Optimization of Biomethane Production via Fermentation of Chicken Manure Using Marine Sediment: A Modeling Approach Using Response Surface Methodology.

In this study, marine sediment (MS) was successfully used as a source of methanogenic bacteria for the anaerobic digestion (AD) of chicken manure (CM). Using MS showed high production in liquid and semi-solid conditions. Even in solid conditions, 169.3 mL/g volatile solids of chicken manure (VS-CM) was produced, despite the accumulation of ammonia (4.2 g NH3-N/kg CM). To the best of our knowledge, this is the highest methane production from CM alone, without pretreatment, in solid conditions (20%). Comparing MS to Ozouh sludge (excess activated sewage sludge) (OS), using OS under semi-solid conditions resulted in higher methane production, while using MS resulted in more ammonia tolerance (301 mL/gVS-CM at 8.58 g NH3-N/kg). Production optimization was carried out via a response surface methodology (RDM) model involving four independent variables (inoculum ratio, total solid content, NaCl concentration, and incubation time). Optimized methane production (324.36 mL/gVS-CM) was at a CM:MS ratio of 1:2.5 with no NaCl supplementation, 10% total solid content, and an incubation time of 45 days.

Plasmodium yoelii Erythrocyte Binding Like Protein Interacts With Basigin, an Erythrocyte Surface Protein.

Erythrocyte recognition and invasion is critical for the intra-erythrocytic development of Plasmodium spp. parasites. The multistep invasion process involves specific interactions between parasite ligands and erythrocyte receptors. Erythrocyte-binding-like (EBL) proteins, type I integral transmembrane proteins released from the merozoite micronemes, are known to play an important role in the initiation and formation of tight junctions between the apical end of the merozoite and the erythrocyte surface. In Plasmodium yoelii EBL (PyEBL), a single amino acid substitution in the putative Duffy binding domain dramatically changes parasite growth rate and virulence. This suggests that PyEBL is important for modulating the virulence of P. yoelii parasites. Based on these observations, we sought to elucidate the receptor of PyEBL that mediates its role as an invasion ligand. Using the eukaryotic wheat germ cell-free system, we systematically developed and screened a library of mouse erythrocyte proteins against native PyEBL using AlphaScreen technology. We report that PyEBL specifically interacts with basigin, an erythrocyte surface protein. We further confirmed that the N-terminal cysteine-rich Duffy binding-like region (EBL region 2), is responsible for the interaction, and that the binding is not affected by the C351Y mutation, which was previously shown to modulate virulence of P. yoelii. The identification of basigin as the putative PyEBL receptor offers new insights into the role of this molecule and provides an important base for in-depth studies towards developing novel interventions against malaria.

The malaria parasite RhopH protein complex interacts with erythrocyte calmyrin identified from a comprehensive erythrocyte protein library.

Malaria merozoite apical organelles; microneme and rhoptry secreted proteins play functional roles during and following invasion of host erythrocytes. Among numerous proteins, the rhoptries discharge high molecular weight proteins known as RhopH complex. Recent reports suggest that the RhopH complex is essential for growth and survival of the malaria parasite within erythrocytes. However, an in-depth understanding of the host-parasite molecular interactions is indispensable. Here we utilized a comprehensive mouse erythrocyte protein library consisting of 443 proteins produced by a wheat germ cell-free system, combined with AlphaScreen technology to identify mouse erythrocyte calmyrin as an interacting molecule of the rodent malaria parasite Plasmodium yoelii RhopH complex (PyRhopH). The PyRhopH interaction was dependent on the calmyrin N-terminus and divalent cation capacity. The finding unveils a recommendable and invaluable usefulness of our comprehensive mouse erythrocyte protein library together with the AlphaScreen technology in investigating a wide-range of host-parasite molecular interactions.

Improved methanization and microbial diversity during batch mode cultivation with repetition of substrate addition using defined organic matter and marine sediment inoculum at seawater salinity.

