Gut microorganisms of Locusta migratoria in various life stages and its possible influence on cellulose digestibility.

Locusta migratoria is an important phytophagous pest, and its gut microbial communities play an important role in cellulose degradation. In this study, the gut microbial and cellulose digestibility dynamics of Locusta migratoria were jointly analyzed using high-throughput sequencing and anthrone colorimetry. The results showed that the gut microbial diversity and cellulose digestibility across life stages were dynamically changing. The species richness of gut bacteria was significantly higher in eggs than in larvae and imago, the species richness and cellulose digestibility of gut bacteria were significantly higher in early larvae (first and second instars) than in late larvae (third to fifth instars), and the diversity of gut bacteria and cellulose digestibility were significantly higher in imago than in late larvae. There is a correlation between the dynamics of gut bacterial communities and cellulose digestibility. Enterobacter, Lactococcus, and Pseudomonas are the most abundant genera throughout all life stages. Six strains of highly efficient cellulolytic bacteria were screened, which were dominant gut bacteria. Carboxymethyl cellulase activity (CMCA) and filter paper activity (FPA) experiments revealed that Pseudomonas had the highest cellulase enzyme activity. This study provides a new way for the screening of cellulolytic bacteria and lays the foundation for developing insects with significant biomass into cellulose-degrading bioreactors. IMPORTANCE: Cellulose is the most abundant and cheapest renewable resource in nature, but its degradation is difficult, so finding efficient cellulose degradation methods is an urgent challenge. Locusta migratoria is a large group of agricultural pests, and the large number of microorganisms that inhabit their intestinal tracts play an important role in cellulose degradation. We analyzed the dynamics of Locusta migratoria gut microbial communities and cellulose digestibility using a combination of high-throughput sequencing technology and anthrone colorimetry. The results revealed that the gut microbial diversity and cellulose digestibility were dynamically changed at different life stages. In addition, we explored the intestinal bacterial community of Locusta migratoria across life stages and its correlation with cellulose digestibility. The dominant bacterial genera at different life stages of Locusta migratoria were uncovered and their carboxymethyl cellulase activity (CMCA) and filter paper activity (FPA) were determined. This study provides a new avenue for screening cellulolytic bacteria and lays the foundation for developing insects with significant biomass into cellulose-degrading bioreactors.

Introduction of Cellulolytic Bacterium Bacillus velezensis Z2.6 and Its Cellulase Production Optimization.

Enzyme-production microorganisms typically occupy a dominant position in composting, where cellulolytic microorganisms actively engage in the breakdown of lignocellulose. Exploring strains with high yields of cellulose-degrading enzymes holds substantial significance for the industrial production of related enzymes and the advancement of clean bioenergy. This study was inclined to screen cellulolytic bacteria, conduct genome analysis, mine cellulase-related genes, and optimize cellulase production. The potential carboxymethylcellulose-hydrolyzing bacterial strain Z2.6 was isolated from the maturation phase of pig manure-based compost with algae residuals as the feedstock and identified as Bacillus velezensis. In the draft genome of strain Z2.6, 31 related cellulolytic genes were annotated by the CAZy database, and further validation by cloning documented the existence of an endo-1,4-ß-D-glucanase (EC 3.2.1.4) belonging to the GH5 family and a ß-glucosidase (EC 3.2.1.21) belonging to the GH1 family, which are predominant types of cellulases. Through the exploration of ten factors in fermentation medium with Plackett-Burman and Box-Behnken design methodologies, maximum cellulase activity was predicted to reach 2.98 U/mL theoretically. The optimal conditions achieving this response were determined as 1.09% CMC-Na, 2.30% salinity, and 1.23% tryptone. Validation under these specified conditions yielded a cellulose activity of 3.02 U/mL, demonstrating a 3.43-fold degree of optimization. In conclusion, this comprehensive study underscored the significant capabilities of strain Z2.6 in lignocellulolytic saccharification and its potentialities for future in-depth exploration in biomass conversion.

Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-ß-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two ß-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-ß-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-ß-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Cellulolytic Bacillus cereus produces a variety of short-chain fatty acids and has potential as a probiotic.

