Recent Development of Anaerobic Digestion Processesfor Energy Recovery from Wastes
Naomichi Nishio1* and Yutaka Nakashimada1
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan1
Received 16 August 2006/Accepted 29 November 2006
Anaerobic digestion leads to the overall gasification of organic wastewaters and wastes, andproduces methane and carbon dioxide; this gasification contributes to reducing organic matterand recovering energy from organic carbons. Here, we propose three new processes and demon-strate the effectiveness of each process. By using complete anaerobic organic matter removal proc-ess (CARP), in which diluted wastewaters such as sewage and effluent from a methane fermenta-tion digester were treated under anaerobic condition for post-treatment, the chemical oxygen de-mand (COD) in wastewater was decreased to less than 20 ppm. The dry ammonia-methane two-stage fermentation process (Am-Met process) is useful for the anaerobic treatment of nitrogen-rich wastes such as waste excess sludge, cow feces, chicken feces, and food waste without the dilu-tion of the ammonia produced by water or carbon-rich wastes. The hydrogen-methane two-stagefermentation (Hy-Met process), in which the hydrogen produced in the first stage is used for afuel cell system to generate electricity and the methane produced in the second stage is used togenerate heat energy to heat the two reactors and satisfy heat requirements, is useful for the treat-ment of sugar-rich wastewaters, bread wastes, and biodiesel wastewaters.
Anaerobic digestion leads to the overall gasification of or-ganic wastewaters to methane and carbon dioxide. Althoughanaerobic digestion processes have been carried out for de-cades, interest in the economical recovery of fuel methanegas from industrial and agricultural surpluses has recentlyincreased owing to the changing socio economical situationin the world. To achieve rapid and effective anaerobic diges-tion, some processes have been developed, including the up-flow anaerobic filter process (UAFP) (1), upflow anaerobicsludge blanket (UASB) (2), anaerobic attached film expandedbed reactor (AAFEB) (3) and anaerobic fluidized bed reac-tor (AFBR) (4) to improve cell retention, and the two-phasedigestion process (5) to optimize acidogenesis and metha-nogenesis. However, to enhance these processes, it is neces-sary to determine their applicability to other types of waste-water, such as those containing recalcitrant and toxic com-pounds, and high solid organic materials.
Anaerobic digestion is useful for decreasing the amountof organic solid waste and recovering energy. Although cel-lulose materials are the most abundant biomass resources,the rates of their degradation and methane production aremuch lower than those of other biomasses. Therefore, a ba-
sic study of an artificial microbial consortium is needed.Furthermore, organic waste solids sometimes include highamounts of salts. For example, soy sauce refuse is producedas a by-product of the process of making soy sauce in facto-ries (6), and contains 10% sodium chloride. Inland sea sedi-ments in some places in Japan have been polluted by organicmatter containing 3% sodium chloride. Thus, it is necessaryto examine the possibility and potential of methane produc-tion in such high-salt-containing organic matter. Throughoutthe world, there is a great deal of waste-activated sludge,which includes discharge from municipal wastewater treat-ment centers and feces from cows, pigs and chickens. Someof these feces include nitrogen-rich compounds such as pro-teins, nucleotides and uric acid that inhibit methane produc-tion after being converted to ammonia. A method of achiev-ing efficient anaerobic digestion under dry conditions (watercontent less than 80%) needs to be developed.
Hydrogen can be used to produce clean energy and canbe produced by photosynthetic and fermentative microor-ganisms under anaerobic conditions in pure cultures (7–9).Methanogenic ecosystems consist of hydrolysis (first stage),acidogenesis (second stage), and methanogenesis (thirdstage). Although hydrogen can be produced in the hydroly-sis and acidogenesis stages, there have been few reports todate focusing on hydrogen production in these steps. If hy-
drogen production at a high rate with high yield can beachieved, the produced hydrogen could be connected direct-ly to fuel cells without the need to reform the methane pro-duced during methanogenesis.
Although many researchers have already reported high-rate methane production and high- yield or high-rate hydro-gen production carried out by isolated strict and facultativeanaerobes and microbial consortia (10), in this review, wewill describe our own research in this field and introducenew anaerobic microbial wastewater and waste treatmentprocesses for energy recovery; these processes are summa-rized in Table 1.
