The Potential of Digested Sludge-Assimilating Microflora for Biogas Production from Food Processing Wastes.

Food processing wastes (FPWs) are residues generated in food manufacturing, and their composition varies depending on the type of food product being manufactured. Therefore, selecting and acclimatizing seed microflora during the initiation of biogas production is crucial for optimal outcomes. The present study examined the biogas production capabilities of digested sludge-assimilating and biogas-yielding soil (DABYS) and enteric (DABYE) microflorae when used as seed cultures for biogas production from FPWs. After subculturing and feeding these microbial seeds with various FPWs, we assessed their biogas-producing abilities. The subcultures produced biogas from many FPWs, except orange peel, suggesting that the heterogeneity of the bacterial members in the seed microflora facilitates quick adaptation to FPWs. Microflorae fed with animal-derived FPWs contained several methanogenic archaeal families and produced methane. In contrast, microflorae fed with vegetable-, fruit-, and crop-derived FPWs generated hydrogen, and methanogenic archaeal populations were diminished by repeated subculturing. The subcultured microflorae appear to hydrolyze carbohydrates and protein in FPWs using cellulase, pectinase, or protease. Despite needing enhancements in biogas yield for future industrial scale-up, the DABYS and DABYE microflorae demonstrate robust adaptability to various FPWs.

Microalga-bacteria Community with High Level Carbon Dioxide Acclimation and Nitrogen-fixing Ability.

Microalgal conversion of high-level CO2 in industrial flue gas to value-added products is attractive technology for mitigating global warming. However, reduction of microalgal production costs for medium ingredients, particularly nitrogen salts, is essential. The use of atmospheric nitrogen as a nitrogen source for microalgal cultivation will dramatically reduce its production costs. We attempted to enrich a microalga-bacteria community, which fixes both CO2 and atmospheric nitrogen under high level CO2. By cultivating biofilm recovered from the surface of cobbles in a riverbank, a microalgal flora which grows in a nitrogen salts-free medium under 10% CO2 was enriched, and the coccoid microalgal strain MP5 was isolated from it. Phylogenetic analysis revealed that the strain MP5 belongs to the genus Coelastrella, and the closest known species was C. terrestris. With PCR-DGGE analysis, it was found that the enriched microalgal community includes bacteria, some of which are suggested diazotrophs. The addition of bactericides in culture medium inhibited MP5 growth, even though the strain MP5 is eukaryotic. Growth of bacteria-free MP5 was stimulated by addition of Agrobacterium sp. isolates in nitrogen salts-free medium, suggesting that MP5 and the bacteria have responsibility for photosynthetic carbon fixation and nitrogen fixation, respectively.

Nitrogen-assisted lipid production by biofilms of aerial microalga Coccomyxa subellipsoidea KGU-D001 in the aerial phase.

The aerial microalga (Coccomyxa subellipsoidea KGU-D001) was photoautotrophically cultured under aerial phase conditions, and the influence of the nitrogen source on lipid production was investigated. Coccomyxa biofilms were introduced onto cotton wool using pure water (i.e., nutrient depletion) or a nitrogen-containing solution in a Petri dish, and they were cultured for 14 days under aerial phase conditions. The biomass in the biofilm increased by more than 150% in 14 days under nutrient depleted conditions and then increased further by approximately 30% following the addition of a nitrogen source. The lipid content rose under both nutrient depletion and nitrogen-added conditions, increasing by 170 and 150% in 14 days, respectively. The protein and sugar contents were also monitored and analyzed. In the presence of a nitrogen source, C. subellipsoidea undergo cell division in a relatively short time span, and biomass and lipids can be synthesized under both nutrient depleted and nitrogen-added conditions.

Production of polyglutamic acid-like mucilage protein by Peribacillus simplex strain 8h.