Cellulolytic bacteria ferment dietary fiber into short-chain fatty acids, which play an important role in improving fiber utilization and maintaining intestinal health. Safe and effective cellulolytic bacteria are highly promising probiotic candidates. In this study, we isolated three strains of Bacillus cereus, which exhibited cellulolytic properties, from Kele pig feces. To assess the genetic basis of cellulose degradation by the isolates, whole-genome sequencing was used to detect functional genes associated with cellulose metabolism. Subsequently, we identified that the B. cereus CL2 strain was safe in mice by monitoring body weight changes, performing histopathologic evaluations, and determining routine blood indices. We next evaluated the biological characteristics of the CL2 strain in terms of its growth, tolerance, and antibiotic susceptibility, with a focus on its ability to produce short-chain fatty acids. Finally, the intestinal flora structure of the experimental animals was analyzed to assess the intestinal environment compatibility of the CL2 strain. In this study, we isolated a cellulolytic B. cereus CL2, which has multiple cellulolytic functional genes and favorable biological characteristics, from the feces of Kele pigs. Moreover, CL2 could produce a variety of short-chain fatty acids and does not significantly affect the diversity of the intestinal flora. In summary, the cellulolytic bacterium B. cereus CL2 is a promising strain for use as a commercial probiotic or in feed supplement. IMPORTANCE: Short-chain fatty acids are crucial constituents of the intestinal tract, playing an important and beneficial role in preserving the functional integrity of the intestinal barrier and modulating both immune responses and the structure of the intestinal flora. In the intestine, short-chain fatty acids are mainly produced by bacterial fermentation of cellulose. Therefore, we believe that safe and efficient cellulolytic bacteria have the potential to be novel probiotics. In this study, we systematically evaluated the safety and biological characteristics of the cellulolytic bacterium B. cereus CL2 and provide evidence for its use as a probiotic.

The Establishment of Fibrolytic Bacteria in the Foal Gastrointestinal Tract Is Related to the Occurrence of Coprophagy by Foals.

The consumption of maternal feces (coprophagy) is commonly observed in healthy foals and is a proposed contributor to microbial colonization of the foal's gastrointestinal tract (GIT). This study investigated the role of coprophagy in the establishment of fibrolytic bacteria in the foal GIT. Nine thoroughbred mares were dosed with chromic oxide, an indigestible marker, as a method to detect the occurrence of coprophagy by their foals. Foal fecal samples were collected from 12 h to 21 d after birth to measure chromic oxide and neutral detergent fiber (NDF) and to enumerate cellulolytic bacteria using culture-based techniques. Milk yield was estimated at 7 and 14 d postpartum. Coprophagy was detected as early as 3 d after birth and detected in all foals by 7 d of age. There were strong relationships between coprophagy and cellulolytic bacteria and NDF in foal feces at 7 d of age (r = 0.9703 and r = 0.7878, respectively; p < 0.05). Fecal NDF and chromic oxide concentrations were negatively related to milk yield (r = -0.8144 and r = -0.6966, respectively; p < 0.05), suggesting milk availability affected the incidence of coprophagy. Based on the relationships identified, maternal feces are an important source of fiber and live microbes for the foal, contributing to the development of the microbial community.

Supplementing branched-chain volatile fatty acids in dual-flow cultures varying in dietary forage and corn oil concentrations. II: Biohydrogenation and incorporation into bacterial lipids.