ADVANCED UASB REACTORS
Alternative use of UASB reactor for sewage treatmentIn sewage treatment, chemical oxygen demand (COD, ap-proximately 400 ppm) and biochemical oxygen demand(BOD) in influent have been decreased to less than 20 ppmunder an aerobic condition using the activated sludge pro-cess. However, the disadvantages of this process are that (i)it requires aeration energy, that is, electricity needed to sup-ply air and that (ii) excess activated sludge is produced, e.g.,50 g of sludge from 100 g of organic carbon.
The UASB method has been developed as an efficientanaerobic wastewater treatment process. However, it is dif-ficult to remove organic carbon to less than 20 ppm by thisanaerobic process. To overcome this problem, a novel sys-tem consisting of a UASB anaerobic pretreatment unit and aDHS (down-flow hanging sponge cube) aerobic post-treat-ment unit was proposed by Harada et al. (11) for a low-costand easy-maintenance process; the system requires neitherexternal aeration input nor excess sludge withdrawal.
The process performance of the afore mentioned novelsewage treatment system using the sulfur reduction-oxida-tion reaction of microbes in the UASB pretreatment unitwas investigated in a pilot-scale reactor with actual sewagefor over 600 d at the Higashi-Hiroshima Sewage TreatmentCenter (12). The reactor system consisted of a denitrifica-tion reactor (1.4 m3), a UASB reactor (8.4 m3), and a DHS re-actor (13.9 m 3) with a recirculation line. As shown in Fig. 1,the total BOD in the influent (128 ppm) decreased to 39 ppmin the UASB effluent, and 7 ppm in the DHS effluent with alow degree of suspended solid (SS) accumulation, namely,2–3% of the inlet SS level, under the conditions of overallhydraulic retention times (HRTs) of 12–24 h and a recircu-lation ratios of 0.3–2 throughout the experimental period.
These results revealed that sulfate-reducing bacteria, notmethanogens, contributed to the degradation of organic mat-ter in the UASB reactor even at low temperatures (less than10°C).
Complete anaerobic treatment of high-strength waste-water (CARP process) In conventional wastewater treat-ment processes, methane fermentation processes such asUASB are commonly used for the biological treatment ofhigh-strength wastewater (e.g., COD = 4000 ppm) and forenergy recovery by recovering methane. Such processeshave been used widely in Japan for wastewaters from food-processing factories that produce commodities such as beer,sugar, soft drinks, and potatoes. However, owing to the lowCOD and BOD removal efficiencies of such processes (lessthan 90%), aerobic treatment (the activated sludge process)has been generally used as a post-treatment to further de-crease the COD and BOD level to less than 20 ppm. How-
TABLE 1. Summary of processes described in this review
Process Background Improvement Accomplishment
CARP Difficulties in decreasing COD and BOD to less than 20 ppm in anaerobic processes
Use of denitrifying and sulfate-reducing bacteria in addition to methanogens
Reducing BODs to 10–20 ppm from 100–200 ppm in methanogenic UASB
AM-MET Difficulties in treating solid wastes con-taining large amount of organic nitro-gen such as dehydrated waste activated sludge in anaerobic process
Ammonia fermentation and physico-chemical removal of free ammonia from organic waste
Sustainable methane production from ammonia-removed dehydrated activated sludge
HY-MET Need for hydrogen and utilization of effluent containing by-products after hydrogen fermentation
Use of methane fermentation process at subsequent stage of hydrogen fermenta-tion
Improved energy recovery by connect-ing hydrogen fermentation to solublizla-tion of solid materials
FIG. 1. Process performance of UASB-DHS system for sewagetreatment (from Ref. 12).
RECENT DEVELOPMENT OF ANAEROBIC DIGESTIONVOL. 103, 2007 107
ever, this activated sludge process requires a high energyconsumption for aeration, as mentioned above. Therefore,if anaerobic treatment is possible as a post-treatment, themethane fermentation process will become more useful.Therefore, methane fermentation following the anaerobicpost-treatment process (complete anaerobic organic matterremoval process, CARP) was carried out. By using CARP,the COD in wastewater (initially 4000 ppm) can be decreasedto 20 ppm after these anaerobic treatments.
