Effects of formic acid and corn flour supplementation of banana pseudostem silages on nutritional quality of silage,growth,digestion,rumen fermentation and cellulolytic bacterial community of Nubian black goats

The objective of this study was to evaluate the effects of adding formic acid and corn flour supplementation to banana pseudostem silages on the nutritional quality of these silages,growth,digestion,rumen fermentation and cellulolytic bacterial community of Nubian black goats fed these silages.Banana pseudostem silage was prepared either conventionally without any additives(CON)or mixed with 0.6% formic acid(F),10% corn flour(C),or both(F+C).Four experimental diets containing 40% of the corresponding silages were designed with roughage to concentrate ratio of 50:50(dry matter(DM)basis).A total of 48 Nubian black castrated goats(body weight(BW),(22.64±1.82)kg;4-mon-old)were randomized into one of the four treatment groups with 12 replicates of one castrated goat per replicate for each treatment in a completely randomized design.Each group was fed on one of the four experimental diets for 40 days.A factorial arrangement of treatments of 2(formic acid levels:0 and 0.6%)×2(corn flour:0 and 10%)was adopted.Formic acid supplementation increased(P<0.05)average daily gain,as well as lactic acid,propionate and butyric acid and water-soluble carbohydrate concentrations,but decreased(P<0.05)the feed conversion rate,pH value,acetate/propionate ratio,and butyric acid concentration relative to the CON group.Corn flour supplementation increased(P<0.05)the apparent digestibility of crude protein,neutral detergent fiber,and non-fibrous carbohydrate and Fibrobacter succinogenes,Ruminococcus albus,and Butyrivibrio fibrisolvens populations,but decreased(P<0.05)the Ruminococcus flavefaciens population relative to the CON group.There were no F×C treatment interactions(P>0.05)for any of the other indices except for the apparent digestibility of non-fibrous carbohydrate(NFC)(P<0.05).The results demonstrated that adding 0.6% formic acid and 10% corn flour supplementation to banana pseudostem silages improved the nutritional quality of these silages and enhanced the growth performance of Nubian black castrated goats by improving apparent nutrient digestibility,and regulating ruminal fermentation and bacteria populations.

Effects of Facultative Anaerobic Cellulolytic Bacteria and Nitrogen-fixing Bacteria Isolated from Cow Rumen Fluid on Rumen Fermentation and Dry Matter Degradation in Vitro

The purpose of the paper was to study the effects of cellulolytic bacteria(CB)mixed with nitrogen-fixing bacteria(NFB)on fermentation in vitro.Nine CB strains and seven NFB strains were isolated from numen fluid of three Holstein cows.Based onhigher activity of cellulose or nitrogenase,three CB types[CB-2(KT725624),CB-5(KT725623)and CB-6(KT725622)]and one NFB type[NFB-3(KT735054)]were screened out,respectively.Two parts were included in this study.The first part explored the optimal mixed ratio of CB to NFB through inoculating filter paper medium with the bacteria of 2×10^5 cfu·mL^-1.According to CMCase and FPase activities in the medium,the ratio of 4 to 1 was proven to be more effective.In the second part,rumen fermentation in vitro was conducted at 4:1 of CB to NFB,aiming at studying the effects of mixed bacteria.Six groups were classified,namely,control group(no bacteria),Group 1(CB-2+NFB-3),Group 2(CB-5+NFB-3),Group 3(CB-6+NFB-3),Group 4(NFB-3)and Group 5(CB-6).All the experimental groups had the same amount of bacteria(4×10^6 cfu·mL^-1)in the fermentation liquid.Samples were collected at 2,4,8,12 and 24 h of incubation.Compared with the groups with CB or NFB alone,gas production,dry matter degradability and bacterial protein expressions in the mixed groups increased.However,NH3-N concentration decreased and p H was stable.Meanwhile,related values among three mixed groups were significantly different;values in Group 2 were the best.