The activation of microbes, which are needed to initiate continuous methane production, can be accomplished by fed-batch methanization. In the present study, marine sediment inoculum was activated by batch mode methanization with repetition of substrate addition using defined organic matter from sugar, protein, or fat at seawater salinity to investigate the potential for application of the activation method to various types of saline waste and microbial community compositions. All substrates had methane potentials close to the theoretical value except for bovine serum albumin (BSA) whose methane potential was lower, but the maximum methane potential reached the value during repeated methanization. Beta diversity analysis revealed that substrate (especially BSA)-fed and non-fed cultures had distinct microbial community compositions. Bacterial members depended on substrate. Thus, marine sediment inocula activated via the methanization method can be used to effectively treat various types of saline waste.

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.

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.

Semi-continuous methane production from undiluted brown algae using a halophilic marine microbial community.

Acclimated marine sediment-derived culture was used for semi-continuous methane production from materials equivalent to raw brown algae, without dilution of salinity and without nutrient supply, under 3 consecutive conditions of varying organic loading rates (OLRs) and hydraulic retention time (HRT). Methane production was stable at 2.0gVS/kg/day (39-day HRT); however, it became unstable at 2.9gVS/kg/day (28-day HRT) due to acetate and propionate accumulation. OLR subsequently decreased to 1.7gVS/kg/day (46-day HRT), stabilizing methane production beyond steady state. Methane yield was above 300mL/g VS at all OLRs. These results indicated that the acclimated marine sediment culture was able to produce methane semi-continuously from raw brown algae without dilution and nutrient supply under steady state. Microbial community analysis suggested that hydrogenotrophic methanogens predominated among archaea during unstable methane production, implying a partial shift of the methanogenic pathway from acetoclastic methanogenesis to acetate oxidation.

Effect of salinity on methanogenic propionate degradation by acclimated marine sediment-derived culture.

Degradation of propionate under high salinity is needed for biomethane production from salt-containing feedstocks. In this study, marine sediment-derived culture was evaluated to determine the effect of salinity on methanogenic propionate degradation. Microbes in marine sediments were subjected to fed-batch cultivation on propionate for developing acclimatized cultures. The rate of propionate degradation increased eightfold during 10 rounds of cultivation. Microbial community composition was determined through pyrosequencing of 16S rRNA gene amplicons after 10 rounds of cultivation. Taxa analysis was conducted for the reads obtained by pyrosequencing. Known propionate degraders were undetectable in the acclimated culture. Comparison of bacterial taxa in the original sediment with those in the acclimated culture revealed that the populations of four bacterial taxa were significantly increased during acclimation. Methanolobus was the predominant archaea genus in the acclimated culture. The propionate degradation rate of the acclimated culture was not affected by salinity of up to equivalent of 1.9 % NaCl. The rate decreased at higher salinity levels and was more than 50 % of the maximum rate even at equivalent of 4.3 % NaCl.

Dysgonomonas alginatilytica sp. nov., an alginate-degrading bacterium isolated from a microbial consortium.

Gram-stain-negative, facultatively anaerobic, non-motile, non-spore-forming, rod-shaped bacterium, designated strain HUA-2T, was isolated from an alginate-degrading microbial consortium. Strain HUA-2T was related to Dysgonomonas capnocytophagoides JCM 16697T, Dysgonomonas macrotermitis JCM 19375T and Dysgonomonas mossii CCUG 43457T with 95.1 %, 94.1 % and 92.1 % 16S rRNA gene sequence similarity, respectively. The optimal growth temperature and pH for strain HUA-2T were 35 °C and pH 8.0, respectively. Enzyme production, major fermentation products from glucose, and the major cellular fatty acids were different from those of D. capnocytophagoides CCUG 17966T or other members of the genus Dysgonomonas. Therefore, strain HUA-2T is proposed to represent a novel species of the genus Dysgonomonas, for which we propose the name Dysgonomonas alginatilytica sp. nov. The type strain is HUA-2T ( = DSM 100214T = HUT 8134T).

