supplemented with ATP produced by the bacterium to further mix each bacterial community. By culturing, almost theoretical amount of hydrogen can be stably produced.
【００２９】光合成微生物に属する微細藻類、特に緑藻
類、藍藻類の中には水素を生産するものが多く、通常、
緑藻類は水素生産にヒドロゲナーゼ、藍藻類はニトロゲ
ナーゼを利用すると云われ、その代表的な属種には次の
ようなものがある。 ＊緑藻 ・セネデスムス（Scenedesmus) ・クロレラ（Chlorella) ・クラミドモナス (Chlamydomonas) ＊藍藻 ・アナベナ（Anabaena) ・ノストック（Nostoc) ・オシラトリア (Oscillatoria) 藍藻の多くは他の微細藻類とは異なり、窒素固定能力を
有し、窒素ガスが存在しない時にのみ光水素生産を行な
うと云われる。Many microalgae belonging to photosynthetic microorganisms, especially green algae and cyanobacteria, produce hydrogen, and
It is said that green algae utilize hydrogenase for hydrogen production, and cyanobacteria utilize nitrogenase. Typical genera include the following. ＊ Green algae ・ Scenedesmus ・ Chlorella ・ Chlamydomonas ＊ Cyanobacteria ・ Anabaena ・ Nostoc ・ Oscillatoria Many cyanobacteria are different from other microalgae and have nitrogen-fixing ability. And it is said to carry out photohydrogen production only when nitrogen gas is not present.
【００３０】藍藻は基本的には独立栄養的な生き方が主
流であるが、同時に従属栄養的な生き方をするものが多
く、微細藻類のなかでは特殊な存在として位置ずけされ
ている。例えば、 無酸素状態で光エネルギーが与えられると、硫化水
素を酸化して光合成を行なう。 多くの藍藻は水素分子を酸化し、得られる電子を利
用して光合成を行なう。 また、グルコースのような有機物を酸化して光合成
を行なう能力がある。 酸素が存在しない状態で有機物を酸化分解し、生ず
るエネルギーをＡＴＰという形で利用し、過剰分は菌体
外に放出し、他の吸エルゴン反応に供給する。 藍藻が有機物を酸化分解する過程は、通常の細菌類
と同じ代謝経路を取り、例えば嫌気暗の条件では解糖
系、ＴＣＡサイクル系により有機物を異化代謝し、この
過程でＡＴＰを生産し、同時に反応系の酸化還元状態を
調整するためにＮＡＤ（Ｐ）Ｈが生産される。Basically, cyanobacteria mainly have an autotrophic way of life, but at the same time, many of them have a heterotrophic way of life, and are positioned as a special existence among microalgae. For example, when light energy is given in anoxic state, hydrogen sulfide is oxidized to perform photosynthesis. Many cyanobacteria oxidize hydrogen molecules and utilize the resulting electrons for photosynthesis. It also has the ability to oxidize organic substances such as glucose to perform photosynthesis. The organic matter is oxidatively decomposed in the absence of oxygen, and the resulting energy is utilized in the form of ATP. The excess is released outside the cells and supplied to other sucking ergon reaction. The process by which cyanobacteria oxidize and decompose organic matter takes the same metabolic pathway as ordinary bacteria, for example, under anaerobic dark conditions, catabolism and metabolism of organic matter by glycolysis and TCA cycle system, and at the same time ATP is produced, NAD (P) H is produced in order to adjust the redox state of the reaction system.
【００３１】藍藻類は環境変動に対して順応性、対応性
に優れている。これらの中でもオッシラトリア（Oscill
atoria) はその性能が特に優れており、本発明のクロス
トリジウム（Clostridium)属との混合共生培養による水
素生産の相手として限りない利点を発現するので、この
属種の藍藻を選択した。オッシラトリア（Oscillatori
a) 属に関する研究は、「光合成微生物による水素生
産」（鈴木周一編；バイオマス・エネルギー変換、講談
社・サイエンチフィック、ｐｐ．１９４〜２２８〔１９
８３〕）に総括・報告されている。水素生産能力の強力
なオッシラトリア（Oscillatoria) として、オッシラト
リア（Oscillatoria) sp.Miami BG7が知られており、嫌
気明の培養条件ではあるが、次のような研究報告があ
る。 ＊嫌気明での水素生産反応式 グリコゲン＋ (n-1)Ｈ2 Ｏ → ｎＣ6 Ｈ12Ｏ6 Ｃ6 Ｈ12Ｏ6 ＋６Ｈ2 Ｏ → ６ＣＯ2 ＋１２Ｈ2 ＊水素生産能力 ・浮遊菌体 △Ｈ2 ＝５．５μｍｏｌ／ｍｇ・ｐｒｏｔｅｉｎ・ｈｒ ・固定化菌体 △Ｈ2 ＝２２０〜３００ｍｌ／ｇ・ｃｅｌｌ・ｄａｙCyanobacteria are excellent in adaptability and adaptability to environmental changes. Among these, Oscillatoria
atoria) is particularly excellent in its performance and expresses unlimited advantages as a partner for hydrogen production by the mixed co-cultivation with the genus Clostridium of the present invention, and thus, cyanobacteria of this genus was selected. Oscillatori
a) Research on genera is described in “Hydrogen Production by Photosynthetic Microorganisms” (edited by Shuichi Suzuki; Biomass / Energy Conversion, Kodansha Scientific, pp. 194-228 [19]).