The Cellulolytic Bacteria <i>R. albus</i>for Improving the Efficiency of Microbial Fuel Cell

The current study has been undertaken to examine the beneficial effect in the power output of a microbial fuel cell (MFC) by adding cellulolytic bacteria Ruminococcus albus (R. albus) into the anodic chamber. Mediator-less H-type MFCs were set up where the anode chamber contained anaerobic digester microorganisms as inocula on finely ground pine tree (Avicel) at 2% (w/v) and the cathode chamber of 10mM phosphate buffered saline conductive solution, both separated by a cation exchange membrane. The functioning of the MFCs for generation of electrical power and the amounts of gaseous byproducts was monitored over a 9-day period. The addition of cellulolytic bacteria caused an increase of average power density from 7.9 m W/m2 to19.5 m W/m2, about 245% increase over a 9-day period. For both groups of MFCs;with R. albus and the control, the head space gases collected were methane and CO2. While the methane: CO2 ratios were found unchanged at 1.7:1 throughout the 9 days of operation, the total gas production increased from 248 mL to 319 mL due to the presence of R. albus addition. This study confirms that whereas the biocatalytic activity of anode microbial population determines the energy production, the addition of external cellulolytic bacteria into anode microbial population can improve and extend the biomass utilization.

Analysis of cellulolytic and hemicellulolytic enzyme activity within the Tipula abdominalis （Diptera： Tipulidae） larval gut and characterization of Crocebacterium ilecola gen. nov., sp. nov., isolated from the Tipula abdominalis larval hindgut

In forested stream ecosystems of the north and eastern United States, larvae of the aquatic crane fly Tipula abdominalis are important shredders of leaf litter detritus. T. abdominalis larvae harbor a dense and diverse microbial community in their hindgut that may aide in the degradation oflignocellulose. In this study, the activities ofcellulolytic and hemicellulolytic enzymes were demonstrated from hindgut extracts and from bacterial isolates using model sugar substrates. One of the bacterial isolates was further characterized as a member of the family Microbacteriaceae. Taxonomic position of the isolate within this family was determined by a polyphasic approach, as is commonly employed for the separation of genera within the family Microbacteriaceae. The bacterial isolate is Gramtype positive, motile, non-sporulating, and rod-shaped. The G ＋ C content of the DNA is 64.9 mol%. The cell wall contains B2y type peptidoglycan, D- and L-diaminobutyric acid as the diamino acid, and rhamnose as the predominant sugar. The predominant fatty acids are 12-methyltetradecanoic acid （ai-C15：0） and 14-methylhexadecanoic acid （ai-C17：0）. The isolate forms a distinct lineage within the family Microbacteriaceae, as determined by 16S rRNA sequence analysis. We propose the name Crocebacterium ilecola gen. nov., sp. nov., to accommodate this bacterial isolate. The type species is T202T （ATCC BAA-1359; GenBank Accession DQ826511）.

Grazing greatly reduces the temporal stability of soil cellulolytic fungal community in a steppe on the Tibetan Plateau

Excessive livestock grazing degrades grasslands ecosystem stability and sustainability by reducing soil organic matter and plant productivity. However, the effects of grazing on soil cellulolytic fungi, an important indicator of the degradation process for soil organic matter,remain less well understood. Using T-RFLP and sequencing methods, we investigated the effects of grazing on the temporal changes of cellulolytic fungal abundance and community structure in dry steppe soils during the growing months from May to September, on the Tibetan Plateau using T-RFLP and sequencing methods. The results demonstrated that the abundance of soil cellulolytic fungi under grazing treatment changed significantly from month to month, and was positively correlated with dissolved organic carbon(DOC) and soil temperature, but negatively correlated with soil p H. Contrastingly, cellulolytic fungal abundance did not change within the fencing treatment(ungrazed conditions). Cellulolytic fungal community structure changed significantly in the growing months in grazed soils,but did not change in fenced soils. Grazing played a key role in determining the community structure of soil cellulolytic fungi by explaining 8.1% of the variation, while p H and DOC explained 4.1% and 4.0%, respectively. Phylogenetically, the cellulolytic fungi were primarily affiliated with Ascomycota(69.65% in relative abundance) and Basidiomycota(30.35%).Therefore, grazing substantially reduced the stability of soil cellulolytic fungal abundance and community structure, as compared with the fencing treatment. Our finding provides a new insight into the responses of organic matter-decomposing microbes for grassland managements.