Improved methane production from brown algae under high salinity by fed-batch acclimation.

Here, a methanogenic microbial community was developed from marine sediments to have improved methane productivity from brown algae under high salinity. Fed-batch cultivation was conducted by adding dry seaweed at 1wt% total solid (TS) based on the liquid weight of the NaCl-containing sediment per round of cultivation. The methane production rate and level of salinity increased 8-fold and 1.6-fold, respectively, at the 10th round of cultivation. Moreover, the rate of methane production remained high, even at the 10th round of cultivation, with accumulation of salts derived from 10wt% TS of seaweed. The salinity of the 10th-round culture was equivalent to 5% NaCl. The improved methane production was attributed to enhanced acetoclastic methanogenesis because acetate became rapidly converted to methane during cultivation. The family Fusobacteriaceae and the genus Methanosaeta, the acetoclastic methanogen, predominated in bacteria and archaea, respectively, after the cultivation.

Evaluation of marine sediments as microbial sources for methane production from brown algae under high salinity.

Various marine sediments were evaluated as promising microbial sources for methane fermentation of Saccharina japonica, a brown alga, at seawater salinity. All marine sediments tested produced mainly acetate among volatile fatty acids. One marine sediment completely converted the produced volatile fatty acids to methane in a short period. Archaeal community analysis revealed that acetoclastic methanogens belonging to the Methanosarcina genus dominated after cultivation. Measurement of the specific conversion rate at each step of methane production under saline conditions demonstrated that the marine sediments had higher conversion rates of butyrate and acetate than mesophilic methanogenic granules. These results clearly show that marine sediments can be used as microbial sources for methane production from algae under high-salt conditions without dilution.

Combined pretreatment using ozonolysis and wet-disk milling to improve enzymatic saccharification of Japanese cedar.

Ozonolysis and subsequent wet-disk milling (DM) were carried out on Japanese cedar (Cryptomeria japonica) to improve sugar production by enzymatic saccharification. When the moisture content reached more than 40%, ozone consumption decreased, resulting in less delignification. Ozone treatment removed mainly lignin, but also small amounts of polysaccharides. The application of DM following the ozone treatment further increased glucose and xylose yields, but had no significant effect on mannose yield, due to the loss of mannan in the ozone-treated product and the lack of mannose-releasing activity in the hemicellulase used. Sugar concentration increased with substrate concentration, when a constant ratio of enzyme to substrate was used.

Improvement of enzymatic saccharification of sugarcane bagasse by dilute-alkali-catalyzed hydrothermal treatment and subsequent disk milling.

Dilute-alkali-catalyzed hydrothermal treatment (HT) was conducted to improve the enzymatic degradability of sugarcane bagasse. Wet-disk milling (DM) was also performed after HT. Sodium carbonate with 0-6% concentration on dry weight basis of bagasse was used as the alkali catalyst. A content of more than 4% of the alkali catalyst was necessary for producing a higher amount of glucose than that produced after HT without an alkali catalyst. HT with 6% of the alkali catalyst, which decreased the pH to the neutral region, retained more xylan and less lignin than HT without an alkali. Subsequent DM improved the enzymatic degradability further and increased the specific surface area. For a substrate concentration of 10%, the amounts of glucose and xylose produced were 344 and 188 mg/g-bagasse, respectively. These values corresponded to yields of 77% and 67% on the basis of the glucan and xylan contents in raw bagasse, respectively.

Characterization of arylsulfatase formed by derepressed synthesis in Citrobacter braakii.

Arylsulfatase activity was detected in a bacterial strain, Citrobacter braakii 69-b, isolated from soil by enrichment cultivation using porcine gastric mucin. The production of arylsulfatase was derepressed markedly in a synthetic medium by the addition of tyramine. The purified enzyme hydrolyzed 4-nitrophenyl sulfate, 4-nitrocatechol sulfate, and 3-indoxyl sulfate, and was classified as type I arylsulfatase.

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus.

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.