83]). Oscillatoria sp. Miami BG7 is known as Oscillatoria having a strong hydrogen production capacity, and the following research reports have been made under anaerobic culture conditions. ＊ Anaerobic hydrogen production reaction formula Glycogen + (n-1) H 2 O → nC 6 H 12 O 6 C 6 H 12 O 6 + 6H 2 O → 6CO 2 + 12H 2 ＊ Hydrogen production capacity ・ Floating cells △ H 2 = 5.5 μmol / mg · protein · hr · Immobilized cells ΔH 2 = 220 to 300 ml / g · cell · day
【００３２】このＢＧ７は異型細胞を持たず、その水素
生成系はニトロゲナーゼとヒドロゲナーゼの両方の酵素
に触媒され、大部分の水素生成反応はニトロゲナーゼに
よって触媒されるが、約１０〜２０％の水素生成はヒド
ロゲナーゼによるものと推定されると結論している。ま
た、ＢＧ７は、従来、水素生産に用いられていたアナベ
ナ（Anabaena) 属、ノストック（Nostoc) 属などに比較
して吸収型ヒドロゲナーゼ活性を持たないという特異的
な性質があるので、水素生産の面から有利であるとい
う。本発明は、藍藻に嫌気明における水素生産を期待し
ているのではなく、嫌気暗におけるＡＴＰ生産に期待す
る新規のエネルギー生産技術である。このオッシラトリ
ア（Oscillatoria) 属は、前記した通り、光エネルギー
が存在しない、所謂、嫌気暗の条件下でも環境変動に対
して順応性があり、基質として糖類、低級脂肪酸類を異
化及び資化して、通常の微生物が嫌気性、好気性で有機
物を分解すると同様な代謝への容易な転換が可能であ
り、解糖系、ＴＣＡサイクル系で高エネルギー物質ＡＴ
ＰとＮＡＤの還元型を生成し、最終的に有機物を理論的
に完全分解する。This BG7 does not have atypical cells, its hydrogen production system is catalyzed by both enzymes of nitrogenase and hydrogenase, and most hydrogen production reactions are catalyzed by nitrogenase, but about 10 to 20% of hydrogen production. Is presumed to be due to hydrogenase. In addition, BG7 has a specific property that it does not have the absorption hydrogenase activity as compared with the genus Anabaena and the genus Nostoc which have been conventionally used for hydrogen production, and therefore, BG7 has a specific property of hydrogen production. It is said to be advantageous from the aspect. The present invention is a novel energy production technology that does not expect cyanobacteria to produce hydrogen in anaerobic light, but expects ATP production in anaerobic darkness. As described above, the genus Oscillatoria is adaptable to environmental changes even under conditions of so-called anaerobic darkness in which light energy does not exist, and catabolizes and assimilates sugars and lower fatty acids as substrates, When ordinary microorganisms are anaerobic or aerobic and decompose organic substances, they can be easily converted into the same kind of metabolism, which is a high energy substance AT in glycolysis and TCA cycle systems.
The reduced forms of P and NAD are produced, and finally the organic matter is theoretically completely decomposed.
【００３３】この原核生物であるオッシラトリア（Osci
llatoria) 属が嫌気暗条件で有機物を分解しＡＴＰ，Ｎ
ＡＤ（Ｐ）Ｈを生産するモデルは図１の通りである。オ
ッシラトリア（Oscillatoria) 属による嫌気暗での有機
物分解過程は、解糖系、ＴＣＡサイクル系、及びチトク
ローム（Cytochrome) 系による電子伝達系によって構成
されているが、多糖類を基質とした場合、解糖系におい
ては３ＡＴＰ、ＴＣＡサイクル系では２ＡＴＰ、合計５
ＡＴＰの生産が予測される。本発明で主役を演ずる偏性
嫌気性水素生産菌クロストリジウム（Clostridium)属
は、式４にグルコースを基質とした場合の水素発酵反応
式のように、異化工程におけるＡＴＰ生産量が少量であ
るために吸エルゴン反応であり、水素発生量が少ないだ
けでなく、発酵消化液に多量の低級脂肪酸が蓄積する。This prokaryote, Oscitria
(Llatoria) genus decomposes organic matter under anaerobic and dark conditions to produce ATP, N
A model for producing AD (P) H is shown in FIG. The process of decomposing organic matter in the genus Oscillatoria under anaerobic darkness is composed of a glycolytic system, a TCA cycle system, and an electron transfer system of the Cytochrome system. When a polysaccharide is used as a substrate, glycolysis is performed. 3 ATP in the system, 2 ATP in the TCA cycle system, total 5
ATP production is predicted. The obligate anaerobic hydrogen-producing bacterium Clostridium, which plays the main role in the present invention, has a small amount of ATP produced in the catabolism step, as in the hydrogen fermentation reaction formula when glucose is used as the substrate in Formula 4. It is an absorption ergon reaction and not only produces a small amount of hydrogen but also accumulates a large amount of lower fatty acids in the fermentation digestive juice.
【００３４】この水素発酵反応式を完結させるために
は、表１に示したように約２．２ｍｏｌのＡＴＰが不足
する。従って本発明のもう一つの構成要因であるクロス
トリジウム（Clostridium)属と光合成微生物の一種であ
る藍藻オッシラトリア（Oscillatoria) 属を混合し、共
生培養することにより式４の水素生産反応式を完結する
に十分なＡＴＰが供給され、式５の生物反応が遅退なく
進行し、嫌気暗条件で式６が完結し、理論的にグルコー
ス１モル当り１２モルの水素を生産することができる。
このように、クロストリジウム（Clostridium)属群と水
素生産性藍藻オッシラトリア（Oscillatoria) 属群を嫌
気暗で混合培養することにより、従来、嫌気明条件下で
水からの水素生産でしか達成できなかった１２モルの水
素生産を達成することが可能となり、同時に水域の強烈
な汚濁源である有機性固形物をも分解することができる
ようになり、実用化の障害が完全に消去された。In order to complete this hydrogen fermentation reaction formula, about 2.2 mol of ATP is insufficient as shown in Table 1. Therefore, it is sufficient to complete the hydrogen production reaction formula of Formula 4 by mixing the genus Clostridium, which is another constituent factor of the present invention, and the genus Oscillatoria, a kind of photosynthetic microorganism, and co-culturing. ATP is supplied, the biological reaction of Formula 5 proceeds without delay, Formula 6 is completed under anaerobic dark conditions, and theoretically 12 mol of hydrogen can be produced per mol of glucose.