In CARP as shown in Fig. 2, three types of microorgan-ism should be functioning. These are methane-producingmicrobes that can convert COD and BOD to methane andcarbon dioxide in methanogenic reactor 1. Denitrifying mi-crobes that convert nitrate to nitrogen gas (13) and sulfate-reducing microbes that convert sulfate to sulfide (14) main-ly act as BOD and COD eliminators in anaerobic reactor 2.This process, however, requires aerobic reactor 3 for post-treatment to recycle the sulfate from the sulfide produced bythe sulfur-oxidizing microbes and to recycle the nitrate fromthe ammonia produced by nitrifying microbes: this post-treatment contributes to the removal of ammonia. The sul-fate-reducing and sulfur-oxidizing microbes can functionunder not only eutrophic but also oligotrophic conditions(15). They contribute to the removal of organic matter atlow (less than 20 ppm) BODs and CODs, and can also func-tion at low temperatures (16).
CARP was used for wastewater treatment dischargedfrom food processing plants using three reactors sequential-ly. Reactor 1 is a mesophilic methanogenic UASB reactor(35°C), in which methane can be produced. Reactor 2 is asecond UASB reactor under ambient temperature in whichnitrate or sulfate reduction mainly occurs but methane pro-duction is not expected. Reactor 3 is a DHS aerobic reactorused to convert ammonia to nitrate and sulfide to sulfate. Aportion of the effluent from reactor 3 is recycled to reactor 2,in which COD and BOD are decreased as a result of nitratebeing reduced to nitrogen. Sulfate can be supplemented intothe stream for recycling sulfate to activate sulfate-reducingbacteria optionally. The influent BOD and SS level were ad-
justed to approximately 2000 ppm and 500 ppm, respective-ly. The temperatures in reactors 2 (denitrifying/sulfate-reduc-ing) and 3 (nitrification/sulfate-oxidizing) were below 10°C,and sometimes close to 1°C during operation days 150 to350 owing to the fact that it was winter. However, the BODdecreased to within the rage of 100–200 ppm in the metha-nogenic UASB effluent and to within the range of 10–20ppm in the final effluent, respectively (Fig. 3). Moreover,
FIG. 2. Schematic diagram of complete anaerobic organic matter removal process (CARP).
FIG. 3. Process performance of CARP (UASB-UASB/DHS system)for sugary wastewater treatment.
NISHIO AND NAKASHIMADA J. BIOSCI. BIOENG.,108
even during winter, the final BOD decreased to 1 ppm. Themethane conversion efficiency was approximately 80%.
In conclusion, CARP is useful for wastewater treatmentwithout the need to conduct the activated sludge process aspost-treatment.
METHANE PRODUCTION FROMSOLID MATERIALS
Methane fermentation has been used for anaerobic waste-water treatment and energy recovery. In particular, the UASBprocess has been developed for the high-rate methane fer-mentation of high-strength wastewater (17), in which micro-bial granule formation including those of acidogens andmethanogens is a key factor. However, this process is appli-cable only in wastewater containing a low SS level. It is,therefore, difficult to apply a UASB system to solid biomassdirectly, and pretreatment to liquefy the SSs is required.
Cellulosic materials Although cellulose and hemicel-lulose are the main components of biomass, it is difficult totreat them directly using a high-rate methane fermentationprocess. Nakashimada et al. (18) investigated the direct con-version of cellulose to methane using defined methanogensand the anaerobic fungus Neocallimastix frontalis, which isfound in ruminants and produces cellulolytic and hemicel-lulolytic enzymes such as cellulase and xylanase (19). Thisfungus can degrade biomass and produce acetate, formate,H
2/CO
2, lactate, and a small amount of ethanol, all of which
are suitable substrates for methanogens and methanogenicconsortia (20–23). As shown in Fig. 4, cocultures of N.frontalis with Methanobacterium formicicum and Meth-anothaeta concilii were successfully performed to producemethane. When cellulose solution was added 5 times to abioreactor in which 24 g l–1 cellulose in total had beenadded, formate and hydrogen accumulations were not ob-
served. After 24 d, 150 mM CH4 was produced and 57 mM
acetate remained in the medium. Then the accumulated ace-tate was consumed while methane formation occurred, andafter 55 d, 178 mM CH
4 was produced.
Coastal sea sediment The recent environmental pol-lution of coastal seawater has resulted in the accumulationof large amounts of organic sediment in mud in some areasat the bottom of Hiroshima Bay in Japan. In particular, mudlayers 2–3 m thick have frequently been observed in oysterfarming areas owing to large amounts of organic matter be-ing continuously excreted by oysters.