To maintain membrane homeostasis, ruminal bacteria synthesize branched-chain fatty acids (BCFA) or their derivatives (vinyl ethers) that are recovered during methylation procedures as branched-chain aldehydes (BCALD). Many strains of cellulolytic bacteria require 1 or more branched-chain volatile fatty acid (BCVFA). Therefore, the objective of this study was to investigate BCVFA incorporation into bacterial lipids under different dietary conditions. The study was an incomplete block design with 8 continuous culture fermenters used in 4 periods with treatments (n = 4) arranged as a 2 × 2 × 2 factorial. The factors were high (HF) or low forage (LF, 67 or 33% forage, 33:67 alfalfa:orchardgrass), without or with supplemental corn oil (CO; 3% dry matter, 1.5% linoleic fatty acid), and without or with 2.15 mmol/d (5 mg/d 13C each of isovalerate, isobutyrate, and 2-methylbutyrate). After methylation of bacterial pellets collected from each fermenter's effluent, fatty acids and fatty aldehydes were separated before analysis by gas chromatography and isotope ratio mass spectrometry. Supplementation of BCVFA did not influence biohydrogenation extent. Label was only recovered in branched-chain lipids. Lower forage inclusion decreased BCFA in bacterial fatty acid profile from 9.45% with HF to 7.06% with LF and decreased BCALD in bacterial aldehyde profile from 55.4% with HF to 51.4% with LF. Supplemental CO tended to decrease iso even-chain BCFA and decreased iso even-chain BCALD in their bacterial lipid profiles. The main 18:1 isomer was cis-9 18:1, which increased (P < 0.01) by 25% from CO (data not shown). Dose recovery in bacterial lipids was 43.3% lower with LF than HF. Supplemental CO decreased recovery in the HF diet but increased recovery with LF (diet × CO interaction). Recovery from anteiso odd-chain BCFA and BCALD was the greatest; therefore, 2-methylbutyrate was the BCVFA primer most used for branched-chain lipid synthesis. Recovery in iso odd-chain fatty acids (isovalerate as primer) was greater than label recovery in iso even-chain fatty acids (isobutyrate as primer). Fatty aldehydes were less than 6% of total bacterial lipids, but 26.0% of 13C recovered in lipids were recovered in BCALD because greater than 50% of aldehydes were branched-chain. Because BCFA and BCALD are important in the function and growth of bacteria, especially cellulolytics, BCVFA supplementation can support the rumen microbial consortium, increasing fiber degradation and efficiency of microbial protein synthesis.

Polyclonal antibodies inhibit growth of key cellulolytic rumen bacterial species.

Antibodies targeting specific bacterial species could allow for modification of the rumen microbial population to enhance rumen fermentation. However, there is limited knowledge of targeted antibody effects on rumen bacteria. Therefore, our objective was to develop efficacious polyclonal antibodies to inhibit the growth of targeted cellulolytic bacteria from the rumen. Egg-derived, polyclonal antibodies were developed against pure cultures of Ruminococcus albus 7 (anti-RA7), Ruminococcus albus 8 (anti-RA8), and Fibrobacter succinogenes S85 (anti-FS85). Antibodies were added to a cellobiose-containing growth medium for each of the three targeted species. Antibody efficacy was determined via inoculation time (0 h and 4 h) and dose response. Antibody doses included: 0 (CON), 1.3 × 10-4 (LO), 0.013 (MD), and 1.3 (HI) mg antibody per ml of medium. Each targeted species inoculated at 0 h with HI of their respective antibody had decreased (P < 0.01) final optical density and total acetate concentration after a 52 h growth period when compared with CON or LO. Live/dead stains of R. albus 7 and F. succinogenes S85 dosed at 0 h with HI of their respective antibody indicated a decrease (≥ 96%; P < 0.05) in live bacterial cells during the mid-log phase compared with CON or LO. Addition of HI of anti-FS85 at 0 h in F. succinogenes S85 cultures reduced (P < 0.01) total substrate disappearance over 52 h by at least 48% when compared with CON or LO. Cross-reactivity was assessed by adding HI at 0 h to non-targeted bacterial species. Addition of anti-RA8 or anti-RA7 to F. succinogenes S85 cultures did not affect (P ≥ 0.45) total acetate accumulation after 52 h incubation, indicating that antibodies have less of an inhibitory effect on non-target strains. Addition of anti-FS85 to non-cellulolytic strains did not affect (P ≥ 0.89) OD, substrate disappearance, or total VFA concentrations, providing further evidence of specificity against fiber-degrading bacteria. Western blotting with anti-FS85 indicated selective binding to F. succinogenes S85 proteins. Identification by LC-MS/MS of 8 selected protein spots indicated 7 were outer membrane proteins. Overall, polyclonal antibodies were more efficacious at inhibiting the growth of targeted cellulolytic bacteria than non-targeted bacteria. Validated polyclonal antibodies could serve as an effective approach to modify rumen bacterial populations.

Biodegradation of willow sawdust by novel cellulase-producing bacterial consortium from wood-feeding termites for enhancing methane production.