As described above, by cultivating the Clostridium genus group and the hydrogen-producing cyanobacterium Oscillatoria genus group in a mixed culture in an anaerobic dark condition, hydrogen production from water was conventionally achieved only under anaerobic light conditions. It has become possible to achieve molar hydrogen production, and at the same time, to decompose organic solid matter, which is a source of intense pollution in water bodies, completely eliminating obstacles to practical use.
【００３５】同属種の藍藻は淡水性、海産性とも各水域
に広く分布しており、本発明者は長野県、島根県、鹿児
島県の天然湖沼から強力な水素生産能力を持つ淡水性、
汽水性のオッシラトリア（Oscillatoria) 属群を採取し
た。即ち本発明は特定菌株の性状に依存するものではな
い。淡水性オッシラトリア（Oscillatoria) 属の嫌気暗
条件における集積培養はＡＴＣＣの６１６液体培地を基
本培地としたが、これに有機物としてグルコース３，０
００ｍｇ／リットル、酵母抽出液（エキス）１，５００
ｍｇ／リットルを加えた。オッシラトリア（Oscillator
ia) 属の菌体集積物は前記の湖沼水を１リットル採取し
て遠心分離器（３，０００Ｇ、１０分間）で濃縮し、こ
の濃縮物を２リットルの嫌気暗培養装置に接種し、これ
に前記の培養液を１リットル張込み、クロマチウム（Ch
romatium) 属の培養と同様に気相部の空気を窒素ガスで
置換し、３０℃で回分的に培養した。培養を開始してか
ら数日経過してから徐々にガス発生が認められたが、次
第にガス発生が活発となり、発生ガス組成をガスクロマ
トグラフで測定した結果、クロマチウム（Chromatium)
属よりは水素ガスの発生量が多かったが、それ以外のガ
スは殆ど炭酸ガスによって占められていた。Cyanobacteria of the same genus are widely distributed in each body of water, both freshwater and marine. The present inventor has found that freshwater having a strong hydrogen production capacity from natural lakes in Nagano, Shimane and Kagoshima prefectures,
A brackish-water group of the genus Oscillatoria was collected. That is, the present invention does not depend on the properties of the specific strain. In the enrichment culture of freshwater Oscillatoria under anaerobic dark conditions, ATCC 616 liquid medium was used as a basic medium, and glucose 3,0 was added as an organic matter.
00 mg / liter, yeast extract (extract) 1,500
mg / l was added. Oscillator
As for the ia) cell aggregate, 1 liter of the lake water was sampled and concentrated with a centrifuge (3,000 G, 10 minutes), and the concentrate was inoculated into a 2 liter anaerobic dark culture device. Add 1 liter of the above culture solution to
Similarly to the culture of the genus romatium, the air in the gas phase was replaced with nitrogen gas, and the culture was carried out batchwise at 30 ° C. Gas generation was gradually observed several days after the start of culture, but the gas generation gradually became active, and the gas composition was measured by a gas chromatograph. Chromatium
The amount of hydrogen gas generated was higher than that of the genera, but the other gases were mostly occupied by carbon dioxide.
【００３６】以上の半定量的所見は淡水性オッシラトリ
ア（Oscillatoria) 属の混合細菌群について得られたも
のであり、純粋に分離した単一菌に関して得られたもの
ではない。然し、実際の濃厚廃水、各種汚泥等の有機性
廃棄物を対象としてクロストリジウム（Clostridium)属
とオッシラトリア（Oscillatoria) 属の混合共生培養に
より水素生産を行なう場合には、クロマチウム（Chroma
tium) 属との混合共生培養に関する項で述べたように、
純菌同士の混合培養を行なうよりも、それぞれ各種の菌
種が混合した、所謂細菌群集を混合培養するほうが幅広
い生態系を構築することができ、外部からの環境変動に
対して順応性、適応性が強く、それぞれの微生物群の機
能の相互補完、機能の増殖が期待される。従って、本発
明では、意識的に特定の純粋菌の混合共生培養を避け、
各種菌群の混合培養を選択した。The above semi-quantitative findings have been obtained with a mixed bacterial population of the freshwater genus Oscillatoria, not with a purely isolated single bacterium. However, when hydrogen is produced by mixed co-cultivation of Clostridium and Oscillatoria for organic waste such as concentrated wastewater and various sludges, Chromatium (Chromatium)
As mentioned in the section on mixed co-cultivation with genus tium,
It is possible to construct a wider ecosystem by mixing and culturing so-called bacterial communities in which various bacterial species are mixed, rather than performing mixed culture of pure bacteria, and it is possible to adapt and adapt to environmental changes from the outside. It has strong properties and is expected to complement each other's microbial functions and to multiply their functions. Therefore, in the present invention, intentionally avoiding mixed co-cultivation of specific pure bacteria,
Mixed cultures of various bacterial groups were selected.
【００３７】前記の３湖沼の淡水性、汽水性オッシラト
リア（Oscillatoria) 属をＡＴＣＣ６１６培養液で培養
した結果、次に示すオッシラトリア（Oscillatoria) 属
の類縁菌らしき藍藻類が検出された。 ＊汽水性オッシラトリア（Oscillatoria) 属 ・Oscillatoria sp. (ATCC 29215) ・Oscillatoria sp. (ATCC 29135) ＊淡水性オッシラトリア（Oscillatoria) 属 ・Oscillatoria sp. (ATCC 29205) ・Oscillatoria sp. (ATCC 27935) 以上、本発明は、偏性嫌気性水素生産菌クロストリジウ
ム（Clostridium)属による水素発酵がＡＴＰ生産量不足
により吸エルゴン反応となり、従って有機物の完全分解
が完結しないために水素生産量が少ないだけでなく、発
酵消化液に多量の低級脂肪酸が蓄積するために２次処理
が不可欠であるという宿命的な欠陥を根本的に解消する
事を目的として発明された革新的な水素生産技術、方法
である。As a result of culturing the freshwater and brackish water genus Oscillatoria of the above-mentioned three lakes in the ATCC616 culture solution, the following cyanobacteria of the genus Oscillatoria were detected. * Brackish water genus Oscillatoria-Oscillatoria sp. (ATCC 29215) -Oscillatoria sp. (ATCC 29135) * Fresh water genus Oscillatoria-Oscillatoria sp. (ATCC 29205) -Oscillatoria sp. (ATCC 27935) and above INDUSTRIAL APPLICABILITY According to the present invention, hydrogen fermentation by an obligate anaerobic hydrogen-producing bacterium, Clostridium, becomes an adsorption ergon reaction due to a shortage of ATP production amount. It is an innovative hydrogen production technology and method invented with the purpose of fundamentally eliminating the fatal defect that secondary treatment is indispensable for accumulating a large amount of lower fatty acids in digestive juice.
