Dry Co-Digestion of Poultry Manure with Agriculture Wastes.

This study tested the effect on thermophilic and mesophilic digestion of poultry manure (PM) or treated poultry manure (TPM) by the addition of agriculture wastes (AWS) as a co-substrate under dry conditions. PM was co-digested with a mixture of AWS consisting of coconut waste, cassava waste, and coffee grounds. Results were increased methane content in biogas, with decreased ammonia accumulation and volatile acids. The highest performance occurred under mesophilic conditions, with a 63 and 41.3 % increase in methane production from addition of AWS to TPM (562 vs. 344 mL g VS(-1) from control) and PM (406 vs. 287 mL g VS(-1) from control), respectively. Thermophilic conditions showed lower performance than mesophilic conditions. Addition of AWS increased methane production by 150 and 69.6 % from PM (323.4 vs. 129 mL g VS(-1) from control) and TPM (297.6 vs. 175.5 mL g VS(-1) from control), respectively. In all experiments, 100 % acetate produced was degraded to methane. Maximum ammonia accumulation was lowered to 43.7 % by mixing of AWS (range 5.35-8.55 vs. 7.81-12.28 g N kg(-1) bed). The pH was held at 7.3-8.8, a range suitable for methanogenesis.

Bacterial community structure and predicted alginate metabolic pathway in an alginate-degrading bacterial consortium.

Methane fermentation is one of the effective approaches for utilization of brown algae; however, this process is limited by the microbial capability to degrade alginate, a main polysaccharide found in these algae. Despite its potential, little is known about anaerobic microbial degradation of alginate. Here we constructed a bacterial consortium able to anaerobically degrade alginate. Taxonomic classification of 16S rRNA gene, based on high-throughput sequencing data, revealed that this consortium included two dominant strains, designated HUA-1 and HUA-2; these strains were related to Clostridiaceae bacterium SK082 (99%) and Dysgonomonas capnocytophagoides (95%), respectively. Alginate lyase activity and metagenomic analyses, based on high-throughput sequencing data, revealed that this bacterial consortium possessed putative genes related to a predicted alginate metabolic pathway. However, HUA-1 and 2 did not grow on agar medium with alginate by using roll-tube method, suggesting the existence of bacterial interactions like symbiosis for anaerobic alginate degradation.

Characterization of a halotolerant acetoclastic methanogen highly enriched from marine sediment and its application in removal of acetate.

A marine sediment collected from Hiroshima Bay was cultured in artificial seawater, containing 0.51 M NaCl and 60 mM acetate and was found to exhibit active methane production at 37°C. Following four successive serial dilutions of cultures in medium containing 0.51 M NaCl, 60 mM acetate, and antibiotics, the well-acclimated methanogen was found to exhibit growth over a range of NaCl concentration (between 0 M and 2.06 M). The specific growth rates of the highly enriched methanogen, termed strain HA, in the absence of NaCl and in the presence of 1.54 M NaCl were estimated to be 0.037 h(-1) and 0.027 h(-1), respectively. The pH and temperature for optimum growth were determined to be 7.0-8.8 and 37°C, respectively. Although cells that had morphology similar to Methanosaeta sp. became dominant in the culture, methane production was still detected in the medium containing 0.51 M NaCl and other substrates such as methanol, formate, and methylamine, indicating contamination with other methanogens. The phylogenetic tree based on 16S rRNA gene sequences revealed that the strain HA was closely related to Methanosaeta harundinacea 6Ac and 8Ac(T), with sequence similarity of 98% and 97%, respectively. The continuous removal of acetate with upflow anaerobic filter reactor for industrial use of strain HA determined a methane production rate of 70 mM/d under condition of 0.51 M NaCl and successful methane production even under 1.54 M NaCl.

Enhancement of methane production from co-digestion of chicken manure with agricultural wastes.

The potential for methane production from semi-solid chicken manure (CM) and mixture of agricultural wastes (AWS) in a co-digestion process has been experimentally evaluated at thermophilic and mesophilic temperatures. To the best of author(')s knowledge, it is the first time that CM is co-digested with mixture of AWS consisting of coconut waste, cassava waste, and coffee grounds. Two types of anaerobic digestion processes (AD process) were used, process 1 (P1) using fresh CM (FCM) and process 2 (P2) using treated CM (TCM), ammonia stripped CM, were conducted. Methane production in P1 was increased by 93% and 50% compared to control (no AWS added) with maximum methane production of 502 and 506 mL g(-1)VS obtained at 55°C and 35°C, respectively. Additionally, 42% increase in methane production was observed with maximum volume of 695 mL g(-1)VS comparing P2 test with P2 control under 55°C. Ammonia accumulation was reduced by 39% and 32% in P1 and P2 tests.