This study was designed to develop a cellulase-producing bacterial consortium (CBC) from wood-feeding termites that could effectively degrade willow sawdust (WSD) and consequently enhance methane production. The bacterial strains Shewanella sp. SSA-1557, Bacillus cereus SSA-1558, and Pseudomonas mosselii SSA-1568 exhibited significant cellulolytic activity. Their CBC consortium showed positive effects on cellulose bioconversion, resulting in accelerated WSD degradation. After nine days of pretreatment, the WSD had lost 63%, 50%, and 28% of its cellulose, hemicellulose, and lignin, respectively. The hydrolysis rate of treated WSD (352 mg/g) was much higher than that of untreated WSD (15.2 mg/g). The highest biogas production (66.1 NL/kg VS) with 66% methane was observed in the anaerobic digester M-2, which contained a combination of pretreated WSD and cattle dung in a 50/50 ratio. The findings will enrich knowledge for the development of cellulolytic bacterial consortia from termite guts for biological wood pretreatment in lignocellulosic anaerobic digestion biorefineries.

Influence of the autochthonous cellulolytic bacteria on the domestic compost process improvement.

This work aimed to determine the influence of the inoculation of autochthonous cellulolytic bacteria on the composting process without any modifications of physical or chemical parameters. Bacteria with cellulolytic abilities were isolated from composted material containing food and plant leftovers and identified as Bacillus licheniformis, Bacillus altitudinis, and Lysinibacillus xylanilyticus. The experimental composter containing garden and household wastes was inoculated with bio-vaccine prepared as a mixture of isolated cellulolytic bacterial strains and composted for the next 96 days parallelly to the control composter without the inoculation. During the experiment, changes in temperature, humidity, the content of the humic acids (HAs), organic carbon, nitrogen, and C : N ratio were determined. As the particular microbial groups play a key role in the composting process, the biodiversity of the microorganisms present in the composter as well as the number of psychrophilic, mesophilic, and sporeforming microorganisms, Actinomycetes, and fungi were analyzed. The changes in the abundance of particular bacterial groups were convergent with temperature changes in the temperature of composting material. The composting material inoculated with autochthonous microorganisms was characterized by higher HA content and lower biodiversity. The inoculation with autochthonous microorganisms positively influenced the composting material in the corners for the entire process and in the middle of the container for 61 days. Thus, the effect of inoculation depended on the localization of the process inside the container subjected to biopreparation.

Assessing milk response to different combinations of branched-chain volatile fatty acids and valerate in Jersey cows.

Some cellulolytic bacteria require 1 or more branched-chain volatile fatty acids (BCVFA) for the synthesis of branched-chain AA and branched-chain long-chain fatty acids because they are not able to uptake branched-chain AA or lack 1 or more enzymes to synthesize branched-chain AA de novo. Supplemental BCVFA and valerate were included previously as a feed additive that was later removed from the market; these older studies and more current studies have noted improvements in neutral detergent fiber digestibility and milk efficiency. However, most studies provided a single BCVFA or else isobutyrate (IB), 2-methylbutyrate (MB), isovalerate, and valerate altogether without exploring optimal combinations. Our objective was to determine a combination of isoacids that is optimal for milk production. Sixty (28 primiparous and 32 multiparous) lactating Jersey cows (106 ± 54 days in milk) were blocked and assigned randomly to either a control (CON) treatment without any isoacids, MB [12.3 mmol/kg dry matter (DM)], MB + IB (7.7 and 12.6 mmol/kg DM of MB and IB, respectively), or all 4 isoacids (6.2, 7.3, 4.2, and 5.1 mmol/kg DM of MB, IB, isovalerate, and valerate, respectively). Cattle were fed the CON treatment for a 2-wk period, then were assigned randomly within a block to treatments for 8 wk (n = 15). There was a trend for an interaction of supplement and parity for milk components. There were no differences in components for primiparous cows, whereas MB + IB tended to increase protein concentration by 0.04 and 0.08 percentage units in multiparous cows compared with the CON and MB treatments, respectively. Feeding MB + IB increased fat concentration by 0.23 to 0.31 percentage units compared with all other treatments in multiparous cows. Milk yield and dry matter intake (DMI) did not change with treatment. Treatment interacted with week for milk net energy for lactation/DMI; MB + IB tended to increase milk net energy of lactation/DMI by 0.10 Mcal/kg compared with MB and approached a trend for CON, mainly during the early weeks of the treatment period, whereas differences decreased during the last 2 wk of the treatment period. Cows fed MB had the highest 15:0 anteiso fatty acids in the total milk fatty acid profile, which was greater than that for CON or MB + IB cows, but not cows supplemented with isoacids. Cows fed MB alone had the numerically lowest milk net energy for lactation/DMI. The combination of MB + IB appeared optimal for increasing feed efficiency in our study and was not at the expense of average daily gain. Further research is needed for evaluating how potential changes in supplemental isoacid dosage should vary under differing dietary conditions.