Mud sediment can be treated by anaerobic acidogenicfermentation followed by the cultivation of photosyntheticbacteria (24). In the acidogenic fermentation, almost all ofthe fatty acids produced were acetate molecules. The com-bined addition of vitamins such as nicotinic acid, thiamineand biotin markedly induced the production of acetate fromsea mud sediment. Furthermore, by using the UASB reactor,which included methanogenic sludge that was acclimated tothe mud sediment, the post acidogenic fermentation liquorcan be treated as mentioned above (25). When a two-stageUASB reactor system was used for the high-rate anaerobictreatment of mud sediment, acetic acid from the mud sedi-ment was present in the effluent of the acidogenic reactorin each run, yielding approximately 110 mM methane from278 g wet wt. mud/l/4 d for each run (Fig. 5).
Traditional Japanese foods Solid wastes are dis-charged during the processes of making traditional Japanesefoods, for example, tofu and soy sauce, and traditional drinkssuch as sake and shochu, and we aimed to treat these solidwastes by methane fermentation.
Soy sauce refuse (SSR), similar to bean curd refuse, is a
FIG. 4. Fed-batch culture for methane production with a tri-cul-ture of N. frontalis, M. formicicum, and M. concilii in cellulose me-dium at 39 °C (from Ref. 18). Symbols: closed squares, hydrogen; opencircles, methane; open squares, lactate; closed circles, acetate; opentriangles, ethanol; closed triangles, formate; and open diamonds, pH.
FIG. 5. Profiles of methane production, acetic acid concentrationand pH during methane fermentation of mud sediment in a two-stageUASB reactor system at 37 °C (from Ref. 25). Symbols: open triangles,pH in acidogenic reactor; open squares, pH in methanogenic reactoreffluent: closed triangles, acetic acid concentration in acidogenic reac-tor effluent; closed squares, acetic acid concentration in methanogenicreactor effluent; and closed circles, methane production in methano-genic reactor.
RECENT DEVELOPMENT OF ANAEROBIC DIGESTIONVOL. 103, 2007 109
highly nutritious biomass, but its utilization is difficultowing to its high salt content of 10% (w/w). Some of thistype of refuse is currently being used as animal feed for cat-tle, but demand for it may not continue. In Japan, over100,000 t of SSR is produced per year; therefore, the treat-ment of this solid waste is also an important goal and majorchallenge. Although anaerobic microbial digestion is oftena successful treatment for wastewater in the food industry,no such method that can be applied to solid and high-saltwastes such as SSR has been reported. Nagai et al. (26) re-ported that thermophilic methanogenic sludge obtained froma municipal wastewater treatment plant could be used asseed to decrease SS level in SSR and produce methane suc-cessfully. At 25 g wet wt./l SSR, the production of 120 mMCH
4 and 50% (w/v) decrease in SS level were observed after
35 d. The acclimation of the sludge to the waste was effec-tive for increasing methane production rate in the pH-con-trolled fed-batch culture using a stirred tank reactor (Fig. 6).
DRY METHANE FERMENTATIONOF ORGANIC SOLID WASTES
(AM-MET PROCESS)
Because the recycling of solid wastes is vigorously pro-moted in Japan and the need to recover energy from organicwastes is increasing, a dry digestion plant, which has beendeveloped for a KOMPOGAS process (27), has been oper-ating for about two years in Kyoto (http://takuma.co.jp/news/2001/20010921.html). Three types of waste (i.e., garbageand leftovers from hotels, yard waste, and used paper) mixedat various ratios are used to control the C/N ratio. The planthas maintained stable operations with the mixture, generat-ing biogas by the decomposition of volatile solid (VS) at arate of about 820 N m3 per ton of VS.
Because the aerobic activated sludge process is commonin Japan, an abundant amount of excess sludge (2,000,000 tas dry sludge) has been discharged annually. Although someof this sludge has been treated by methane fermentation to
reduce the amounts of the excess sludge after the condensa-tion of the excess sludge (approximately 4% solid content),most of the sludge is dumped after dehydration (approxi-mately 20% solid content) or incineration. If the dehydratedexcess sludge can be treated directly by methane fermenta-tion, dry methane fermentation (water content: 80%) will bemore useful because the plant scale could reduce by one-fifth.