Preparation and properties of poly(lactic acid)/cellulolytic enzyme lignin/PGMA ternary blends

Poly（lactic acid）-based ternary blends consisting of poly（lactic acid）（PLA）,cellulolytic enzyme lignin（CEL）,and polyolefine grafting maleic anhydride（PGMA） were prepared by extrusion blending and the mechanical properties and the morphology of the ternary blends were investigated.It was found that the mechanical properties varied with various loading of the components in the blends.Compared to neat PLA,the tensile strength and the Young＇s modulus of the ternary blends were decreased,but the elongation at break and the impact strength were effectively improved.Scanning electron microscope observations revealed that the CEL plays a bridging role between PLA and PGMA,enhancing the miscibility between them and resulting in the improvement of ductility and toughness of the ternary blends.Considering the cost and performance,we obtained the optimal blend PLA/CEL/ PGMA（80/20/20,w/w/w）,of which the impact strength and the elongation at break were doubled as that of neat PLA,and the tensile strength remained moderate.

Exploration of the key functional proteins from an efficient cellulolytic microbial consortium using dilution-to-extinction approach

In the present study, the cellulose binding proteins（CBPs） secreted by a putative cellulolytic microbial consortium were isolated and purified by affinity digestion. The purified CBPs were subsequently separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis（SDS-PAGE）. Using mass spectrometric analyses, eight CBPs were identified and annotated to be similar to known proteins secreted by Clostridium clariflavum DSM 19732 and Paenibacillus sp. W-61. In addition, in combination with dilution-to-extinction approach and zymogram analysis technique, CBPs 6（97 k Da） and 12（52 k Da） were confirmed to be the key functional proteins that influence cellulolytic activities. Moreover, structural domain analyses and enzymatic activity detection indicated that CBPs 6 and 12 contained glycoside hydrolase families（GH） 9 and 48 catalytic modules, which both revealed endoglucandase and xylanase activities. It was suggested that the coexistence of GH9 and GH48 catalytic domains present in these two proteins could synergistically promote the efficient degradation of cellulose.

Identification of Antarctic culturable bacteria able to produce diverse enzymes of potential biotechnological interest

It is estimated that more than three quarters of the Earth＇s biosphere is in perennially cold environments. Despite the extreme environmental conditions of desiccation and freezing, microbes can colonize these habitats through the adaptation of metabolic functions and the synthesis of structurally adapted enzymes. Enzymes within psychrophilic microbes exhibit high specific activity at low and moderate temperature, with low thermostability. In this study we used a classic microbiological approach to isolate Antarctic bacteria with cellulolytic, lipolytic, and ligninolytic activities. From 15 different environmental samples, we generated a collection of approximately 800 bacterial isolates that could grow on R2A or Marine medium at 4℃. This collection was then screened for the presence of the three types of activity at 4℃. We found that 47.7% of the isolates displayed lipolytic activity, 10.2% had cellulase/xylanase activity, and 7.7% showed guaiacol oxidase activity. Of these, 10% displayed two different types of activity, while 0.25% displayed all three types of activity. Our results indicate that cold environments represent outstanding resources for bioprospecting and the study of enzymatic adaptation.

Distribution of Myxomycetes on Varied Leaf Litter Types in a Mixed Forest in Warm-Temperate Western Japan

Myxomycete assemblages were compared on various leaf litters of different vegetation types in a local mixed forest consisting of deciduous and evergreen trees in western Japan. A total of 33 myxomycete species were recorded and associated with the chemical and biological environments of leaf litters under natural condition of the forest floor. Different myxomycete assemblages were found on different sites under the dominant trees in a short distance apart (300 m). A site of Prunus jamasakura tree yielded 21 species, a Quercus glauca tree yielded 20 species, an Ilex pedunculosa tree yielded 12 species, and two Quercus serrata trees yielded 13 and 14 species, respectively. Non-metric multidimensional scaling demonstrated that the myxomycete assemblages were closely related to the litter types of deciduous and evergreen trees, and both litter pH and cellulolytic activity influenced distribution of myxomycete species. Species richness was higher in leaf litters with higher pH than in leaf litters with more acidic pH such as I. pedunculosa litter. The dominant tree litter and litter pH strongly influenced the species distribution of foliicolous myxomycetes in a local mixed forest.