Characterization of Potential Virulence, Resistance to Antibiotics and Heavy Metals, and Biofilm-Forming Capabilities of Soil Lignocellulolytic Bacteria.

Soil bacteria participate in self-immobilization processes for survival, persistence, and production of virulence factors in some niches or hosts through their capacities for autoaggregation, cell surface hydrophobicity, biofilm formation, and antibiotic and heavy metal resistance. This study investigated potential virulence, antibiotic and heavy metal resistance, solvent adhesion, and biofilm-forming capabilities of six cellulolytic bacteria isolated from soil samples: Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Strains were subjected to phenotypic methods, including heavy metal and antibiotic susceptibility and virulence factors (protease, lipase, capsule production, autoaggregation, hydrophobicity, and biofilm formation). The effect of ciprofloxacin was also investigated on bacterial susceptibility over time, cell membrane, and biofilm formation. Strains MKAL2, MKAL5, and MKAL6 exhibited protease and lipase activities, while only MKAL6 produced capsules. All strains were capable of aggregating, forming biofilm, and adhering to solvents. Strains tolerated high amounts of chromium, lead, zinc, nickel, and manganese and were resistant to lincomycin. Ciprofloxacin exhibited bactericidal activity against these strains. Although the phenotypic evaluation of virulence factors of bacteria can indicate their pathogenic nature, an in-depth genetic study of virulence, antibiotic and heavy metal resistance genes is required.

Characterization of Cellulose-Degrading Bacteria Isolated from Soil and the Optimization of Their Culture Conditions for Cellulase Production.

The characterization of bacteria with hydrolytic potential significantly contributes to the industries. Six cellulose-degrading bacteria were isolated from mixture soil samples collected at Kingfisher Lake and the University of Manitoba campus by Congo red method using carboxymethyl cellulose agar medium and identified as Paenarthrobacter sp. MKAL1, Hymenobacter sp. MKAL2, Mycobacterium sp. MKAL3, Stenotrophomonas sp. MKAL4, Chryseobacterium sp. MKAL5, and Bacillus sp. MKAL6. Their cellulase production was optimized by controlling different environmental and nutritional factors such as pH, temperature, incubation period, substrate concentration, nitrogen, and carbon sources using the dinitrosalicylic acid and response surface methods. Except for Paenarthrobacter sp. MKAL1, all strains are motile. Only Bacillus sp. MKAL6 was non-salt-tolerant and showed gelatinase activity. Sucrose enhanced higher cellulase activity of 78.87 ± 4.71 to 190.30 ± 6.42 U/mL in these strains at their optimum pH (5-6) and temperature (35-40 °C). The molecular weights of these cellulases were about 25 kDa. These bacterial strains could be promising biocatalysts for converting cellulose into glucose for industrial purposes.

Silage Fermentation Quality, Anthocyanin Stability, and in vitro Rumen Fermentation Characteristic of Ferrous Sulfate Heptahydrate-Treated Black Cane (Saccharum sinensis R.).