Polyglutamic acid (PGA), a protein in the mucilage of PGA-producing Bacillus spp., has expected applications in medical and biotechnological industries. Although the Bacillaceae family contains over 100 genera, research on bacterial PGA has exclusively focused on the genus Bacillus, especially B. subtilis var. natto and B. licheniformis. In the present study, indigenous Bacillaceae family strains were isolated from withered leaves and soil samples and screened for PGA production. As a result of the screening, the strain 8h was found to produce a mucilage possessing greater viscosity than PGA of B. subtilis var. natto (natto PGA). Biochemical analyses revealed that the 8h mucilage contains 63% protein and 37% polysaccharide, while mucilage of B. subtilis var. natto is composed of 61% protein and 39% polysaccharide. The most plentiful amino acid in 8h mucilage protein was glutamate (43%, mol/mol), which is similar to that of natto PGA, suggesting that it possesses characteristics of PGA. Although natto mucilage contains fructan, glucan was found as the polysaccharide of 8h mucilage. While phylogenetic studies indicated that the strain 8h belongs to Peribacillus simplex, the yield of the viscous mucilage by strain 8h was significantly higher than P. simplex type strain, suggesting that 8h is a mucilage-overproducing strain of P. simplex. Interestingly, 8h mucilage protein was found to contain more hydrophobic amino acid residues than natto PGA, suggesting that its amphiphilicity is suitable as a drug carrier and adjuvant. The present study is the first report of viscous mucilage and PGA-like protein produced by the genus Peribacillus.

Isolation and Characterization of Basidiomycetous Yeasts Capable of Producing Phytase under Oligotrophic Conditions.

Phytic acid is an organic phosphorus source naturally produced by plants as phosphorus stock and can be an alternative to rock phosphate, which is a dwindling resource globally. However, phytic acid is insoluble, owing to its binding to divalent metals and is, thus, not readily bioavailable for plants and monogastric livestock. Therefore, the enzyme phytase is indispensable for hydrolyzing phytic acid to liberate free phosphates for nutritional availability, making the screening of novel phytase-producing microbes an attractive research focus to agriculture and animal feed industries. In the present study, a soil-extract-based culture medium was supplemented with phytic acid as the sole phosphorus source and oligotrophic phytase-producing strains, which had not been previously studied, were isolated. Four fungal strains with phytic acid, assimilation activities were isolated. They were found to produce phytase in the culture supernatants and phylogenetic analysis identified three strains as basidiomycetous yeasts (Saitozyma, Leucosporidium, and Malassezia) and one strain as an ascomycetous fungus (Chaetocapnodium). The optimal pH for phytase activity of the strains was 6.0-7.0, suggesting that they are suitable for industrial applications as feed supplements or fertilizer additives for farmland.

Microflora communities which can convert digested sludge to biogas.

In the present study, we developed several microflora communities that utilize digested sludge (DS), the recalcitrant waste product of anaerobic digestion, as a substrate for biogas production with the aim of their future application to DS recycling. Strict enrichment with DS as the sole nutrient source was introduced to culture microbes from soil and herbivore dung samples; microflora communities promoting stable levels of biogas production were obtained. The average methane and hydrogen yield from soil-derived microflora were 4.86 and 0.94 ml per 1.0 g DS, respectively. Notably, two microflora communities enriched from a riverbank sediment produced 20.79 ml and 14.10 ml methane from 1.0 g DS. By contrast, the methane and hydrogen yield for herbivore dung-derived microfloras were on average 1.31 ml and 1.87 ml per 1.0 g DS, respectively. Potent hydrogen-biogas producers were obtained from rabbit (4.12 ml per 1.0 g DS), goat (3.16 ml per 1.0 g DS), and sheep dung (2.52 ml per 1.0 g DS). The cultured microflora communities included representatives from the eubacterial genera, Clostridiaceae and Eubacteriaceae together with several anaerobic genera. Pseudomonas spp. are found in the riverbank sediment-derived microfloras, suggesting that the floras employ syntrophic acetate oxidation and hydrogentrophic methanogenesis (SAO-HM) pathway for methane production. The methanogenic microflora communities were dominated by bacteria from the Methanobacteriaceae family and unclassified archaea. Moreover, ascomycetous fungi and protists were found, implying that they act as oxygen scavengers and bacterial grazers, respectively. Enzymatic analysis suggested that the microfloras hydrolyze DS via cellulase, chitinase, and protease activities.

