Genome-guided analysis of transformation efficiency and carbon dioxide assimilation by Moorella thermoacetica Y72.

We determined a draft genome sequence for Moorella thermoacetica strain Y72, a syngas-assimilating bacterium with high transformation efficiency. This strain was confirmed to be M. thermoacetica because its overall genome sequence characteristics were similar to those of M. thermoacetica strain ATCC39073. Y72 was confirmed to carry all the genes encoding the enzymes in the reductive acetyl-CoA pathway, with very high similarities to those of ATCC39073. In addition, it was confirmed to assimilate carbon dioxide using this pathway. However, although both Y72 and ATCC39073 carried common genes encoding several enzymes related to the reductive tricarboxylic acid (TCA) cycle, their gene sets were different. Our results suggested that the reason for higher transformation efficiency in Y72 than that in ATCC39073, a reference strain of M. thermoacetica, may be that Y72 possesses only 2 sets of genes considered to be involved in a restriction-modification system, which was half of those found in ATCC39073.

High-coverage gene expression profiling analysis of the cellulase-producing fungus Acremonium cellulolyticus cultured using different carbon sources.

The gene expression of a cellulase-producing fungus, Acremonium cellulolyticus, was investigated after culturing with three different carbon sources: glycerol, lactose, and Solka-Floc powdered cellulose (SF). High-coverage gene expression profiling (HiCEP) analysis, a method requiring no prior sequence knowledge, was used to screen genes upregulated at the early stage of cellulase production. SF was used as a strong inducer of cellulase production, lactose was used as an inducer of the expression of cellulase genes at the early stage of the culture, and glycerol was used as a negative control. Approximately 15,000 transcript-derived fragments (TDFs) were detected in each sample prepared from the culture grown for 16 h. Based on the expression profiles of the cultured cells, 36 fragments upregulated in both the SF and lactose cultures were selected and sequenced. The deduced gene products of 31 TDFs were likely related to biomass degradation, sugar metabolism, transcriptional regulation, protein modification and metabolism, cell wall recycling, fatty acid and polyketide biosynthesis, and other functions. Quantitative real-time reverse-transcriptase polymerase chain reaction analysis verified that almost all of the transcripts obtained by HiCEP analysis were upregulated in the SF and lactose cultures grown for 18 h. Some of the TDFs in the SF culture were further upregulated over the course of 72 h. The gene products from these TDFs would provide insight into improving the cellulase productivity of A. cellulolyticus.

Combination of hot compressed water treatment and wet disk milling for high sugar recovery yield in enzymatic hydrolysis of rice straw.

Rice straw has attracted significant interest in Japan as a potential raw material for biorefineries. Combination of hot-compressed water treatment (HCWT) and wet disk milling (WDM) was investigated to improve the enzymatic digestibility of rice straw and enhance sugar recovery yield. Rice straw, cut to <3 mm, was autoclaved at 121, 135, and 150 °C for 60 min, and subsequently treated by wet disk milling. WDM with HCWT at 135 °C for 60 min produced maximum xylose and glucose yields of 79% and 90%, respectively, at 10 FPU/g-substrate cellulase loading. Autoclaving at 150 °C leaked a 35% arabinose effluence in the liquid phase. Hydrolysis via WDM with HCWT required a lower enzyme loading (5 FPU/g-substrate) than either pretreatment process in isolation for >70% xylose and 80% glucose yield. Economical analysis indicate that enzymes cost for ethanol production is reduced by 19-67% by WDM with HCWT.

Production and characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice straw subjected to various pretreatments as the carbon source.

Cellulases and hemicellulases are key enzymes in the production of alternative fuels and chemicals from lignocellulosic biomass-an abundant renewable resource. Carbon source selection is an important factor in the production of cellulases and hemicellulases. Rice straw--a potential ethanol source--has recently gained considerable interest in Asian countries. Here, we investigated the production of cellulases by using rice straw subjected to various pretreatments as substrates in order to produce cellulases at low costs; we also identified the enzymes' characteristics. Rice straw cutter milled to <3mm was pretreated by wet disk milling, dry ball milling, or hot-compressed water treatment (HCWT). Pretreated rice straw and commercial cellulose, Solka Floc (SF), were used as carbon sources for cellulase production by the fungus Acremonium cellulolyticus. Filter paper cellulase, ß-xylanase, and ß-xylosidase production from ball- and disk-milled samples were higher than those from SF. Enzymatic activity was absent in cultures where HCWT rice straw was used as carbon source. Wet disk-milled rice straw cultures were more suitable for enzymatic hydrolysis of pretreated rice straw than SF cultures. Thus, wet disk milling may be a suitable pretreatment for producing substrates for enzymatic hydrolysis and generating inexpensive carbon sources for cellulase production.

Enhancement on biodegradation and anaerobic digestion efficiency of activated sludge using a dual irradiation process.

A dual irradiation process involving aerobic thermophilic irradiation pretreatment (ATIP) and intermittent irradiation anaerobic digestion was developed to improve the digestion of waste-activated sludge. First, the effect of ATIP on further anaerobic digestion of activated sludge in batch mode was investigated. When exposed to ATIP for 24 h, the digestion reactor gave the highest methane yield, removed the most dissolved organic carbon (DOC) and showed the most effective reduction of VS compared to other irradiation times. This process was further enhanced by using an anaerobic fluidised-bed reactor packed with carbon felt in semi-continuous mode for digesting the pretreated activated sludge under intermittent irradiation conditions. Dual irradiation for 24 h followed by 60 min of anaerobic irradiation processing per day turned out to be optimal. This resulted in 65.3% of VS reduction, 83.9% of DOC removal ratio and 538 ml/g-VS of methane yield.