Pretreatment of lignocellulose agricultural biomass with iron prior to ensiling is required to accelerate biomass breakdown during fermentation, which could result in functional microorganisms and chemicals that reduce nutrition loss, harmful substances, and improve animal performance. The objective of this study was to investigate the effects of increasing dilutions of ferrous sulfate heptahydrate (FS) pretreatment at fresh matter concentrations of 0, 0.015, and 0.030% on the fermentation quality of black cane (BC) silage, anthocyanin stability, ruminal biogas, rumen fermentation profile, and microbial community. Pre-ensiled and silage materials were evaluated. High moisture, fiber, anthocyanin, and lignification of biomass, as well as undesirable ensiling microorganisms, were found in BC' pre-ensiled form. Increasing dilutions of FS incorporated into silages were observed to linearly decrease dry matter, anthocyanin, and nutritive value losses. The lignin values decreased linearly as the percentage of FS increased up to 0.030%. Given that the ruminants were fed pre-ensiled materials, BC silage treated with 0.030% FS dilution had comparable results to pre-ensiled BC in terms of increasing in vitro volatile fatty acid concentrations, maintaining total gas production, and reducing methane production, when compared to other FS-treated silages. In addition, BC silage treated with a 0.030% FS dilution inhibited methanogenic bacteria and regulated cellulolytic bacteria in rumen fluid. Overall, the anthocyanin content of BC remained constant throughout the rumen fermentation process after increasing dilutions of FS, indicating that BC is a viable ruminant feedstock and that pretreatment of BC with dilute FS-assisted ensiling at 0.030% could be used to generate ruminant diets.

Screening and identification of cellulose-degrading bacteria from soil and leaves at Kerman province, Iran.

Cellulosic biomass is considered one of the most promising sources for the production of alternative renewable bioenergy and other valuable products. Identification and optimization of strains with high enzymatic activity that can overcome constraints imposed by the cellulosic structure is an essential step in the development of new biotechnologies. The aim of this study was to isolate and identify thermophilic (50 °C) and mesophilic (37 °C) cellulolytic bacteria from soil and leaves samples at Kerman, Iran. Degrader bacteria were isolated using serial dilution and pour plate method. Media contained carboxymethylcellulose (CMC), and filter paper was used as sources of carbon. Totally 22 mesophilic and 17 thermophilic bacterial strains which produced clear zones were further identified by morphological and biochemical tests. Screening of purified bacteria was performed to identify cellulase-producing bacteria by Congo red test. These bacteria were compared to each other based on cellulase activity, the percentage of growth, and extracellular protein amounts. The strains with the highest enzymatic activity were determined by the DNS method. The isolated US5 and US7 grew rapidly, and produced cellulase. The US5 created the largest clear zones (7 mm). Besides, these strains were selected for analysis of 16S rRNA sequence. The results showed that selected bacteria strains belong to Brevibacillus borstelensis. The B. borstelensis strains isolated in this study showed a suitable cellulase enzyme activity.

Tropical Cellulolytic Bacteria: Potential Utilization of Sugarcane Bagasse as Low-Cost Carbon Source in Aquaculture.

Sugarcane bagasse (SB), as a major by-product of sugarcane, is one of the most abundant organic matter and characterized by cheap and easily available carbon source in Hainan Island, China. The objective of this study was to isolate tropical cellulolytic bacteria from Hainan Island and demonstrate their prospects of utilization of SB as a low-cost carbon source to greatly reduce the cost of aquaculture. A total of 97 cellulolytic marine bacteria were isolated, of which, 58 cellulolytic marine bacteria displayed the hydrolysis capacity (HC) of more than 1, while 28 cellulolytic marine bacteria displayed more than 2. Of the 28 tropical cellulolytic bacterial strains with HC more than 2, Microbulbifer sp. CFW-C18 and Vibrio sp. MW-M19 exhibited excellent SB decomposition in a small-scale laboratory simulation of shrimp aquaculture, up to 75.31 and 74.35%, respectively, and both of them were safe for shrimps. Meanwhile, both of CFW-C18 and MW-M19 besides displaying low multiple antibiotic resistance (MAR) index, also increased the C/N ratio (CFW-C18: C/N ratio of 14.34; MW-M19: C/N ratio of 14.75) of the small-scale laboratory simulation of shrimp aquaculture by decreasing the nitrogen content after a supplement of SB for 15 days. More importantly, CFW-C18 and MW-M19 displayed a relatively low MAR index, 0.47 and 0.1, respectively, especially MW-M19, with the lowest MAR index (0.1), which was resistant to only three antibiotics, streptomycin, amikacin, and levofloxacin, indicating that this strain was safe and non-drug resistance for further use. Overall, tropical cellulolytic bacteria isolated from Hainan Island, especially CFW-C18 and MW-M19, will provide the proficient candidates as probiotics for further construction of the recirculating aquaculture system based on the supplement of low-cost external carbon source-SB.