【００３８】この技術的な問題点を抜本的に改革、改善
するために本発明では、偏性嫌気性水素生産菌クロスト
リジウム（Clostridium)属と光合成微生物に属する光合
成細菌クロマチウム（Chromatium) 属及び／又は藍藻オ
ッシラトリア（Oscillatoria) 属を嫌気暗条件で混合共
生培養し、クロストリジウム（Clostridium)属だけの生
産量では水素発酵反応を完結するに不足するＡＴＰ量を
光合成微生物のＡＴＰ生産量で補完し、前記の生物反応
を完結せしめ、汚染源となる有機物を分解すると同時に
大量の水素を生産する事を目的とした新規の発想、思想
による革新的なエネルギー生産方法である。本発明が、
光合成微生物に水素生産を期待しているのではなく、Ａ
ＴＰ生産を期待しているという事実を認識する必要があ
る。In order to drastically reform and improve this technical problem, the present invention provides an obligate anaerobic hydrogen-producing bacterium, Clostridium, and a photosynthetic bacterium, Chromatium, and / or a photosynthetic bacterium belonging to a photosynthetic microorganism. The cyanobacterium Oscillatoria genus is mixed and co-cultivated in an anaerobic dark condition, and the amount of ATP produced by the genus Clostridium is insufficient to complete the hydrogen fermentation reaction, and the amount of ATP produced by the photosynthetic microorganism is supplemented by It is an innovative energy production method based on a new idea and idea aimed at completing biological reactions and decomposing organic substances that are pollution sources, and at the same time producing a large amount of hydrogen. The present invention
Rather than expecting photosynthetic microorganisms to produce hydrogen,
It is necessary to recognize the fact that we are expecting TP production.
【００３９】[0039]
【実施例】以下に、本発明を実施例により具体的に説明
するが、本発明はこれらの実施例に限定されるものでは
ない。 実施例１：ロドシュードモナス（Rhodopseudomonas)
属、クロマチウム（Chromatium) 属群、及びオッシラト
リア（Oscillatoria) 属群の硫化物耐性濃厚有機性廃
水、各種汚泥等の有機性廃棄物には殆ど例外なく可成り
高濃度の硫化物を含んでいる。従って、有機性廃棄物を
基質として、本発明の水素生産法により安定して多量の
水素を生産するには、本発明に関与する微生物が硫化物
耐性を保有していなければ現実的に成り立たない。有機
物が多量に存在する環境に野性的に生息している偏性嫌
気性の従属栄養性水素生産菌クロストリジウム（Clostr
idium)属は本来硫化物耐性が強いが、該菌と混合共生培
養する光合成微生物は基本的には硫化物に対して感受性
が強く、硫化物耐性が弱い。各微生物の供試試料のう
ち、クロマチウム（Chromatium) 属群、及びオッシラト
リア（Oscillatoria) 属群の試料、使用培地、培養条
件、嫌気暗培養装置は次に記載したとおりであり、ロド
シュードモナス（Rhodopseudomonas) 属の各条件と共
に、表２に総括すると次の通りである。EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples. Example 1: Rhodopseudomonas
Almost all organic wastes such as sulfide-resistant concentrated organic wastewater and various sludges of the genera, Chromatium group, and Oscillatoria group contain a considerably high concentration of sulfide. Therefore, in order to stably produce a large amount of hydrogen by the hydrogen production method of the present invention using organic waste as a substrate, it is not practically possible unless the microorganism involved in the present invention has sulfide resistance. . The obligately anaerobic heterotrophic hydrogen-producing bacterium Clostridium (Clostr) that wildly inhabits an environment rich in organic matter
The genus idium) is originally highly resistant to sulfides, but photosynthetic microorganisms mixed and co-cultured with the bacterium are basically sensitive to sulfides and weakly resistant to sulfides. Among the test samples of each microorganism, the samples of Chromatium genus group and Oscillatoria genus group, the culture medium used, the culture conditions, and the anaerobic dark culture device are as described below. Rhodopseudomonas The conditions of each genus are summarized in Table 2 as follows.