Effects of light-emitting diodes (LEDs) on lipid production of the aerial microalga Coccomyxa sp. KGU-D001 under liquid- and aerial-phase conditions.

Algal biofuels are a promising alternative to fossil fuels, but their widespread use is hindered by problems with mass production. Light-emitting diodes (LEDs) with specific light wavelengths could be used as an energy source for algal growth and lipid synthesis. In this study, the effects of light source on the biomass and lipid production of the aerial microalga Coccomyxa sp. KGU-D001 were evaluated using LEDs. The integration of two-phase cultures, including growth and lipid production under the stress of nitrate depletion, was assessed for efficient lipid production under liquid- or aerial-phase conditions. Different wavelengths of light (blue, green, and red) were tested under liquid- and aerial-phase conditions. Under aerial-phase culture, the fatty acid contents in biofilm reached 320 mg g DWC-1 with the red LEDs. In view of these findings, we describe a one-step culture method for growth and lipid accumulation in algal biofilm under aerial-phase culture with red LED irradiation. When Coccomyxa biofilm was cultured on wet cotton wool with BBM in a petri dish under the red LED, it was able to grow and accumulate lipids under the aerial-phase condition. Based on the results of this study, a potential method for a continuous biodiesel production system is proposed.

Atom Probe Tomography Interlaboratory Study on Clustering Analysis in Experimental Data Using the Maximum Separation Distance Approach.

We summarize the findings from an interlaboratory study conducted between ten international research groups and investigate the use of the commonly used maximum separation distance and local concentration thresholding methods for solute clustering quantification. The study objectives are: to bring clarity to the range of applicability of the methods; identify existing and/or needed modifications; and interpretation of past published data. Participants collected experimental data from a proton-irradiated 304 stainless steel and analyzed Cu-rich and Ni-Si rich clusters. The datasets were also analyzed by one researcher to clarify variability originating from different operators. The Cu distribution fulfills the ideal requirements of the maximum separation method (MSM), namely a dilute matrix Cu concentration and concentrated Cu clusters. This enabled a relatively tight distribution of the cluster number density among the participants. By contrast, the group analysis of the Ni-Si rich clusters by the MSM was complicated by a high Ni matrix concentration and by the presence of Si-decorated dislocations, leading to larger variability among researchers. While local concentration filtering could, in principle, tighten the results, the cluster identification step inevitably maintained a high scatter. Recommendations regarding reporting, selection of analysis method, and expected variability when interpreting published data are discussed.

Biochemical characteristics of cellulose and a green alga degradation by Gilvimarinus japonicas 12-2T, and its application potential for seaweed saccharification.

Cellulose is one of the major constituents of seaweeds, but reports of mechanisms in microbial seaweed degradation in marine environment are limited, in contrast to the multitude of reports for lignocellulose degradation in terrestrial environment. We studied the biochemical characteristics for marine cellulolytic bacterium Gilvimarinus japonicas 12-2T in seaweed degradation. The bacterial strain was found to degrade green and red algae, but not brown algae. It was shown that the bacterial strain employs various polysaccharide hydrolases (endocellulase, agarase, carrageenanase, xylanase, and laminarinase) to degrade seaweed polysaccharides. Electrophoretic analysis and peptide sequencing showed that the major protein bands on the electrophoresis gel were homologous to known glucanases and glycoside hydrolases. A seaweed hydrolysate harvested from the bacterial culture was found useful as a substrate for yeasts to produce ethanol. These findings will provide insights into possible seaweed decomposition mechanisms of Gilvimarinus, and its biotechnological potential for ethanol production from inedible seaweeds.

Gilvimarinus japonicus sp. nov., a cellulolytic and agarolytic marine bacterium isolated from coastal debris.

A cellulolytic and agarolytic bacterial strain, designated 12-2T, was isolated from a piece of cotton rope fragment washed ashore on a beach and was studied phenotypically, genotypically and phylogenetically. Analyses of 16S rRNA and gyrB gene sequences and DNA base composition suggested that the strain is a member of the genus Gilvimarinus. However, levels of 16S rRNA and gyrB gene sequence similarity between it and the type strains of Gilvimarinus species were no higher than 97.9 and 78.7 %, respectively, suggesting that the strain is distinct. Moreover, the results of DNA-DNA hybridization experiments and physiological characterization clearly differentiated the strain from its closest neighbours. The strain is therefore considered to represent a novel species of the genus Gilvimarinus, for which the name Gilvimarinus japonicus sp. nov. is proposed. The type strain is 12-2T (=NBRC 111987T=KCTC 52141T).

