Biomethane production and microbial strategies corresponding to high organic loading treatment for molasses wastewater in an upflow anaerobic filter reactor.

Molasses wastewater contains high levels of organic compounds, cations, and anions, causing operational problems for anaerobic biological treatment. In this study, an upflow anaerobic filter (UAF) reactor was employed to establish a high organic loading treatment system for molasses wastewater and further investigated the microbial community dynamics in response to this stressful operation. The biogas production increased with an increase in total organic carbon (TOC) loading rate from 1.0 to 14 g/L/day, and then it decreased with further TOC loading rate addition until 16 g/L/day. The UAF reactor achieved a maximum biogas production of 6800 mL/L/day with a TOC removal efficiency of 66.5% at a TOC loading rate of 14 g/L/day. Further microbial analyses revealed that both the bacterial and archaeal communities developed multiple strategies to maintain stable operation of the reactor at high organic loading (e.g., Proteiniphilum and Defluviitoga maintained high abundances throughout the operation; Tissierella temporarily dominated the bacterial community at TOC loading rates of 8.0 to 14 g/L/day; and multi-trophic Methanosarcina shifted as the dominant methanogen at the TOC loading rates of 8.0 to 16 g/L/day). This study presents insights into a high organic loading molasses wastewater treatment system and the microbial flexibility in methane fermentation in response to process disturbances.

Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability.

Production of ethanol from xylose by recombinant Saccharomyces cerevisiae is suboptimal with slow fermentation rate, compared with that from glucose. In this study, a strain-expressing Scheffersomyces stipitis xylose reductase-xylitol dehydrogenase (XR-XDH) pathway was subjected to adaptive evolution on xylose; this approach generated populations with the significantly improved cell growth and ethanol production rate. Mutants were isolated, and the best one was used for sporulation to generate eight stable mutant strains with improved xylose fermentation ability. They were used in a microarray assay to study the molecular basis of the enhanced phenotype. The enriched transcriptional differences among the eight mutant strains and the native strain revealed novel responses to xylose, which likely contributes to the improved xylose utilization. The upregulated vitamin B1 and B6 biosynthesis indicated that thiamine served as an important cofactor in xylose metabolism and may alleviate the redox stress. The increased expression of genes involved in sulfur amino acid biosynthesis and the decreased expression of genes related to Fe(II) transport may alleviate redox stress as well. Meanwhile, it was remarkable that several glucose-repressible genes, including genes of the galactose metabolism, gluconeogenesis, and ethanol catabolism, had a lower expression level after adaptive evolution. Concomitantly, the expression levels of two regulators of the glucose signaling pathway, Rgs2 and Sip4, decreased, indicating a reshaped signaling pathway to xylose after adaptive evolution. Our findings provide new targets for construction of a superior bioethanol producing strain through inverse metabolic engineering.

Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37.

Industrial yeast strains with good xylose fermentation ability and inhibitor tolerance are important for economical lignocellulosic bioethanol production. The flocculating industrial Saccharomyces cerevisiae strain NAPX37, harboring the xylose reductase-xylitol dehydrogenase (XR-XDH)-based xylose metabolic pathway, displayed efficient xylose fermentation during batch and continuous fermentation. During batch fermentation, the xylose consumption rates at the first 36 h were similar (1.37 g/L/h) when the initial xylose concentrations were 50 and 75 g/L, indicating that xylose fermentation was not inhibited even when the xylose concentration was as high as 75 g/L. The presence of glucose, at concentrations of up to 25 g/L, did not affect xylose consumption rate at the first 36 h. Strain NAPX37 showed stable xylose fermentation capacity during continuous ethanol fermentation using xylose as the sole sugar, for almost 1 year. Fermentation remained stable at a dilution rate of 0.05/h, even though the xylose concentration in the feed was as high as 100 g/L. Aeration rate, xylose concentration, and MgSO4 concentration were found to affect xylose consumption and ethanol yield. When the xylose concentration in the feed was 75 g/L, a high xylose consumption rate of 6.62 g/L/h and an ethanol yield of 0.394 were achieved under an aeration rate of 0.1 vvm, dilution rate of 0.1/h, and 5 mM MgSO4. In addition, strain NAPX37 exhibited good tolerance to inhibitors such as weak acids, furans, and phenolics during xylose fermentation. These findings indicate that strain NAPX37 is a promising candidate for application in the industrial production of lignocellulosic bioethanol.