Host Phylogeny and Diet Shape Gut Microbial Communities Within Bamboo-Feeding Insects.

The gut microbiome plays an important role in a host's development and adaption to its dietary niche. In this study, a group of bamboo-feeding insects are used to explore the potential role of the gut microbiota in the convergent adaptation to extreme diet specialization. Specifically, using a 16S rRNA marker and an Illumina sequencing platform, we profiled the microbial communities of 76 gut samples collected from nine bamboo-feeding insects, including both hemimetabolous (Orthoptera and Hemiptera) and holometabolous (Coleoptera and Lepidoptera) species, which are specialized in three distinct dietary niches: bamboo leaf, shoot, and sap. The gut microbiota of these insects were dominated by Proteobacteria, Firmicutes, and Bacteroidetes and were clustered into solid (leaf and shoot) and liquid (sap) dietary niches. The gut bacterial communities of insects feeding on solid diet overlapped significantly, even though these insects belong to phylogenetically distant lineages representing different orders. In addition, the presence of cellulolytic bacterial communities within the gut microbiota allows bamboo-feeding insects to adapt to a highly specialized, fiber-rich diet. Although both phylogeny and diet can impact the structure and composition of gut microbiomes, phylogeny is the primary driving force underlying the convergent adaptation to a highly specialized diet, especially when the related insect species harbor similar gut microbiomes and share the same dietary niche over evolutionary timescales. These combined findings lay the foundation for future research on how convergent feeding strategies impact the interplays between hosts and their gut microbiomes and how the gut microbiota may facilitate convergent evolution in phylogenetically distant species in adaptation to the shared diet.

The synergistic effect of rumen cellulolytic bacteria and activated carbon on thermophilic digestion of cornstalk.

To explore the bioaugmentation of rumen cellulolytic bacteria (RCB) and activated carbon (AC) on thermophilic digestion of cornstalk, biochemical methane potential tests were carried out. Adding RCB or AC can improve methane production, while simultaneous existence of AC (10 g/L) and RCB (5%) obtained the best performance. The maximum cellulose degradation rate, methane production rate and methane yield were 66.92%, 32.2 L/(kgVS·d), and 144.9 L/kgVS, which increased by 30.23%, 51.17%, and 20.35% compared with control group. The cellulolytic and fermentative bacteria (Hydrogenispora), syntrophic acetate-oxidizing bacteria (norank_o_MBA03), and hydrogenotrophic Methanothermobacter were crucial for thermophilic digestion of cornstalk. The enhancement of AC was due to the enrichment of Hydrogenispora and Methanothermobacter, while RCB can increase the abundance of cellulolytic bacteria (Halocella and norank_o_M55-D21) and mixotrophic Methanosarcina. The synergetic effect of AC and RCB owing to the enriched cellulolytic bacteria, the enhanced syntrophic acetate oxidation and the concentrated carbon metabolic flow to methane.

Effect of parota (Enterolobium cyclocarpum) pod protein supplement on feed intake and digestibility and calf ruminal characteristics.

The objective of this study was to evaluate feed intake and digestibility and ruminal characteristics of development-stage calves fed mulatto II (Brachiaria sp.) grass hay (MGH) and a protein supplement (PS) consisting of increasing levels of the parota (Enterolobium cyclocarpum) pod (PP). We used eight Swiss-zebu calves in growth stage with an average age of 11 months and initial average weight of 157.6 ± 8.5 kg. They were distributed in a repeated 4 × 4 Latin square design with 4 treatments (period 30 days): 0% (PP0), 25% (PP25), 50% (PP50), and 75% (PP75) of the PP. Calves in the PP0 and PP25 treatments had higher intake of PS and MGH as dry matter (DM) than those in the PP50 and PP75 treatments (p < 0.05). Organic matter intake (OMI) of the PP75 calves was lower than that of PP0 and PP25 calves. Crude protein intakes (CPI) of PP0 and PP25 calves were higher than those of PP50 and PP75 calves (p < 0.05). Apparent digestibility of crude protein was higher in the PP0, PP25, and PP50 treatments compared with that in treatment PP75 (p < 0.05). The treatments did not affect total bacterial count, cellulolytic bacterial count, cellulase enzymatic activity, volatile fatty acids, or the acetate/propionate ratio (p > 0.05). Rumen pH in the PP0 calves was higher than that of the PP25 calves, whereas the protozoa count and ammonia content were higher in PP0 calves than in PP75 (p < 0.05). In conclusion, the inclusion of 25% PP in the PS for forage-fed calves is a feeding alternative.