Engineering of a functional thermostable kanamycin resistance marker for use in Moorella thermoacetica ATCC39073.

A transformation system for Moorella thermoacetica ATCC39073 was developed using thermostable kanamycin resistant gene (kanR) derived from the plasmid pJH1 that Streptococcus faecalis harbored. When kanR with its native promoter was introduced into uracil auxotrophic mutant of M. thermoacetica ATCC39073 together with a gene to complement the uracil auxotrophy as a selection marker, it did not give kanamycin resistance due to poor transcription level of kanR. However, the use of glyceraldehyde-3-phosphate dehydrogenase promoter cloned from M. thermoacetica ATCC39073 significantly improved transcription level of kanR and resulted in the cell growth in the presence of more than 150 µg mL(-1) kanamycin. It was also demonstrated that kanR with G3PD promoter can be used as a selection marker for transformation of wild-type strain of M. thermoacetica ATCC39073.

Development of genetic transformation and heterologous expression system in carboxydotrophic thermophilic acetogen Moorella thermoacetica.

To develop a microbial production platform based on hydrogen and carbon dioxide, a genetic transformation system for the thermophilic acetogen Moorella thermoacetica ATCC39073 was developed. The uracil auxotrophic strain dpyrF was constructed by disrupting pyrF for orotate monophosphate decarboxylase. The transformation plasmids were methylated by restriction methylases of M. thermoacetica to avoid the decomposition of introduced plasmids by restriction-modification system. Reintroduction of native pyrF into the mutant by homologous recombination ensured recovery from uracil auxotrophy. To test heterologous gene expression in dpyrF, the lactate dehydrogenase (LDH) gene (T-ldh) from Thermoanaerobacter pseudethanolicus ATCC33223 was electroporated into dpyrF with a promoter of the glyceraldehyde-3-phosphate dehydrogenase (G3PD) gene of M. thermoacetica ATCC39073. The resulting transformant (C31) successfully transcribed T-ldh and exhibited higher LDH activity than ATCC39073 and dpyrF, yielding 6.8 mM of lactate from fructose, whereas ATCC39073 did not produce lactate.

Brewer's yeast cell wall affects microbiota composition and decreases Bacteroides fragilis populations in an anaerobic gut intestinal model.

Brewer's yeast cell wall (BYC) has been reported to have prebiotic activity that improves the microbiotal composition of the human gut. To understand the precise effect of BYC on gut microbiota and its metabolism, we used a three-stage continuous-flow reactor system that mimicked the environment of the large intestine. The reactor system was able to maintain the bacterial community stably for a week. The Bacteroides fragilis population decreased drastically after the addition of BYC into this system while the number of Lactobacillus was stably maintained. In addition, propionate and acetate levels increased drastically. This metabolic change correlated with an increase in a number of specific operational taxonomic units annotated to the genus Veillonella and Megasphaella. These results suggest that BYC affects the composition of microbiota in an in vitro model system.

Thermophilic two-stage dry anaerobic digestion of model garbage with ammonia stripping.

To avoid the inhibition of methane production by ammonia that occurs during the degradation of garbage, anaerobic digestion with prior ammonia production and subsequent stripping was investigated. In the ammonia production phase, the maximum ammonia concentration was approximately 2800 mg N/kg of total wet sludge in the range of 4 days of sludge retention time, indicating that only 43% of total nitrogen in the model garbage was converted to ammonia. The model garbage from which ammonia was produced and stripped was subjected to semi-continuous thermophilic dry anaerobic digestion over 180 days. The gas yield was in the range of 0.68 to 0.75 Nm(3)/kg volatile solid, and it decreased with the decrease of the sludge retention time. The ammonia-nitrogen concentration in the sludge was kept below 3000 mg N/kg total wet sludge. Microbial community structure analysis revealed that the phylum Firmicutes dominated in the ammonia production, but the community structure changed at different sludge retention times. In dry anaerobic digestion, the dominant bacteria shifted from the phylum Thermotogae to Firmicutes. The dominant archaeon was the genus Methanothermobacter, but the ratio of Methanosarcina increased during the process of dry anaerobic digestion.

Improved methane fermentation of chicken manure via ammonia removal by biogas recycle.