Development of a more efficient process for production of fuel ethanol from bamboo.

A process for production of fuel ethanol from bamboo treated with concentrated sulfuric acid has been previously proposed. To improve efficiency of the process, we tested saccharification with 70 weight% (wt%) sulfuric acid, acid-sugar separation by ion exclusion, addition of nutrients to the ethanol fermentation, and bioconversion of xylose to xylitol. A high efficiency of both sugar recovery (82.5 %) and acid recovery (97.5 %) was achieved in the saccharification process and in the continuous acid-sugar separation using a modified anion exchange resin, respectively. Reduction of the amount of mineral salts added to the saccharified liquid after acid-sugar separation did not negatively affect performance of the continuous ethanol fermentation. The ethanol yield and productivity were 93.7 % and 6 g/l h, respectively, at 35 °C and pH 4.0. And the ethanol yield and productivity were almost the same even at pH 3.5. Moreover, the xylose remaining in the fermented mash was efficiently converted to xylitol in batch fermentation by Candida tropicalis strain 2.1776. These results demonstrate a more efficient process for the production of fuel ethanol from bamboo.

Synergistic effects of TAL1 over-expression and PHO13 deletion on the weak acid inhibition of xylose fermentation by industrial Saccharomyces cerevisiae strain.

In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors.

Production of bio-fuel ethanol from distilled grain waste eluted from Chinese spirit making process.

Distilled grain waste eluted from Chinese spirit making is rich in carbohydrates, and could potentially serve as feedstock for the production of bio-fuel ethanol. Our study evaluated two types of saccharification methods that convert distilled grain waste to monosaccharides: enzymatic saccharification and concentrated H2SO4 saccharification. Results showed that enzymatic saccharification performed unsatisfactorily because of inefficient removal of lignin during pretreatment. Concentrated H2SO4 saccharification led to a total sugar recovery efficiency of 79.0 %, and to considerably higher sugar concentrations than enzymatic saccharification. The process of ethanol production from distilled grain waste based on concentrated H2SO4 saccharification was then studied. The process mainly consisted of concentrated H2SO4 saccharification, solid-liquid separation, decoloration, sugar-acid separation, oligosaccharide hydrolysis, and continuous ethanol fermentation. An improved simulated moving bed system was employed to separate sugars from acid after concentrated H2SO4 saccharification, by which 95.8 % of glucose and 85.8 % of xylose went into the sugar-rich fraction, while 83.3 % of H2SO4 went into the acid-rich fraction. A flocculating yeast strain, Saccharomyces cerevisiae KF-7, was used for continuous ethanol fermentation, which produced an ethanol yield of 91.9-98.9 %, based on glucose concentration.

Digestion performance and microbial community in full-scale methane fermentation of stillage from sweet potato-shochu production.

Sweet potato shochu is a traditional Japanese spirit produced mainly in the South Kyushu area in Japan. The amount of stillage reaches approximately 8 x 10(5) tons per year. Wastewater mainly containing stillage from the production of sweet potato-shochu was treated thermophilically in a full-scale treatment plant using fixed-bed reactors (8 reactors x 283 m3). Following the addition of Ni2+ and Co2+, the reactors have been stably operated for six years at a high chemical oxygen demand (COD) loading rate of 14 kg/(m3 x day). Analysis of coenzyme content and microbial communities indicated that similar microbial communities were present in the liquid phase and on the fiber carriers installed in reactors. Bacteria in the phyla Firmicutes as well as Bacteroidetes were dominant bacteria, and Methanosarcina thermophila as well as Methanothermobacter crinale were dominant methanogens in the reactors. This study reveals that stillage from sweet potato-shochu production can be treated effectively in a full-scale fixed-bed reactor under thermophilic conditions with the help of Ni2+ and Co2+. The high diversity of bacterial community and the coexistence of both aceticlastic and hydrogenotrophic methanogens contributed to the excellent fermentation performance.

Improvement of ethanol production from D-lactic acid by constitutive expression of lactate transporter Jen1p in Saccharomyces cerevisiae.