Possible practical utility of an enzyme cocktail produced by sludge-degrading microbes for methane and hydrogen production from digested sludge.

Digested sludge (DS) is a major waste product of anaerobic digestion of sewage sludge and is resistant to biodegradation. In this study, we examined suitability of the hydrolases produced by DS-degrading fungal strains (DS-hydrolases) for methane and hydrogen fermentation from DS. Although the strains are mesophilic, DS-hydrolases showed strong chitinase and keratinase activity at ∼50°C. SDS-PAGE analysis suggested that the strains possess a multienzyme system, which allows the hydrolases of some strains to be stable in a wide range of temperatures. Addition of the DS-hydrolases to a vial-scale anaerobic digester enhanced methane and hydrogen production from DS at pH 9.0 and 5.0, respectively. The hydrogen production was also enhanced by the use of methacrylate ester-precipitated DS as a substrate. Further improvement of culture and reaction conditions may make these hydrolases suitable for production of renewable fuels.

Streptomyces abietis sp. nov., a cellulolytic bacterium isolated from soil of a pine forest.

Cellulolytic bacteria A191(T), A192 and A193 isolated from the soil of Sakhalin fir forest in Hokkaido, Japan were studied phenotypically, genotypically and phylogenetically. Analysis of their 16S rRNA gene and gyrB sequences and DNA base composition suggested that these isolates were conspecific and members of the genus Streptomyces. However, levels of 16S rRNA gene and gyrB sequence similarity between the isolates and the type strains of their closest relatives in the genus Streptomyces were no higher than 97.9 and 95.0 %, respectively, implying that these isolates were distinctive. Moreover, the results of DNA-DNA hybridization experiments and physiological characterization clearly differentiated these isolates from their closest neighbours. It is therefore concluded that these isolates represent a novel species of the genus Streptomyces, for which the name Streptomyces abietis is proposed. The type strain is A191(T) ( = NBRC 109094(T) = DSM 42080(T)).

Cellulolytic microbes in the Yanbaru, a subtropical rainforest with an endemic biota on Okinawa Island, Japan.

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.

Isolation and characterization of aerobic microorganisms with cellulolytic activity in the gut of endogeic earthworms.

The ability of earthworms to decompose lignocellulose involves the assistance of microorganisms in their digestive system. While many studies have revealed a diverse microbiota in the earthworm gut, including aerobic and anaerobic microorganisms, it remains unclear which of these species contribute to lignocellulose digestion. In this study, aerobic microorganisms with cellulolytic activity isolated from the gut of two endogeic earthworms, Amynthas heteropoda (Megascolecidae) and Eisenia fetida (Lumbricidae) were isolated by solid culture of gut homogenates using filter paper as a carbon source. A total of 48 strains, including four bacterial and four fungal genera, were isolated from two earthworm species. Characterization of these strains using enzyme assays showed that the most representative ones had exocellulase and xylanase activities, while some had weak laccase activity. These findings suggest that earthworms digest lignocellulose by exploiting microbial exocellulase and xylanase besides their own endocellulase. Phylogenetic analysis showed that among the cellulolytic isolates in both earthworm species Burkholderia and Chaetomium were the dominant bacterial and fungal members.

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes.

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.

Penicillium strains as dominant degraders in soil for coffee residue, a biological waste unsuitable for fertilization.