This study demonstrates methane fermentation that was carried out along with ammonia striping to avoid ammonia accumulation that significantly inhibited methane production. Ammonia was successfully removed by means of recycling of biogas followed by gas washing in sulfuric acid to capture ammonia, when chicken manure was anaerobically digested for 4 days at 55 degrees C and at an initial pH of 8-9. By using this method, 80% of total nitrogen in chicken manure was converted to ammonia and 82% of the produced ammonia was removed. A bench scale reactor equipped with an ammonia-stripping unit for methane production from chicken manure was developed and operated in repeated batch mode. At an initial pH of 8 and at 55 degrees C, 195 and 157 ml g-VS(-1) of methane was successfully produced from the treated chicken manure and the mixture of treated chicken manure and raw chicken manure in the ratio of 1:1, respectively. In this method, ammonia concentration was maintained at a level lower than 2g-N kg-wet sludge(-1) in the reactor.

Dry mesophilic fermentation of chicken manure for production of methane by repeated batch culture.

The dry fermentation of chicken manure (CM) for production of methane in mesophilic conditions at 37 degrees C was studied under laboratory conditions using a repeated batch culture system. Methane was successfully produced after an acclimation period of about 254 d. A total volume of 4.4 l kg(-1) CM (31 ml g(-1)VS) of methane gas was produced, despite the presence of a high level of ammonia of ca. 8 to 14 g-N kg(-1) CM. This clearly demonstrates that spontaneous acclimation of the methanogenic consortia to high levels of ammonia could occur and result in production of methane even under a high percentage of total solid (25%) and a high level of ammonia.

Dry anaerobic ammonia-methane production from chicken manure.

The effect of temperature on production of ammonia during dry anaerobic fermentation of chicken manure (CM), inoculated with thermophilic methanogenic sludge, was investigated in a batch condition for 8 days. Incubation temperature did not have a significant effect on the production of ammonia. Almost complete inhibition of production of methane occurred at 55 and 65 degrees C while quite low yields of 8.45 and 6.34 ml g(-1) VS (volatile solids) were observed at 35 and 45 degrees C due to a higher accumulation of ammonia. In order to improve the production of methane during dry anaerobic digestion of CM, stripping of ammonia was performed firstly on the CM previously fermented at 65 degrees C for 8 days: the stripping for 1 day at 85 degrees C and pH 10 removed 85.5% of ammonia. The first-batch fermentation of methane for 75 days was conducted next, using the ammonia-stripped CM inoculated with methanogenic sludge at different ratios, (CM: thermophilic sludge) of 1:2, 1:1, and 2:1 on volume per volume basis at both 35 and 55 degrees C. Production of methane improved and was higher than that of the control (without stripping of ammonia) but the yield of 20.4 ml g(-1) VS was still low, so second stripping of ammonia was conducted, which resulted in 74.7% removal of ammonia. A great improvement in the production of methane of 103.5 ml g(-1) VS was achieved during the second batch for 55 days.

Ammonia-methane two-stage anaerobic digestion of dehydrated waste-activated sludge.

The study investigated methane production from dehydrated waste-activated sludge (DWAS) with approximately 80% water content under thermophilic conditions. The repeated batch-wise treatment of DWAS using methanogenic sludge unacclimated to high concentrations of ammonia, increased the ammonia production up to 7,600 mg N per kilogram total wet sludge of total ammonia concentration, and stopped the methane production. Investigation revealed that the loading ratio of DWAS for methanogenic sludge influences anaerobic digestion. Methane production significantly decreased and ammonia concentration increased with the increase in loading ratio of DWAS. Since the semicontinuous culture revealed that approximately 50% of organic nitrogen in DWAS converted to ammonia at sludge retention time (SRT) after 4 days at 37 degrees C and 1.33 days at 55 degrees C, the previous stripping of the ammonia produced from DWAS was carried out. The stripping of ammonia increased methane production significantly. This ammonia-methane two-stage anaerobic digestion demonstrated a successful methane production at SRT 20 days in the semicontinuous operation using a laboratory-scale reactor system.

Degradation of glyoxylate and glycolate with ATP synthesis by a thermophilic anaerobic bacterium, Moorella sp. strain HUC22-1.