To improve ethanol production from D-lactate, Jen1p, a monocarboxylate-proton symporter, was constitutively expressed in Saccharomyces cerevisiae NAM34-4C. The mutant produced 2.4 g/L of ethanol, approximately 2.4 times higher than that of the wild-type strain. A monocarboxylate/proton symporter gene (JEN1) null mutant was also constructed. It produced 0.19 g/L of ethanol, 5 times lower than that of the wild-type strain.

Efficient production of bioethanol from corn stover by pretreatment with a combination of sulfuric acid and sodium hydroxide.

Corn stover is the most abundant agricultural residue in China and a valuable reservoir for bioethanol production. In this study, we proposed a process for producing bioethanol from corn stover; the pretreatment prior to presaccharification, followed by simultaneous saccharification and fermentation (SSF) by using a flocculating Saccharomyces cerevisiae strain, was optimized. Pretreatment with acid-alkali combination (1% H2SO4, 150 °C, 10 min, followed by 1% NaOH, 80°C, 60 min) resulted in efficient lignin removal and excellent recovery of xylose and glucose. A glucose recovery efficiency of 92.3% was obtained by enzymatic saccharification, when the pretreated solid load was 15%. SSF was carried out at 35 °C for 36 hr after presaccharification at 50 °C for 24 hr, and an ethanol yield of 88.2% was achieved at a solid load of 15% and an enzyme dosage of 15 FPU/g pretreated corn stover.

Pretreatment followed by anaerobic digestion of secondary sludge for reduction of sewage sludge volume.

The influence of two pretreatment methods, thermal treatment and low-pressure wet oxidation, on the sludge digestion efficiency was examined. Batch thermophilic anaerobic digestion was used to evaluate the effectiveness of the pretreatment methods in terms of volatile suspended solids (VSS) digestion efficiency and gas production. The results showed that the gas production was not proportional to the VSS degradation efficiency of either thermal treatment or low-pressure wet oxidation. Low-pressure wet oxidation treatment at 150 °C along with 40% of the theoretical oxygen required to oxidize organic carbon gave the highest gas production and the VSS digestion efficiency of 77% at a VSS loading rate of 8 g l(-1) d(-1). The digestion efficiency was about 30% higher than that of thermophilic anaerobic digestion without sludge pretreatment. Sewage sludge could be treated effectively at a high VSS digestion efficiency with this pretreatment followed by thermophilic anaerobic digestion.

A secreted placental alkaline phosphatase-based reporter assay system for screening of compounds acting at an octopamine receptor stably expressed in a mammalian cell line.

Octopamine receptors are attractive insecticide targets. To screen compounds acting at octopamine receptors simply and rapidly, we constructed a chemiluminescent reporter gene assay system that detects secreted placental alkaline phosphatase transcriptionally regulated by the cAMP response element for a silkworm octopamine receptor. This system proved useful in high-throughput screening to develop octopamine receptor-specific insecticides.

Effect of distillery sewage sludge addition on performance and bacterial community dynamics during distilled grain waste composting.

This study evaluated the dynamics of physicochemical characteristics and bacterial communities during the co-composting of distilled grain waste (DGW) and distillery sewage sludge (SS), with DGW mono-composting as a control. Results showed that co-composting with SS significantly improved DGW degradation efficiency (61.38% vs. 54.13%) and end-product quality (seed germination index: 129.82% vs. 113.61%; N + P2O5 + K2O: 9.08% vs. 5.28%), compared to DGW mono-composting. Microbial community analysis revealed that co-composting accelerated the bacterial community succession rate and enhanced the abundance of the phyla Proteobacteria, Firmicutes, Chloroflexi, and Deinococcota by 45.86%, 4.38%, 37.49%, and 15.29%, respectively. Network analysis showed that DGW-SS co-composting altered the interactions among the bacterial genera and improved bacterial community stability. Spearman correlation analysis indicated that the correlation between bacterial genera and environmental factors was more significant in DGW-SS co-composting. Therefore, co-composting of DGW and SS is a suitable strategy for the treatment of solid byproducts from spirit distilleries.

Bacterial Community Structure and Metabolic Function Succession During the Composting of Distilled Grain Waste.