【００４０】[0040]
【表２】 [Table 2]
【００４１】硫黄泉から得た淡水性クロマチウム（Chro
matium) 属群は、同温泉水を数リットル採取し、遠心分
離器で分離濃縮した菌体群を２リットルの嫌気暗培養槽
に接種し、表２に示した培養液を１リットル張込み、気
相部の空気を窒素ガスで置換したのち、３０℃の温度条
件で２０日間回分培養を行なった。ロドバクター カプ
スラタス（Rhodobacter capsulatus) ＡＴＣＣ １１１
６６株はＡＴＣＣから購入し、ＡＴＣＣ１１３９培地に
表２に示した濃度のグルコースと酵母エキスを加え、ス
ラント培養した菌体を液体培養液１リットルに１白金耳
接種し、培養条件、方法はクロマチウム（Chromatium)
属群の培養に準じて行なった。オッシラトリア（Oscill
atoria) 属群の培養も前記に準じて行なったが、ロドバ
クター カプスラタス（Rhodobacter capsulatus) ＡＴ
ＣＣ １１１６６株、及びオッシラトリア（Oscillator
ia) 属群の嫌気暗培養では、当初は硫黄イオンは添加せ
ず、回分培養を開始してから１０日目にＮａ2 Ｓ・９Ｈ
2 Ｏを硫黄イオンとして１，０００ｍｇ／リットルとな
るように注入し、全ての細菌について２０日間における
菌数変動（菌体濃度）を測定した。回分培養実験の結果
を図２に示した。図において、●−●はクロマチウム属
群（ａ）、〇−〇はオッシラトリア属群（ｂ）、▲−▲
はロドバクター カプスラタスＡＴＣＣ １１１６６株
（ｃ）である。Freshwater chromatium (Chro
matium) genus group, several liters of the same hot spring water was collected, and the bacterial cell group separated and concentrated by a centrifuge was inoculated into a 2 liter anaerobic dark culture tank, and 1 liter of the culture solution shown in Table 2 was added. After replacing the air in the gas phase with nitrogen gas, batch culture was performed for 20 days at a temperature of 30 ° C. Rhodobacter capsulatus ATCC 111
The 66 strains were purchased from ATCC, glucose and yeast extract having the concentrations shown in Table 2 were added to ATCC1139 medium, and 1 platinum loop of the slant-cultured cells was inoculated into 1 liter of liquid culture solution. The culture conditions and method were chromatium ( Chromatium)
It was performed according to the culture of the genus group. Ossillatoria
The culture of the genus atoria was also carried out according to the above, but Rhodobacter capsulatus AT
CC 11166 strain and Oscillator
In the anaerobic dark culture of the ia) genus group, sulfur ion was not added at the beginning, and Na 2 S · 9H was added 10 days after the batch culture was started.
2 O was injected as sulfur ion at a concentration of 1,000 mg / liter, and changes in the number of bacteria (concentration of bacterial cells) over 20 days were measured for all bacteria. The results of the batch culture experiment are shown in FIG. In the figure, ●-● is a group of the genus Chromatium (a), ◯-◯ is a group of the genus Ossillatoria (b), and
Is Rhodobacter capsulatus ATCC 11166 strain (c).
【００４２】（１）当然ではあるが、クロマチウム（Ch
romatium) 属は紅色硫黄細菌とも云われる光合成細菌で
あり、硫化物を電子供与体として利用できるので、嫌気
暗、従属栄養条件で有機物を異化、資化しながら順調に
増殖する。従って、本発明のクロストリジウム（Clostr
idium)との嫌気暗条件での混合共生培養は成立する。 （２）水素生産性藍藻であるオッシラトリア（Oscillat
oria) 属も嫌気暗、従属栄養条件で比較的順調に増殖す
る。然し、硫化物の影響を若干受けて増殖阻害が認めら
れるが、次第に硫化物の毒性に馴養され、一旦減衰した
増殖速度に回復傾向が認められる。 （３）光合成細菌であるロドバクター カプスラタス
（Rhodobacter capsulatus) ＡＴＣＣ １１１６６株も
嫌気暗、従属栄養条件で増殖するが、硫化物の毒性を強
く受け、その後に回復傾向が認められず、硫化物により
不可逆的な活性阻害（増殖阻害）を受ける。 （４）以上の実験結果より、偏性嫌気性の従属栄養水素
生産菌クロストリジウム（Clostridium)属との嫌気暗条
件における混合共生関係はクロマチウム（Chromatium)
属群とオッシラトリア（Oscillatoria) 属群は構築でき
るが、ロドバクター（Rhodobacter ）属は構築できない
事が明確となった。(1) Naturally, Chromium (Ch
The genus romatium) is a photosynthetic bacterium also called purple sulfur bacterium, and it can utilize sulfide as an electron donor, so that it grows smoothly while catabolizing and assimilating organic matter under anaerobic and heterotrophic conditions. Therefore, the Clostridium of the present invention (Clostr
Mixed symbiotic culture with anaerobic dark conditions is established. (2) Oscillat, a hydrogen-producing cyanobacteria
Oria) also grows relatively smoothly under anaerobic and heterotrophic conditions. However, although the growth inhibition was observed under the influence of sulfide, the growth rate was gradually acclimated to the toxicity of sulfide, and the growth rate once attenuated showed a tendency to recover. (3) The photosynthetic bacterium Rhodobacter capsulatus ATCC 11166 strain also grows under anaerobic darkness and heterotrophic conditions, but was strongly toxic to sulfide and showed no recovery tendency after that, and was irreversible by sulfide. Receive specific activity inhibition (growth inhibition). (4) From the above experimental results, the mixed symbiotic relationship between the obligate anaerobic heterotrophic hydrogen-producing bacterium Clostridium and genus Clostridium under anaerobic dark conditions is Chromatium.
It was clarified that the genera group and the Oscillatoria genera group can be constructed, but the Rhodobacter genera cannot be constructed.
【００４３】実施例２：偏性嫌気性の従属栄養水素生産
菌単独、及び本発明方法による水素生産の比較実験 国内、Ｈ県の某市に建設されている標準都市下水を対象
とした下水処理場の最初沈殿池汚泥（生汚泥）を供試試
料として、本発明方法とクロストリジウム（Clostridiu
m)属との水素生産量について比較検討した。採取した最
初沈殿池汚泥から予め土砂分（傾斜法で除去）、粗大固
形物を除去し、大型冷蔵庫に５℃で保管し、１日当りの
使用量をその都度取出し、有効容量１リットルの嫌気暗
培養槽（Fill and Draw 方式の半連続培養槽・・・容積
２リットル）に供給した。実験期間中に使用した供試最
初沈殿池の理化学的性状（平均値）は表３の通りであ
る。Example 2: Comparative experiment of hydrogen production by an obligately anaerobic heterotrophic hydrogen-producing bacterium alone and by the method of the present invention Sewage treatment for standard municipal sewage constructed in a certain city of H prefecture, Japan The method of the present invention and Clostridium (Clostridiu) were used as the test sample of the first sedimentation tank sludge (raw sludge) in the plant.
m) Hydrogen production with genus was compared and examined. Sediment (removal by slanting method) and coarse solids were removed from the collected first settling tank sludge in advance, and stored in a large refrigerator at 5 ° C, and the daily usage amount was extracted each time, and an effective volume of 1 liter of anaerobic dark It was supplied to a culture tank (semi-continuous culture tank of Fill and Draw method ... Volume 2 liters). Table 3 shows the physicochemical properties (average value) of the first sedimentation tank used during the experiment.