The thermophilic homoacetogenic bacterium Moorella sp. strain HUC22-1 ferments glyoxylate to acetate roughly according to the reaction 2 glyoxylate --> acetate + 2 CO(2). A batch culture with glyoxylate and yeast extract yielded 11.7 g per mol of cells per substrate, which was much higher than that obtained with H(2) plus CO(2). Crude extracts of glyoxylate-grown cells catalyzed the ADP- and NADP-dependent condensation of glyoxylate and acetyl coenzyme A (acetyl-CoA) to pyruvate and CO(2) and converted pyruvate to acetyl-CoA and CO(2), which are the key reactions of the malyl-CoA pathway. ATP generation was also detected during the key enzyme reactions of this pathway. Furthermore, this bacterium consumed l-malate, an intermediate in the malyl-CoA pathway, and produced acetate. These findings suggest that Moorella sp. strain HUC22-1 can generate ATP by substrate-level phosphorylation during glyoxylate catabolism through the malyl-CoA pathway.

Recent development of anaerobic digestion processes for energy recovery from wastes.

Anaerobic digestion leads to the overall gasification of organic wastewaters and wastes, and produces methane and carbon dioxide; this gasification contributes to reducing organic matter and recovering energy from organic carbons. Here, we propose three new processes and demonstrate the effectiveness of each process. By using complete anaerobic organic matter removal process (CARP), in which diluted wastewaters such as sewage and effluent from a methane fermentation digester were treated under anaerobic condition for post-treatment, the chemical oxygen demand (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 dilution of the ammonia produced by water or carbon-rich wastes. The hydrogen-methane two-stage fermentation (Hy-Met process), in which the hydrogen produced in the first stage is used for a fuel cell system to generate electricity and the methane produced in the second stage is used to generate heat energy to heat the two reactors and satisfy heat requirements, is useful for the treatment of sugar-rich wastewaters, bread wastes, and biodiesel wastewaters.

Characterization of enzymes involved in the ethanol production of Moorella sp. HUC22-1.

Since the thermophilic bacterium Moorella sp. HUC22-1 produces 120 mM acetate and 5.2 mM ethanol from H(2)-CO(2), several candidate genes, which were predicted to code for three alcohol dehydrogenases (AdhA, B, C) and one acetaldehyde dehydrogenase (Aldh), were cloned from HUC22-1. The cloned genes were subcloned into a His-tagged expression vector and expressed in Escherichia coli. Recombinant AdhA and B were both dependent on NADP(H) but independent of NAD(H), and their reduction activities from aldehyde to alcohol were higher than their oxidation activities. In contrast with AdhA and B, no activity of AdhC was observed in either reaction. On the other hand, Aldh was active toward both NADP(H) and NAD(H). The enzyme activity of Aldh was directed toward the thioester cleavage and the thioester condensation. When 50 microg of AdhA and 50 microg Aldh were added to the buffer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60 degrees C, 1.6 mM ethanol was produced from 3 mM acetyl-CoA after 90 min. Expression analysis of the mRNAs revealed that the expression level of aldh was threefold higher in the H(2)-CO(2) culture than that in the fructose culture, but levels of adhA, B and C were decreased.

Feasibility study of a pilot-scale sewage treatment system combining an up-flow anaerobic sludge blanket (UASB) and an aerated fixed bed (AFB) reactor at ambient temperature.

A feasibility test of a 17 m3-pilot-scale sewage treatment system was carried out by continuous feeding of raw municipal sewage under ambient temperature conditions. The system consisted of a UASB and an aerated fixed bed reactor. Some of the effluent from the fixed bed reactor was returned to the UASB influent in order to provide a sulfate source. The total BOD of 148-162 mg l(-1) in the influent was reduced to a more desirable 11-25 mg l(-1) in the final effluent. The levels of methane-producing activity from acetate and H2/CO2 gas at 10 degrees C were only 2% and 0% of those at 35 degrees C, respectively. On the other hand, the sulfate-reducing activity levels of the UASB sludge were relatively high at 10 degrees C, for example, 18% for acetate and 9% for H2/CO2 gas, compared to the activity levels at 35 degrees C. Therefore, BOD oxidization by sulfate reduction in the UASB was greater than that by methane production under low temperature conditions. This sulfate-reducing activity tended to be proportional to the copy number of adenosine-5'-phosphosulfate (APS) reductase genes in DNA extracted from the sludge.

Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process.