Distilled grain waste (DGW) can be converted to organic fertilizer via aerobic composting process without inoculating exogenous microorganisms. To illustrate the material conversion mechanism, this study investigated the dynamic changes of bacterial community structure and metabolic function involved in DGW composting. Results showed that a significant increase in microbial community alpha diversity was observed during DGW composting. Moreover, unique community structures occurred at each composting stage. The dominant phyla were Firmicutes, Proteobacteria, Actinobacteriota, Bacteroidota, Myxococcota, and Chloroflexi, whose abundance varied according to different composting stages. Keystone microbes can be selected as biomarkers for each stage, and Microbispora, Chryseolinea, Steroidobacter, Truepera, and Luteimonas indicating compost maturity. Co-occurrence network analysis revealed a significant relationship between keystone microbes and environmental factors. The carbohydrate and amino acid metabolism were confirmed as the primary metabolic pathways by metabolic function profiles. Furthermore, nitrogen metabolism pathway analysis indicated that denitrification and NH3 volatilization induced higher nitrogen loss during DGW composting. This study can provide new understanding of the microbiota for organic matter and nitrogen conversion in the composting process of DGW.

Characteristic microbial community of a dry thermophilic methanogenic digester: its long-term stability and change with feeding.

Thermophilic dry anaerobic digestion of sludge for cellulose methanization was acclimated at 53 °C for nearly 5 years using a waste paper-based medium. The stability of the microbial community structure and the microbial community responsible for the cellulose methanization were studied by 16S rRNA gene-based clone library analysis. The microbial community structure remained stable during the long-term acclimation period. Hydrogenotrophic methanogens dominated in methanogens and Methanothermobacter, Methanobacterium, Methanoculleus, and Methanosarcina were responsible for the methane production. Bacteria showed relatively high diversity and distributed mainly in the phyla Firmicutes, Bacteroidetes, and Synergistetes. Ninety percent of operational taxonomic units (OTUs) were affiliated with the phylum Firmicutes, indicating the crucial roles of this phylum in the digestion. Relatives of Clostridium stercorarium, Clostridium thermocellum, and Halocella cellulosilytica were dominant cellulose degraders. The acclimated stable sludge was used to treat garbage stillage discharged from a fuel ethanol production process, and the shift of microbial communities with the change of feed was analyzed. Both archaeal and bacterial communities had obviously changed: Methanoculleus spp. and Methanothermobacter spp. and the protein- and fatty acid-degrading bacteria became dominant. Accumulation of ammonia as well as volatile fatty acids led to the inhibition of microbial activity and finally resulted in the deterioration of methane fermentation of the garbage stillage.

[Surveillance of antimicrobial activity of Pseudomonas aeruginosa isolated in the Kinki district].

The antimicrobial activity of 18 antimicrobial agents were measured for the 500 Pseudomonas aeruginosa strains that had been isolated from various clinical specimens in 17 medical institutions in the Kinki district from April to July of 2008. The antimicrobial activity was excellent in the order of tobramycin (TOB), arbekacin (ABK), doripenem (DRPM), gentamicin (GM) and amikacin (AMK). Susceptible rate that was interpreted by Clinical and Laboratory Standards Institute (CLSI) was high in the order of AMK, TOB, tazobactam/piperacillin (TAZ/PIPC), DRPM, ABK. Also, the difference in susceptible rate was observed between departments, materials and institutions. Multidrug resistant strains were only 12 (2.4%) but strains that had resistance to 2 agents were 48 (9.6%), therefore, implementation of further surveillance should be continued.

Potential for reduced water consumption in biorefining of lignocellulosic biomass to bioethanol and biogas.

Increasing ethanol demand and public concerns about environmental protection promote the production of lignocellulosic bioethanol. Compared to that of starch- and sugar-based bioethanol production, the production of lignocellulosic bioethanol is water-intensive. A large amount of water is consumed during pretreatment, detoxification, saccharification, and fermentation. Water is a limited resource, and very high water consumption limits the industrial production of lignocellulosic bioethanol and decreases its environmental feasibility. In this review, we focused on the potential for reducing water consumption during the production of lignocellulosic bioethanol by performing pretreatment and fermentation at high solid loading, omitting water washing after pretreatment, and recycling wastewater by integrating bioethanol production and anaerobic digestion. In addition, the feasibility of these approaches and their research progress were discussed. This comprehensive review is expected to draw attention to water competition between bioethanol production and human use.

Biochar addition reduces nitrogen loss and accelerates composting process by affecting the core microbial community during distilled grain waste composting.