【００４４】[0044]
【表３】 （註）＊１：グルコース換算。 単位はｐＨ以外は全てｍｇ／リットル 硫化物はＳ2-として３００ｍｇ／リットル前後であったが、Ｎａ2 Ｓ・９Ｈ2 Ｏを添加して５００ｍｇ／リットルに調整した。[Table 3] (Note) * 1: Glucose conversion. All the units were mg / liter except for pH. Sulfide was around 300 mg / liter as S 2- , but was adjusted to 500 mg / liter by adding Na 2 S.9H 2 O.
【００４５】偏性嫌気性の従属栄養水素生産菌としては
ＡＴＣＣからクロストリジウム ブチリカム（Clostrid
ium butyricum)ＡＴＣＣ８５９を購入し、培地ＡＴＣＣ
３８の液体培地を用いて３０℃で増量培養した。実験系
列は下記に示した３系列であり、それぞれに主たる培養
条件を付記した。 （１）菌種クロストリジウム ブチリカム（Clostridiu
m butyricum)ＡＴＣＣ８５９（対照実験） ＊培養日数 ： １０日（嫌気暗培養槽の有効容積 １
リットル） ＊培養温度 ： ３０℃ ＊混合培養液のｐＨ ： （初発８．０）発酵期間中は
無調整 ＊培地ＡＴＣＣ３８で増量した培養液を、実験開始時に
２００ｍｌ注入。 （２）クロストリジウム ブチリカム（Clostridium bu
tyricum)ＡＴＣＣ８５９＋クロマチウム（Chromatium)
属群 ＊培養日数 ： １０日 ＊培養温度 ： ３０℃ ＊混合培養液のｐＨ ： 発酵期間中は無調整 ＊実施例１記載の増量培養液を、実験開始に２００ｍｌ
注入 （３）クロストリジウム ブチリカム（Clostridium bu
tyricum)ＡＴＣＣ８５９＋オッシラトリア（Oscillator
ia) 属群 ＊全ての条件は（２）に準ずる。As an obligate anaerobic heterotrophic hydrogen-producing bacterium, from ATCC, Clostridium butyricum (Clostrid
ium butyricum) ATCC 859 was purchased and the medium ATCC
Expand culture was carried out at 30 ° C. using 38 liquid media. The experimental series are the three series shown below, and the main culture conditions are added to each. (1) Bacterial species Clostridium butyricum
m butyricum) ATCC 859 (control experiment) * Number of culture days: 10 days (effective volume of anaerobic dark culture tank 1
Liter) * Culturing temperature: 30 ° C * pH of mixed culture solution: (Initiation 8.0) No adjustment during fermentation period * 200 ml of culture solution increased in medium ATCC38 is injected at the start of the experiment. (2) Clostridium bucuricum
tyricum) ATCC 859 + Chromatium
Genus group * Number of culture days: 10 days * Culture temperature: 30 ° C * pH of mixed culture solution: No adjustment during fermentation period * 200 ml of the expanded culture solution described in Example 1 was used for starting the experiment.
Injection (3) Clostridium bucuricum
tyricum) ATCC 859 + Oscillator
ia) Genus group * All conditions are based on (2).
【００４６】比較実験は、まず、表３の水質を有する供
試汚泥を（１）については８００ｍｌ、（２）、（３）
については６００ｍｌ嫌気暗培養槽に張込み、それぞれ
に菌体増殖液を規定量注入して全量を１リットル（嫌気
暗培養槽の容積は２リットル）とした。実験開始から１
週間ほどは基質を供給すること無く、３０℃の温度で培
養し、ガス発生に増大傾向が認められるまでそのまま放
置した。その後は、最終負荷条件の１／２程度となるよ
うに供試試料を半連続式に注入、引抜きを継続し、ガス
発生を観察しながら当初に設定した負荷条件となるよう
に供給量を増加した（約１月後）。日常観察で水素発酵
がほぼ定常状態に達したとみなせる時点から、隔日に水
素発生量を正確に計測し、一方発酵消化液の全糖量（グ
ルコース換算）を測定し、除去全糖量を求めてｍｏｌ・
Ｈ2 ／ｍｏｌ・Ｃ6 Ｈ12Ｏ6 を算出した。この比較検証
実験から得られた結果を図３に示した。図３において、
●−●はクロストリジウム（１）で発酵期間のｐＨ≒
６．４、▲−▲はクロストリジウム＋クロマチウム
（２）で発酵期間のｐＨ≒７．３、〇−〇はクロストリ
ジウム＋オッシラトリア（３）で発酵期間のｐＨ≒７．
３である。In the comparative experiment, first, the sample sludge having the water quality shown in Table 3 was 800 ml for (1), (2), (3).
For 600 ml, the cells were filled in a 600 ml anaerobic dark culture tank, and a prescribed amount of the bacterial cell growth liquid was injected into each to make the total volume 1 liter (the volume of the anaerobic dark culture tank was 2 liters). 1 from the start of the experiment
The substrate was not supplied for about a week, the culture was carried out at a temperature of 30 ° C., and the culture was left as it was until an increase tendency in gas generation was observed. After that, the test sample was semi-continuously injected and withdrawn so that it was about half of the final load condition, and withdrawal was continued, and the supply amount was increased so that the initially set load condition was observed while observing gas generation. Yes (about one month later). From the time when hydrogen fermentation can be considered to have reached a nearly steady state by daily observation, the amount of hydrogen generated is accurately measured every other day, while the total sugar amount (glucose conversion) of the fermentation digestion liquid is measured to obtain the total sugar amount removed. Mol ·
Was calculated H 2 / mol · C 6 H 12 O 6. The results obtained from this comparative verification experiment are shown in FIG. In FIG.