H2 and ethanol production from glycerol-containing wastes discharged after a manufacturing process for biodiesel fuel (biodiesel wastes) using Enterobacter aerogenes HU-101 was evaluated. The biodiesel wastes should be diluted with a synthetic medium to increase the rate of glycerol utilization and the addition of yeast extract and tryptone to the synthetic medium accelerated the production of H2 and ethanol. The yields of H2 and ethanol decreased with an increase in the concentrations of biodiesel wastes and commercially available glycerol (pure glycerol). Furthermore, the rates of H2 and ethanol production from biodiesel wastes were much lower than those at the same concentration of pure glycerol, partially due to a high salt content in the wastes. In continuous culture with a packed-bed reactor using self-immobilized cells, the maximum rate of H2 production from pure glycerol was 80 mmol/l/h yielding ethanol at 0.8 mol/mol-glycerol, while that from biodiesel wastes was only 30 mmol/l/h. However, using porous ceramics as a support material to fix cells in the reactor, the maximum H2 production rate from biodiesel wastes reached 63 mmol/l/h obtaining an ethanol yield of 0.85 mol/mol-glycerol.

Acetate and ethanol production from H2 and CO2 by Moorella sp. using a repeated batch culture.

The growth inhibition of Moorella sp. HUC22-1 by undissociated acetic acid was analyzed using a non-competitive inhibition model coupled with a pH inhibition model. In the cells grown on H2 and CO2, the inhibition constant, K(p) of the undissociated acetic acid was 6.2 mM (164 mM as the total acetate at pH 6.2, pKa = 4.795, 55 degrees C), which was 1.5-fold higher than that obtained in cells grown on fructose. When a pH-controlled batch culture was performed using a fermentor at pH 6.2 with H2 and CO2, a maximum of 0.92 g/l of dry cell weight and 339 mM of acetate were produced after 220 h, which were 4.4- and 6.8-fold higher than those produced in the pH-uncontrolled batch culture, respectively. In order to reduce acetate inhibition in the culture medium, a repeated batch culture with cell recycling was performed at a constant pH with H2 and CO2. At a pH of 6.2, the total acetate production reached 840 mmol/l-reactor with 4.7 mmol/l-reactor of total ethanol production after 420 h. When the culture pH was maintained at 5.8, which was the optimum for ethanol production, the total ethanol production reached 15.4 mmol/l-reactor after 430 h, although the total acetate production was decreased to 675 mmol/l-reactor.

High rate production of hydrogen/methane from various substrates and wastes.

To treat soluble and solid wastes and recover energy from them, high rate methane fermentation, especially using the UASB (upflow anaerobic sludge blanket) reactor, and hydrogen fermentation using various microorganisms and microbial consortia have been investigated intensively in Japan. In this chapter, recent works on high rate methane fermentation in Japan are reviewed, focusing on: 1) basic studies into the applicability of the UASB reactor for various substrates such as propionate, lactate, ethanol, glucose and phenol; 2) its applications to unfeasible conditions, such as lipid and protein containing wastes, low temperature and high salt-containing wastes; 3) progress made in the field of advanced UASB reactors, and; 4) research into methane fermentation from solid wastes, such as from cellulosic materials, municipal sewage sludge, and mud sediments. Following this, although hydrogen fermentation with photosynthetic microorganisms or anaerobic bacteria was researched, for this review we have focused on fermentative hydrogen production using strictly or facultative anaerobes and microbial consortia in Japan, since high rate production of hydrogen-methane via a two-stage process was judged to be more attractive for biological hydrogen production and wastewater treatments.

High-yield production of hydrogen by Enterobacter aerogenes mutants with decreased alpha-acetolactate synthase activity.

To enhance hydrogen (H2) production from glucose by Enterobacter aerogenes HU-101, two mutants, strains VP-1 and VP-2, with decreased alpha-acetolactate synthase activity, were isolated using the Voges-Proskauer (VP) test. In pH-uncontrolled batch culture, both mutants showed a lower 2,3-butanediol yield for the glucose consumed than that shown by the wild-type strain, although glucose remained in the medium after 12 h of culture. In the same cultures, compared to the H2 yield of 0.80 mol/mol-glucose of the wild-type strain, strain VP-1 showed a high H2 yield of 1.8 mol/mol-glucose with decreased lactate and increased succinate yields, while strain VP-2 showed an H2 yield of 1.0 mol/mol-glucose with an increased lactate yield. Increasing the phosphate buffer concentration, which contributes to maintaining the pH in the medium, increased the glucose consumption by both strains. However, in a pH-controlled batch culture at neutral pH, the H2 yield of strain VP-1 was decreased to 1.2 mol/mol-glucose due to the accumulation of formate, an intermediate of the H2-producing pathway, with the yield of H2 plus formate being 1.7 mol/mol-glucose.