Thus, the dehydrated excess sludge was fermented semicontinuously to methane under a dry condition (water con-tent = 80%). Although methane was produced at yield of ap-proximately 610 mmol/kg wet wt., this methane productiongradually decreased and eventually ceased (Fig. 7). This isthe reason that the dry fermentation of only excess sludgehas not yet succeeded. However, it is very important to notethat ammonia fermentation almost constantly occurred, in-dicating that the cessation of methane fermentation mightbe due to the accumulation of ammonia produced from pro-tein in the excess sludge.
The ammonia-removed dehydrated excess sludge was fer-mented to methane semi continuously under the same drycondition (water content = 80%) by changing the solid wasteretention time (SRT). As shown in Fig. 8, methane produc-tion began even at an SRT of 20 d. The ammonia concentra-tion was maintained at less than 2000 mg-N/kg wet wt., andtotal organic carbon (TOC) removal efficiencies of 33% and20% were obtained for methane and carbon dioxide, respec-tively.
These results show that the dry methane fermentation of
FIG. 6. Methane fermentation of soy souce refuses in fed batchculture at 50°C. Symbols: open circles, acetate concentration; closedcircles, propionate concentration; open triangles, butyrate concentra-tion; and closed triangles, methane production.
dehydrated excess sludge is possible at a relatively shortSRT, which corresponded to the HRT for the wet methanefermentation, if the ammonia produced is removed. If theammonia produced can be completely removed, the dry fer-mentation of sludge can be possible at shorter SRTs.
The proposed dry ammonia-methane fermentation (Am-Met process) is useful for the anaerobic treatment of nitro-gen-rich wastes such as cow feces, chicken feces, and foodwastes.
Hydrogen can be used to produce clean energy and isgenerally produced by photosynthetic and fermentative mi-croorganisms under anaerobic conditions in pure cultures(7–9). However, the methanogenic ecosystem consists ofthree stages: hydrolysis (first stage), acidogenesis (secondstage) and methanogenesis (third stage). Although hydrogencan be produced in the hydrolysis and acidogenesis stages,there are few reports so far on hydrogen production in thesestages. If high-rate and high-yield hydrogen productions areachieved, it is possible that the hydrogen gas produced canbe directly connected to a fuel cell system without any re-forming being necessary. Furthermore, methane can be pro-duced from fatty acids such as acetate, propionate and bu-tyrate that accumulate in liquid broth after hydrogen fer-mentation.
We have proposed this process as a hydrogen-methanetwo-stage fermentation (Hy-Met) process. In this process,the energy in the hydrogen produced is converted to elec-tricity by the fuel cell system and the produced methane isused to generate heat energy to heat the two reactors and tosatisfy heat requirements.
Potential of fermentative hydrogen production To
gain an understanding of the potential of fermentative hy-drogen production, continuous hydrogen production usingself-flocculating cells of Enterobacter aerogenes HU-101and AY-2 (28, 29) was carried out in a packed-bed reactorunder a glucose-limiting condition. As shown in Fig. 9, themaximum hydrogen production rates were 31 and 58mmol/l ⋅h for HU-101 and AY-2, respectively, at a dilutionrate of 0.67 h–l. High-rate hydrogen production is now pos-sible at an HRT of 1.5 h. In addition, a higher yield, i.e., ap-proximately 2 mol/mol glucose, can also be obtained by mu-tation (30). The fermented broth contained 2,3-butandiol,ethanol, lactate and acetate, which are good substrates forUASB methane fermentation.
Hy-Met process for bread waste In Japan, a total of100,000 t/year solid bread waste is discharged. For thiswaste, the Hy-Met process was applied (31, 32). As shownin Fig. 10, in a batch culture, in which 100 g-wet wt./l breadwaste (43% water content, w/w) was treated at 55°C with10% (w/v) of a thermophilic sludge collected from an anaer-obic digester of sewage sludge at a sewage-treatment plant inHiroshima, Japan, the waste was fermented to hydrogen and
FIG. 8. Semi continuous dry methane fermentation of ammonia-removed dehydrated excess sludge obtained after ammonia fermenta-tion.
FIG. 9. Performance of hydrogen fermentation of mutant AY-2 inpacked-bed reactor (from Ref. 29). Symbols: closed circles, H
2 evolu-
tion rate; closed squares, residual glucose concentration; open circles,optical density of effluent; closed triangles, height of bed of floccu-lated cells in reactor from bottom; and open squares, dilution rate.
FIG. 10. Effects of pH on liquefaction and hydrogen fermentationof bread waste (from Ref. 32).