●-● is Clostridium (1) pH during fermentation ≒
6.4, ▲-▲ are Clostridium + Chromium (2) during the fermentation period pH 7.3, and ◯-◯ are Clostridium + Ossillatoria (3) during the fermentation period pH ≈ 7.
It is 3.
【００４７】実験結果 （１）偏性嫌気性の従属栄養性水素生産菌クロストリジ
ウム ブチリカム（Clostridium butyricum)ＡＴＣＣ８
５９による水素発酵は明らかにＡＴＰ生産量が不足する
ために吸エルゴン反応となり、発酵消化液中に低級脂肪
酸が３，０００〜４０００ｍｇ／リットル蓄積し、水素
化反応は完結しなかった。そのために水素生産量は極端
に少なく、約２０日間の実験期間を通じてＨ2 ／Ｃ6 Ｈ
12Ｏ6 は１．５〜２．５ｍｏｌ／ｍｏｌに過ぎなかっ
た。 （２）これに対して、本発明の水素生産法であるクロス
トリジウム（Clostridium)＋クロマチウム（Chromatiu
m) の混合培養、及びクロストリジウム（Clostridium)
＋オッシラトリア（Oscillatoria) の混合培養における
Ｈ2 ／Ｃ6 Ｈ12Ｏ6は前者の実験では１０〜１１ｍｏｌ
／ｍｏｌ、後者の実験では８．５〜９ｍｏｌ／ｍｏｌの
高い水素発生量が得られた。また、両実験での全糖両の
除去率は８５〜９０％の高い値を示した。 （３）以上の検証実験の結果から、供給基質中に高濃度
の硫化物が存在しても、クロストリジウム（Clostridiu
m)が生産した低級脂肪酸を基質としてクロマチウム（Ch
romatium) 、及びオッシラトリア（Oscillatoria) は過
剰のＡＴＰを生産し、このＡＴＰをクロストリジウム
（Clostridium)の水素生成反応に供給して水素発酵を完
結させるという共役関係が確実に成立することが照明さ
れた。Experimental Results (1) Obligate Anaerobic Heterotrophic Hydrogen-Producing Bacterium Clostridium butyricum ATCC8
The hydrogen fermentation by 59 was obviously an ergon reaction due to insufficient ATP production, 3,000 to 4000 mg / liter of lower fatty acids were accumulated in the fermentation digestion liquid, and the hydrogenation reaction was not completed. Therefore, the hydrogen production is extremely small, and H 2 / C 6 H
12 O 6 was only 1.5 to 2.5 mol / mol. (2) On the other hand, the hydrogen production method of the present invention, Clostridium + Chromatiu
m) mixed culture and Clostridium
H 2 / C 6 H 12 O 6 in the mixed culture of + Oscillatoria was 10 to 11 mol in the former experiment.
/ Mol, in the latter experiment, a high hydrogen generation amount of 8.5 to 9 mol / mol was obtained. In addition, the removal rates of both total sugars in both experiments were as high as 85 to 90%. (3) From the results of the above verification experiments, even if a high concentration of sulfide is present in the feed substrate, Clostridium (Clostridiu)
Chromium (Ch
It was illuminated that romatium and Oscillatoria produce an excess of ATP and supply this ATP to the hydrogen production reaction of Clostridium to complete the hydrogen fermentation.
【００４８】[0048]
【発明の効果】以上詳細に説明、検証したように、本発
明による偏性嫌気性の従属栄養水素生産菌クロストリジ
ウム（Clostridium)属と水素生産性光合成細菌である紅
色硫黄細菌クロマチウム（Chromatium) 属及び／又は藍
藻オッシラトリア（Oscillatoria) 属を、光エネルギー
供給なしの嫌気暗条件で混合共生培養することにより、
硫化物を多量に含む有機性廃棄物から安定して多量の水
素を生産する事が可能となった。この卓越した生物学的
水素生産法の確立により、次に示す格段の効果が得られ
る。 （１）従来のクロストリジウム（Clostridium)属の単独
培養、或いは嫌気明（光エネルギー供給）条件での光合
成微生物からの生物学的水素生産法が経済的理由と水素
生産量、及び安定性の観点から実用化困難であったが、
本発明により実用化が可能となった。Industrial Applicability As described and verified in detail above, the obligately anaerobic heterotrophic hydrogen-producing bacterium of the genus Clostridium and the hydrogen-producing photosynthetic bacterium of the purple sulfur bacterium Chromatium and / Or by culturing the cyanobacterium Oscillatoria genus under co-cultivation under anaerobic dark conditions without light energy supply,
It has become possible to stably produce a large amount of hydrogen from organic waste containing a large amount of sulfide. The establishment of this outstanding biological hydrogen production method has the following remarkable effects. (1) From the viewpoints of economic reason, hydrogen production amount, and stability, the conventional hydrogen cultivation method of Clostridium (Clostridium) single culture or biological hydrogen production method from photosynthetic microorganisms under anaerobic light (light energy supply) conditions It was difficult to put into practical use,
The present invention has enabled practical application.
【００４９】（２）偏性嫌気性の従属栄養水素生産菌と
光合成微生物の混合共生培養により、水素発酵の完結に
不足するＡＴＰが前者に供給されるので、水素発酵及び
／メタン発酵などの所謂嫌気性細菌による発酵消化液に
殆ど低級脂肪酸が分解異化、資化され、発酵消化液の汚
染強度が極端に軽減される。そのために、２次処理を必
要としない嫌気性処理技術が確立、出現される可能性が
高い。 （３）本発明方法に微嫌気減圧発酵法を適用することに
より、本発明の効果は一段と卓越した効果が期待され
る。(2) ATP, which is insufficient for completion of hydrogen fermentation, is supplied to the former by the mixed symbiotic culture of the obligately anaerobic heterotrophic hydrogen-producing bacterium and the photosynthetic microorganism, so-called hydrogen fermentation and / or methane fermentation. Almost all lower fatty acids are decomposed and catabolized and assimilated in the fermentation digestion liquid by anaerobic bacteria, and the contamination intensity of the fermentation digestion liquid is extremely reduced. Therefore, there is a high possibility that an anaerobic treatment technology that does not require secondary treatment will be established and emerged. (3) By applying the slightly anaerobic reduced pressure fermentation method to the method of the present invention, the effect of the present invention is expected to be much more excellent.