Integrated eco-strategies towards sustainable carbon and nitrogen cycling in agriculture.

To meet the ever-growing human demands for food, fuel, and fiber, agricultural activities have dramatically altered the global carbon (C) and nitrogen (N) cycles. These biogeochemical cycles along with water, phosphorus, and sulfur cycles are fundamental features of life on Earth. Human alteration of the global N cycle has had both positive and negative outcomes. To efficiently feed a growing population, crop-livestock production systems have been developed, however, these systems also contribute significantly to environmental pollution and global climate change. Management of agricultural waste (AW) and the application of N fertilizers are central to the issues of greenhouse gas (GHG) emissions and nutrient runoff that contributes to the eutrophication of water bodies. If managed properly, AW can provide nutrients for plants and contribute to the conservation of soil health. In order to achieve the long-term conservation of agricultural production systems, it is important to promote the proper recycling of AW in agroecosystems and to minimize the reliance on chemical N fertilizers. Composting is one of the sustainable and effective approaches for recycling AW in agriculture. However, the conventional composting process is dilatory and produces compost with low N content compared to chemical N fertilizers. For this reason, comprehensive research is required to improve the composting process and the N content of the soil organic amendments. This work aims to explore the beneficial effects of the integrated application of biochar and specific C and N cycling microorganisms to the composting process and the quality of the composted products. In pursuit of replacing chemical N fertilizers with bio/organic fertilizers, we further discussed the power of the combined application of compost, biochar, and N-fixing bacteria in agricultural production systems. The knowledge of smart integration of AW and microorganisms in agriculture could solve the main agricultural and environmental problems associated with human-induced flows of C and N. Building upon the knowledge disseminated in review to further extensive research will pave the way for better management of agricultural production systems and sustainable C and N cycling in agriculture.

High cellulolytic potential of the Ktedonobacteria lineage revealed by genome-wide analysis of CAZymes.

Traditionally, filamentous fungi and actinomycetes are well-known cellulolytic microorganisms that have been utilized in the commercial production of cellulase enzyme cocktails for industrial-scale degradation of plant biomass. Noticeably, the Ktedonobacteria lineage (phylum Chloroflexi) with actinomycetes-like morphology was identified and exhibited diverse carbohydrate utilization or degradation abilities. In this study, we performed genome-wide profiling of carbohydrate-active enzymes (CAZymes) in the filamentous Ktedonobacteria lineage. Numerous CAZymes (153-290 CAZymes, representing 63-131 glycoside hydrolases (GHs) per genome), including complex mixtures of endo- and exo-cellulases, were predicted in 15 available Ktedonobacteria genomes. Of note, 4-28 CAZymes were predicted to be extracellular enzymes, whereas 3-29 CAZymes were appended with carbohydrate-binding modules (CBMs) that may promote their binding to insoluble carbohydrate substrates. This number far exceeded other Chloroflexi lineages and were comparable to the cellulolytic actinomycetes. Six multi-modular extracellular GHs were cloned from the thermophilic Thermosporothrix hazakensis SK20-1T strain and heterologously expressed. The putative endo-glucanases of ThazG5-1, ThazG9, and ThazG12 exhibited strong cellulolytic activity, whereas the putative exo-glucanases ThazG6 and ThazG48 formed weak but observable halos on carboxymethyl cellulose plates, indicating their potential biotechnological application. The purified recombinant ThazG12 had near-neutral pH (optimal 6.0), high thermostability (60°C), and broad specificity against soluble and insoluble polysaccharide substrates. It also represented described a novel thermostable bacterial ß-1,4-glucanase in the GH12 family. Together, this research revealed the underestimated cellulolytic potential of the Ktedonobacteria lineage and highlighted its potential biotechnological utility as a promising microbial resource for the discovery of industrially useful cellulases.