RECENT DEVELOPMENT OF ANAEROBIC DIGESTIONVOL. 103, 2007 111
volatile fatty acids under pH-uncontrolled (initial pH = 7)and pH-controlled conditions at 7 or 5. Although the pH-un-controlled condition yielded only 70 mM H
2 with an 80%
decrease in SS level, the pH 7-controlled condition yielded240 mM H
2 with a 91% decrease in SS level after 24 h. The
culture broth contained 150 mM each of acetate and butyrate,and the TOC concentration was approximately 20,000 ppm.On the other hand, under the pH 5-controlled condition, only100 mM H
2 was produced, and lactate (approximately 220
mM) was mainly produced in the culture broth. Next, cul-ture broth of the hydrogen fermentation of the bread waste,which contained approximately 20,000 ppm of TOC concen-tration, was used for methane production. This culture brothwas diluted to yield TOC concentration of 2000–5000 ppmand supplied continuously to a UASB methane reactor, inwhich acclimatized methanogenic granules were inoculated.When the organic loading rate was increased by increasingthe dilution rate stepwise, the optimum loading rate was9.5 g-TOC/l ⋅d yielding 80% TOC removal, a methane pro-duction rate of 400 mmol/l ⋅d and a methane yield of ap-proximately 0.6 as the carbon base. These results show thatwhen reactor volumes for hydrogen and methane fermenta-tions are set to a ratio of 1:2.1, SS level will be decreasedby 91% at a the loading rate of 29 g-wet wt./l ⋅d, and the hy-drogen and methane yields will be 2.4 mol/kg wet wt. and8.6 mol/kg wet wt., respectively.
The amount of energy recovered from the Hy-Met proc-ess using bread waste was estimated on the basis of theseresults. To treat the waste discharged from one factory at2.67 t/d, a 26.7-m3 hydrogen fermentation reactor in which145 m3 of hydrogen/d will be produced, which correspondsto 214 kwh when the conversion efficiency of the fuel cellsystem is 50%, and a 56-m3 methane fermentation reactorin which 514 m3 methane/d will be produced, which corre-sponds to 530 l of oil/d, are necessary.
Hy-Met process for beer waste Recently, a UASBmethane fermentation process has been used a lot in a beermanufacture factory. However, because the pressed filtratefrom the spent malt in the lauter tun at the beer factory con-tains a high density of suspended matter, such filtrate is dif-ficult to apply to the UASB reactor. Therefore, only the fil-trate obtained from the pressed filtrate after SS removal hasbeen treated using the UASB reactor. The Hy-Met Processwas applied directly to this pressed filtrate (33). When con-tinuous culture for hydrogen fermentation and the follow-ing UASB methane fermentation was carried out at 50°Cand 37°C, respectively, COD removal was more or less thesame compared with the single UASB methane fermenta-tion, which is currently in use in this factory. However, thetotal amount of energy recovered as the sum of hydrogen
and methane increased to 103 kJ/l from 90 kJ/l, which corre-sponds to the amount of the suspended matter solubilizedduring the hydrogen fermentation (Table 2). Our results alsodemonstrated that waste treatment could be carried out with-out the removal of suspended matter from the pressed fil-trate by connecting the hydrogen fermentor prior to UASBmethane fermentation.
Hydrogen-ethanol production from biodiesel waste-water Biodiesel fuels have attracted a great deal ofattention recently because they are an alternative to petro-leum-based fuel, renewable and nontoxic, contribute to afavorable energy balance, and produce less harmful emis-sions than gasoline. Although biodiesel fuels are producedchemically and enzymatically, glycerol is essentially gener-ated as the by-product (34, 35). However, if there is an in-crease in the production of biodiesel fuels in the world, thenthe problem of efficiently treating wastes containing glyc-erol will need to be faced. Because glycerol is the best sub-strate for hydrogen production by E. aerogenes as men-tioned above (36), hydrogen production was examined fromglycerol-containing wastes discharged after the biodieselmanufacturing process. In continuous culture with a porous,ceramic packed-bed reactor as a support material for fixingcells in the reactor using flock-formed E. aerogenes, themaximum hydrogen production rate reached was 63mmol/l/h, giving an ethanol yield of 0.85 mol/mol-glycerolin the culture broth (37). This indicates that we can producebiodiesel, hydrogen, and ethanol from vegetable oils andanimal fats or their wastes.
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