【図１】オッシラトリア属のＡＴＰ、ＮＡＤ（Ｐ）Ｈ生
産の生体機構を示す図。FIG. 1 is a diagram showing a biological mechanism of ATP and NAD (P) H production in the genus Ossillatoria.
【図２】嫌気暗回分培養における硫化物耐性実験結果を
示すグラフ。FIG. 2 is a graph showing the results of sulfide resistance experiments in anaerobic dark batch culture.
【図３】培養方法による水素生産量の比較を示すグラ
フ。FIG. 3 is a graph showing a comparison of hydrogen production amounts according to culture methods.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 (Ｃ１２Ｐ 39/00 Ｃ１２Ｒ 1:145 1:01） (Ｃ１２Ｐ 39/00 Ｃ１２Ｒ 1:145 1:89） (Ｃ１２Ｐ 3/00 Ｃ１２Ｒ 1:145 1:01 1:89） ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification number Office reference number FI technical display location (C12P 39/00 C12R 1: 145 1:01) (C12P 39/00 C12R 1: 145 1:89) ) (C12P 3/00 C12R 1: 145 1:01 1:89)
Claims (1)
する方法において、該有機性廃棄物中で、偏性嫌気性の
従属栄養性水素生産菌であるクロストリジウム（Clostr
idium)属に属する微生物群と、水素生産性光合成微生物
である紅色硫黄細菌クロマチウム（Chromatium) 属及び
／又は藍藻であるオッシラトリア（Oscillatoria) 属に
属する微生物とを、嫌気暗条件下で共生せしめ、混合培
養することを特徴とする水素ガス生産方法。1. A method for producing hydrogen gas from various organic wastes, wherein in the organic wastes, Clostridium (Clostridi), which is an obligate anaerobic heterotrophic hydrogen-producing bacterium, is used.
A microbial group belonging to the genus idium and a microorganism belonging to the genus Oscillatoria of the red-sulfur bacterium Chromatium which is a hydrogen-producing photosynthetic microorganism and / or the genus Oscillatoria which is a cyanobacteria are allowed to coexist under anaerobic dark conditions and mixed. A method for producing hydrogen gas, which comprises culturing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12456295A JPH08294396A (en) | 1995-04-26 | 1995-04-26 | Production of hydrogen gas |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12456295A JPH08294396A (en) | 1995-04-26 | 1995-04-26 | Production of hydrogen gas |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH08294396A true JPH08294396A (en) | 1996-11-12 |
Family
ID=14888558
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12456295A Pending JPH08294396A (en) | 1995-04-26 | 1995-04-26 | Production of hydrogen gas |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH08294396A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100442741B1 (en) * | 2002-04-30 | 2004-08-02 | 한국에너지기술연구원 | Process for hydrogeon production from biological reaction of organic wastes |
| KR100467789B1 (en) * | 2002-04-30 | 2005-01-24 | 한국에너지기술연구원 | Method for hydrogeon production from anaerobic fermentation of organic compoound |
| WO2005087911A1 (en) * | 2004-03-16 | 2005-09-22 | Sharp Kabushiki Kaisha | Microbe culturing apparatus and utilizing the same, hydrogen production apparatus and fuel cell system |
| JP2006101831A (en) * | 2004-10-08 | 2006-04-20 | National Institute Of Advanced Industrial & Technology | Hydrogen production method |
| US7258938B2 (en) | 2001-03-06 | 2007-08-21 | Sharp Kabushiki Kaisha | Polymer electrolyte fuel cell |
| US7432091B2 (en) | 2003-02-24 | 2008-10-07 | Research Institute Of Innovative Technology For The Earth | Highly efficient hydrogen production method using microorganism |
| US8846358B2 (en) | 2008-05-12 | 2014-09-30 | Sharp Kabushiki Kaisha | Method and device for producing hydrogen |
-
1995
- 1995-04-26 JP JP12456295A patent/JPH08294396A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7258938B2 (en) | 2001-03-06 | 2007-08-21 | Sharp Kabushiki Kaisha | Polymer electrolyte fuel cell |
| US7527883B2 (en) | 2001-03-06 | 2009-05-05 | Sharp Kabushiki Kaisha | Polymer electrolyte fuel cell |
| KR100442741B1 (en) * | 2002-04-30 | 2004-08-02 | 한국에너지기술연구원 | Process for hydrogeon production from biological reaction of organic wastes |
| KR100467789B1 (en) * | 2002-04-30 | 2005-01-24 | 한국에너지기술연구원 | Method for hydrogeon production from anaerobic fermentation of organic compoound |
| US7432091B2 (en) | 2003-02-24 | 2008-10-07 | Research Institute Of Innovative Technology For The Earth | Highly efficient hydrogen production method using microorganism |
| WO2005087911A1 (en) * | 2004-03-16 | 2005-09-22 | Sharp Kabushiki Kaisha | Microbe culturing apparatus and utilizing the same, hydrogen production apparatus and fuel cell system |
| JP2006101831A (en) * | 2004-10-08 | 2006-04-20 | National Institute Of Advanced Industrial & Technology | Hydrogen production method |
| JP4576581B2 (en) * | 2004-10-08 | 2010-11-10 | 独立行政法人産業技術総合研究所 | Hydrogen production method |
| US8846358B2 (en) | 2008-05-12 | 2014-09-30 | Sharp Kabushiki Kaisha | Method and device for producing hydrogen |
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