This study evaluated the impact of biochar addition on nitrogen (N) loss and the process period during distilled grain waste (DGW) composting. Results from the five treatments (0, 5, 10, 15, and 20% biochar addition) indicated that 10% biochar addition (DB10) was optimal, resulting in the lowest N loss, 25.69% vs. 40.01% in the control treatment. Moreover, the DGW composting period was shortened by approximately 14 days by biochar addition. The composition of the microbial community was not significantly altered with biochar addition in each phase, however, it did accelerate the microbial succession during DGW composting. N metabolism pathway prediction revealed that biochar addition enhanced nitrification and inhibited denitrification, and the latter phenomenon was the main reason for reducing N loss during DGW composting. Based on the above results, a potential mechanism model for biochar addition to reduce N loss during the DGW composting process was established.

Immunostimulation-Mediated Anti-tumor Activity of Bamboo (Sasa senanensis) Leaf Extracts Obtained Under 'Vigorous' Condition.

Traditional Japanese medicine uses the leaves of Kumaizasa bamboo extracted in hot water at 100°C. For this study, we developed a new, 'vigorous' extraction method involving steps at 100, 121 and 196°C. This procedure not only yielded greater amounts of extract but also with significant increase in immunostimulating activity, which induces activation of human natural killer (NK) cells, macrophages and potent induction of IL-2, IL-12 and IFN-γ in tumor bearing mice. The efficacy of the extract to facilitate phagocytosis and nitric oxide production by mouse peritoneal macrophages was determined and compared with that of 1,3-ß-glucan. Anti-tumor activity was evaluated in vivo in several mouse tumor models (S-180, C38 and Meth-A). Oral administration of the extracts was carried out when tumor reached size of approximately 6 mm at concentrations of 0.05% or higher. The extracts significantly suppressed tumor growth in S-180 and C38 tumor models. Overall survival was significantly prolonged in the treatment group than that of control. Activation of macrophages and NK cells by the extracts suggests that the anti-tumor efficacy of the extract is mediated by immunopotentiation. The extracts resolved into three major fractions (F-I, F-II and F-III) in Sephadex gel chromatography. Fraction F-I consists of 1,3-ß-glucan and stimulated both macrophages and NK cells suggesting that it may be the primary immunopotentiating factor in suppressing cancer. Fraction F-III has potent free radical scavenging effects and may play an important role in cancer prevention. These results warrant further translation and clinical investigations.

Production of bioethanol from unwashed-pretreated rapeseed straw at high solid loading.

Reduction in water consumption and increase in ethanol concentration are two main challenges for bioethanol production from lignocellulosic materials. To address the two challenges, the aim of this work was to study the production of bioethanol from unwashed-pretreated rapeseed straw (RS) at high solid loading. RS pretreated with 1% (w w-1) H2SO4 at 160 °C for 10 min resulted in excellent digestibility and fermentability of pretreated RS. The unwashed-pretreated RS was subjected to presaccharification and fed-batch simultaneous saccharification and fermentation (P-FB-SSF) at a final solid loading of 22% (w w-1). Ethanol concentration and ethanol yield of 53.1 g L-1 (equivalent to 4.1% (w w-1) based on fermentation slurry) and 72.4% were obtained, respectively. In total, 92.1 g water g-1 ethanol was consumed, a much smaller amount than that observed with washing after pretreatment or fermentation performed at lower solid loading.

Insight into the microbiology of nitrogen cycle in the dairy manure composting process revealed by combining high-throughput sequencing and quantitative PCR.

Nitrogen cycling during composting process is not yet fully understood. This study explored the key genes involved in nitrogen cycling during dairy manure composting process using high-throughput sequencing and quantitative PCR technologies. Results showed that nitrogen fixation occurred mainly during the thermophilic and cooling phases, and significantly enhanced the nitrogen content of compost. Thermoclostridium stercorarium was the main diazotroph. Ammonia oxidation occurred during the maturation phase and Nitrosomonas sp. was the most abundant ammonia oxidizing bacteria. Denitrification contributed to the greatest nitrogen loss during the composting process. The nirK community was dominated by Luteimonas sp. and Achromobacter sp., while the nirS community was dominated by Alcaligenes faecalis and Pseudomonas stutzeri. The nosZ community varied in a succession of Halomonas ilicicola, Pseudomonas flexibili and Labrenzia alba dominated communities according to different composting phases. Based on these results, nitrogen cycling models for different phases of the dairy manure composting process were established.