Preliminary Characterization of a Cellulase Producing Bacterial Strain Isolated from a Romanian Hypersaline Lake

Cellulases are a group of enzymes that are used in many biotechnological processes. Since most of the enzymes synthesised by mesophilic microorganisms are unstable in industrial environments, it is necessary to direct research towards extremophile cellulolytic microorganisms because the enzymes synthesised by them are stable and active even in harsh physicochemical conditions. In the present investigation, our aim was to isolate and identify some microbial cellulolytic strains from a hypersaline lake located in Romania and to determine their optimal growth conditions. Of a total of 25 microbial strains isolated, only one extreme halotolerant bacterial strain was able to produce an endoglucanase. Based on molecular identification, we identified this cellulolytic strain as a species of Bacillus genus, most closely related to Bacillus zhangzhouensis. Optimal growth conditions were found to be at 15&deg;C, pH 7.5 and 2 M NaCl. Endoglucanase activity of this bacterial strain is influenced by both salinity and temperature. The most significant endoglucanase activity was detected in the presence of 3 M NaCl, after 72 h of incubation at 15&deg;C. In this situation, the amount of glucose released from a volume of 0.5 mL of 2% (w/v) carboxymethyl cellulose substrate is equivalent to 2.05 mg. In conclusion, this study represents the first preliminary characterization of a B. zhangzhouensis strain that has the ability to degrade cellulose and that demonstrates tolerance to high salt concentrations.

Characterization of glucose isomerase-producing bacteria and optimization of fermentation conditions for producing glucose isomerase using biomass

Glucose isomerase(GI)is an enzyme with high potential applications.Characterization of GI producing bacteria with interesting properties from an industrial point of view is essential.Bacillus sp.,Paenarthrobacter sp.,Chryseobacterium sp.,Hymenobacter sp.,Mycobacterium sp.,and Stenotrophomonas sp.were isolated from soil samples.Optimization of enzyme production yield was investigated in various fermentation conditions using response surface methodology.All isolates exhibited maximum GI activity at 40℃,pH 6–8 after 4 days of incubation.A mixture of peptone/yeast extract or tryptone/peptone enhanced higher enzyme production.The same trend was observed in fermentation medium containing 1%xylose or 2%–2.5%wheat straw.This study advanced the knowledge of these bacterial isolates in promoting wheat straw as feedstock for the bio-based industry.

The scarab gut： A potential bioreactor for bio-fuel production

Cellulose and hemicelluloses are the most prevalent sources of carbon in nature. Currently many approaches employ micro-organisms and their enzyme products to degrade plant feedstocks for production of bioenergy. Scarab larvae are one such model. They consume celluloses from a variety of sources including plant roots, soil organic matter and decaying wood, and are able to extract nutrients and energy from these sources. In this paper, we review the physicochemical properties of the scarab larval gut, the diversity and digestive role that microflora play in the scarab gut and discuss the potential for applying these digestive processes in bioreactors for improving bio-fuel production. Scarab larvae are characterised by their highly alkaline midgut which is dominated by serine proteinase enzymes, and a modified hindgut which harbors the majority of the intestinal microbiota under anaerobic conditions. Evidence suggests that digestion of recalcitrant organic matter in scarab larvae likely results from a combination of endogenous gut proteinases and cellulolytic enzymes produced by symbiotic micro-organisms. Most of the easily digestible proteins are mobilized and absorbed in the midgut by endogenous proteinases. The hindgut contents of scarab larvae are characterized by high concentrations of volatile fatty acids, the presence of fermenting bacteria, and typical anaerobic activities, such as methanogenesis. The hindgut typically contains a wide diversity of micro-organisms, some of which appear to be obligate symbionts with cellulolytic potential. As a result, the scarab larval gut can be regarded as a small bioreactor resembling the rumen of sheep or cattle, where solid food particles composed of cellulose, hemicellulose, pectin and polysaccharides are degraded through enzymatic and fermentation processes. Together these observations suggest scarab larvae have potential to assist the bio-fuel industry by providing new sources of （hemi）cellulolytic bacteria and bacterial （hemi）cellulolytic enzymes.