Wet disk milling pretreatment without sulfuric acid for enzymatic hydrolysis of rice straw.

Rice straw has recently attracted interest in Japan as a potential source of raw material for ethanol production. Wet disk milling, a continuous pretreatment to enhance the enzymatic digestibility of rice straw, was compared with conventional ball milling and hot-compressed water treatment. Pretreated rice straw was evaluated by enzymatic hydrolysis using Acremonium cellulase and characterized by X-ray diffraction and scanning electron microscopy. Glucose and xylose yields by wet disk milling, ball milling, and hot-compressed water treatment were 78.5% and 41.5%, 89.4% and 54.3%, and 70.3% and 88.6%, respectively. Wet disk milling and hot-compressed water treatment increased sugar yields without decreasing their crystallinity. The feature size of the wet disk milled rice straw was similar to that of hot-compressed water-treated rice straw. The energy consumption of wet disk milling was lower than that of other pretreatments. Thus, wet disk milling is an economical, practical pretreatment for the enzymatic hydrolysis of lignocellulosic biomass, especially herbaceous biomass such as rice straw.

Simultaneous methanogenesis and phototrophic bacterial growth in relatively dry sewage sludge under light.

Anaerobically digested sewage sludge with a variety of moisture content, namely 81%, 86%, 90% and 98%, were anaerobically cultured at 35 degrees C under light. Phototrophic bacteria grew in the 86% moisture sludge (bacteriochlorophyll a, 0.46 g/L), 90% sludge (bacteriochlorophyll a, 0.36 g/L) and 98% sludge (bacteriochlorophyll a, 0.04 g/L) with methane production. Phototrophic bacteria could not grow in the 81% moisture sludge (bacteriochlorophyll a 0.004 g/L). Phototrophic bacteria could assimilate about 46% of the extracellular ammonium in the 90% moisture sludge. Phototrophic bacteria utilized organic compounds competing with methanogens; therefore, methane yield from the 90% moisture sludge under the light conditions was lower than that under the dark conditions. Phototrophic bacteria could grow in anaerobically digested sludge with relatively low moisture content, and assimilated extracellular ammonium in the sludge. The quality of digested sludge with phototrophic bacterial biomass for fertilizer could be improved compared with that without phototrophic bacterial biomass.

Performance of a fixed-bed reactor packed with carbon felt during anaerobic digestion of cellulose.

The anaerobic digestion of cellulose was assessed in batch and semi-continuous studies using a carbon felt fixed-bed reactor. In the batch operation, the volatile solids reduction (%) and the cumulative methane production during the mesophilic and thermophilic digestion were 52.2% and 15.9%, 96.7 and 49.2 ml/g-total solid fed, respectively. After 99 days of semi-continuous mesophilic digestion, the degradation of cellulose reached its highest level of 67.6% at the hydraulic retention time of 9 days. The methane production and methane concentration of biogas from the bioreactor were maintained at a steady state. The fixed-bed reactor with carbon felt would be suitable for the efficient anaerobic digestion of cellulose. The biomass distribution in the reactor was, in the liquid phase 0.73 g/l-reactor, in the felt 1.59 g/l-reactor, and on the felt surface 9.86 g/l-reactor, which indicated that most of the microbes were immobilized on the carbon felt fixed-bed in the reactor.

Effect of ammonium addition on methanogenic community in a fluidized bed anaerobic digestion.

After immobilization of anaerobes on carbon felt in a fluidized-bed anaerobic digester at an ammonium concentration of 500 mg N/l, the results of real-time PCR analysis indicated that the cell densities of the immobilized methanogens and bacteria increased compared with those of the free-living methanogens and bacteria in the original anaerobically digested sewage sludge, respectively. The results of the clone analysis of the original sludge suggested that the major methanogens were Methanosaeta sp. and the members of the order Methanomicrobiales, and that after immobilization, these were changed to Methanobacterium and Methanosarcina sp. The results of real-time PCR analysis also showed that the ratio of the Methanosaeta sp. in the methanogenic archaea decreased from 58.2% to 0.3% after the immobilization. Methane production decreased at ammonium concentrations of greater than 6000 mg N/l. The results of real-time PCR analysis indicated that the cell density of the immobilized archaea decreased at ammonium concentrations of greater than 3000 mg N/l. On the other hand, the cell density of the immobilized bacteria did not decrease at an ammonium concentration of 6000 mg N/l, but decreased at that of 9000 mg N/l. The major methanogenic clones immobilized on the carbon felt at an ammonium concentration of 3000 or 6000 mg N/l were Methanobacterium sp. The present results indicated that methanogens were relatively more sensitive to ammonium than bacteria.

Removal of 3-chlorobenzoate using granules in the upflow anaerobic sludge blanket method.

The possibility of removing 3-chlorobenzoate from water using granules in the upflow anaerobic sludge blanket (UASB) method without the addition of a dechlorinating culture was studied on a laboratory scale. After three months of operation, the 3-chlorobenzoate concentration in the effluent decreased to less than 1 mg.l(-1).

Analysis of effluent after anaerobic digestion of liquid phase separated from liquidized garbage.

The organic compositions of the liquid phase separated from liquidized garbage as the influent and its effluent after anaerobic digestion at an overloading rate were analyzed. A large amount of organic acids was found in the effluent. The accumulation of organic acids suggests that the rate of methanogenesis is lower than that of acidogenesis.