AIMS: Coffee residue is an agricultural waste which inhibits the growth of several crops. Therefore coffee residue-degrading microbes in soil were screened, isolated and characterized. METHODS AND RESULTS: Forty isolates were obtained after enrichment culture of soil samples. Seven strains (fast degraders) showed strong degrading activity, while 18 strains (slow degraders) showed weak degrading activity. DNA analysis suggested that the fast degraders are Penicillium, and the slow degraders are Penicillium, Trichoderma/Hypocrea, Fusarium/Gibberella, Phaeoacremonium/Togninia or Acidocella. The all fast degraders are cellulolytic, mannolytic and pectinolytic. CONCLUSIONS: Although it is generally thought that fungi such as Trichoderma contribute largely to aerobic degradation of cellulosic biomass, our data suggested that Penicillium overwhelms them in coffee residue degradation. It was implied that polysaccharides in coffee residue are not degraded independently by different microbes, but degraded simultaneously by strains with cellulolytic, mannolytic and pectinolytic activity. Since there is no report of an ascomycete possessing all the three enzyme activities, the fast degraders are ecologically important and have the potential to be used as producers of the costly enzymes from agricultural wastes. SIGNIFICANCE AND IMPACT OF STUDY: The present results advance our understanding of microbial degradation of a phytotoxic agricultural waste, and offer a new tool for recycling it.

Crystal structure of the complex of porcine pancreatic elastase with TEI-8362.

The crystal structure of porcine pancreatic elastase (PPE) complexed with a new benzoxazinone inhibitor, TEI-8362, of human neutrophil elastase (HNE) was determined at 1.8 A resolution. The hydroxyl oxygen of Ser195 opened the benzoxazinone by nucleophilic attack and formed a covalent bond with the carbonyl carbon. Hydrophobic interaction between the terminal benzene of TEI-8362 and the S4 pocket is reinforced by the side chain of Arg217 and has an impact on the ligand binding conformation. Two additional interactions with the oxyanion hole and His57 are introduced to the benzoxazinone structure of TEI-8362. These combinatorial interactions will also exist in HNE and cause high preference of TEI-8362 for HNE.

Potential of a new biotreatment: Sphingomonas cloacae S-3T degrades nonylphenol in industrial wastewater.

Sphingomonas cloacae S-3(T), a nonylphenol (NP)-degrading bacterium, was evaluated for its utility in the remediation of NP-contaminated wastewater. In flask-scale experiments, S-3(T) cells immobilized on porous polypropylene carriers (beads) efficiently degraded NP to concentrations routinely measured in aquatic environments [a few parts per billion (ppb), or micrograms per liter). Therefore, we constructed and evaluated a laboratory-scale wastewater treatment system with a 3-l carrier-filled column. The system worked properly and consistently removed several hundred ppb of NP to ecologically safe concentrations of less than 10 ppb in industrial wastewater without the addition of nutrients. The effect of wastewater pH on the system performance was also evaluated; and wastewater samples with pH values of 6 or 8 were treated efficiently without pH adjustment. These results suggest that a biotreatment system using NP-degrading bacteria can efficiently remediate industrial wastewater and contribute to the preservation of aquatic environments.

Novosphingobium tardaugens sp. nov., an oestradiol-degrading bacterium isolated from activated sludge of a sewage treatment plant in Tokyo.

An oestradiol-degrading bacterium isolated at a sewage treatment plant in Tokyo was studied phenotypically, genotypically and phylogenetically. Analysis of its 16S rDNA sequence, DNA base composition, whole-cell fatty acid profile and isoprenoid quinone composition, as well as the presence of sphingoglycolipid, revealed that the isolate is a member of the genus Novosphingobium. However, the sequence similarity of its 16S rDNA to those of known Novosphingobium species was no higher than 97%, implying that the isolate is distinctive. The results of DNA-DNA hybridization experiments and physiological characterization also indicated that the isolate represents a novel Novosphingobium species, for which the name Novosphingobium tardaugens sp. nov. is proposed; strain ARI-1T (=JCM 11434T =ATCC BAA-531T =IFO 16725T) is the type strain.

Degradation of 17beta-estradiol by a gram-negative bacterium isolated from activated sludge in a sewage treatment plant in Tokyo, Japan.

A 17beta-estradiol (E2)-degrading bacterium was isolated from activated sludge in a sewage treatment plant in Tokyo, Japan. The isolate was suggested to be a new Novosphingobium species. Gas chromatography-mass spectrometry and (1)H nuclear magnetic resonance analyses of the metabolites of E2 degradation suggested that no toxic products accumulated in the culture medium.