The unusual cellulose utilization system of the aerobic soil bacterium Cytophaga hutchinsonii.

Cellulolytic microorganisms play important roles in global carbon cycling and have evolved diverse strategies to digest cellulose. Some are 'generous,' releasing soluble sugars from cellulose extracellularly to feed both themselves and their neighbors. The gliding soil bacterium Cytophaga hutchinsonii exhibits a more 'selfish' strategy. It digests crystalline cellulose using cell-associated cellulases and releases little soluble sugar outside of the cell. The mechanism of C. hutchinsonii cellulose utilization is still poorly understood. In this review, we discuss novel aspects of the C. hutchinsonii cellulolytic system. Recently developed genetic manipulation tools allowed the identification of proteins involved in C. hutchinsonii cellulose utilization. These include periplasmic and cell-surface endoglucanases and novel cellulose-binding proteins. The recently discovered type IX secretion system is needed for cellulose utilization and appears to deliver some of the cellulolytic enzymes and other proteins to the cell surface. The requirement for periplasmic endoglucanases for cellulose utilization is unusual and suggests that cello-oligomers must be imported across the outer membrane before being further digested. Cellobiohydrolases or other predicted processive cellulases that play important roles in many other cellulolytic bacteria appear to be absent in C. hutchinsonii. Cells of C. hutchinsonii attach to and glide along cellulose fibers, which may allow them to find sites most amenable to attack. A model of C. hutchinsonii cellulose utilization summarizing recent progress is proposed.

Metabolomic profiling of 13C-labelled cellulose digestion in a lower termite: insights into gut symbiont function.

Termites consume an estimated 3-7 billion tonnes of lignocellulose annually, a role in nature which is unique for a single order of invertebrates. Their food is digested with the help of microbial symbionts, a relationship that has been recognized for 200 years and actively researched for at least a century. Although DNA- and RNA-based approaches have greatly refined the details of the process and the identities of the participants, the allocation of roles in space and time remains unclear. To resolve this issue, a pioneer study is reported using metabolomics to chart the in situ catabolism of (13)C-cellulose fed to the dampwood species Hodotermopsis sjostedti. The results confirm that the secretion of endogenous cellulases by the host may be significant to the digestive process and indicate that a major contribution by hindgut bacteria is phosphorolysis of cellodextrins or cellobiose. This study provides evidence that essential amino acid acquisition by termites occurs following the lysis of microbial tissue obtained via proctodaeal trophallaxis.

Stormwater runoff microplastics: Polymer types, particle size, and factors controlling loading rates.

Stormwater runoff is a pathway of entry for microplastics (MPs, plastics <5 mm) into aquatic ecosystems. The objectives of this study were to determine MP size, morphology, chemical composition, and loading across urban storm events. Particles were extracted from stormwater samples collected at outfall locations using wet peroxide oxidation and cellulose digestion followed by analysis via attenuated total reflectance (ATR) FTIR. Concentrations observed were 0.99 ± 1.10 MP/L for 500-1000 µm and 0.41 ± 0.30 MP/L for the 1000-5000 µm size ranges. Seventeen different polymer types were observed. MP particle sizes measured using a FTIR-microscope camera indicated non-target size particles based on sieve-size classification, highlighting a potential source of error in studies reporting concentration by size class. A maximum MP load of 38.3 MP/m2 of upstream catchment was calculated. MP loadings had moderate correlations with both rainfall accumulation and intensity (Kendall τ = 0.54 and 0.42, respectively, both p ≤ 0.005). First flush (i.e. rapid wash-off of pollutants from watershed surfaces during rainfall early stages) was not always observed, and antecedent dry days were not correlated with MP abundance, likely due to the short dry periods between sampling events. Overall, the results presented provide data for risk assessment and mitigation strategies.

Effects of Methylcellulose on Cellulolytic Bacteria Attachment and Rice Straw Degradation in the In vitro Rumen Fermentation.

An in vitro experiment was conducted to evaluate the effect of methylcellulose on the attachment of major cellulolytic bacteria on rice straw and its digestibility. Rice straw was incubated with ruminal mixture with or without 0.1% methylcellulose (MC). The attachment of F. succinogenes, R. flavefaciens and R. albus populations on rice straw was measured using real-time PCR with specific primer sets. Methylcellulose at the level of 0.1% decreased the attachment of all three major cellulolytic bacteria. In particular, MC treatment reduced (p<0.05) attachment of F. succinogenes on rice straw after 10 min of incubation while a significant reduction (p<0.05) in attachment was not observed until 4 h incubation in the case of R. flavefaciens and R. albus. This result indicated F. succinogenes responded to MC more sensitively and earlier than R. flavefaciens and R. albus. Dry matter digestibility of rice straw was subsequently inhibited by 0.1% MC, and there was a significant difference between control and MC treatment (p<0.05). Incubated cultures containing MC had higher pH and lower gas production than controls. Current data clearly indicated that the attachment of F. succinogenes, R. flavefaciens and R. albus on rice straw was inhibited by MC, which apparently reduced rice straw digestion.

An amateur gut microbial configuration formed in giant panda for striving to digest cellulose in bamboo: Systematic evidence from intestinal digestive enzymes, functional genes and microbial structures.

The giant panda has been considered to maximize nutritional intake including protein and soluble carbohydrates in bamboo, but it has spent almost entire life with the high-cellulose diet. Whether giant panda is still helpless about digesting bamboo cellulose or not is always contentious among many researchers around the world. The work has systematically clarified this issue from the perspectives of digestive enzymes, functional genes, and microbial structures in giant panda gut. The intestinal cellulase activities of panda increase with bamboo consumption, performing that the endoglucanase activity of adults reaches 10-fold that of pandas first consuming bamboo. More abundance and types of microbial endoglucanase genes occur in bamboo-diet giant panda gut, and the corresponding GH5 gene cluster is still efficiently transcribed. Gut microbes possessing cellulose-degrading genes, belong to the phylum Firmicutes and some Bacteroidetes, but their structural and functional configurations are insufficient to completely degrade cellulose. Therefore, giant panda is striving to digest cellulose in bamboo, but this adaptation is incomplete. This is probably related to the short straight carnivore-like gut structure of the giant panda, preventing the colonization of some efficient functional but anaerobic-preferred flora.

Comparative analysis of microbiota along the length of the gastrointestinal tract of two tree squirrel species (Sciurus aberti and S. niger) living in sympatry.

Microbiota inhabiting the gastrointestinal (GI) tract of animals has important impacts on many host physiological processes. Although host diet is a major factor influencing the composition of the gut micro-organismal community, few comparative studies have considered how differences in diet influence community composition across the length of the GI tract. We used 16S sequencing to compare the microbiota along the length of the GI tract in Abert's (Sciurus aberti) and fox squirrels (S. niger) living in the same habitat. While fox squirrels are generalist omnivores, the diet of Abert's squirrels is unusually high in plant fiber, particularly in winter when they extensively consume fiber-rich inner bark of ponderosa pine (Pinus ponderosa). Consistent with previous studies, microbiota of the upper GI tract of both species consisted primarily of facultative anaerobes and was less diverse than that of the lower GI tract, which included mainly obligate anaerobes. While we found relatively little differentiation between the species in the microbiota of the upper GI tract, the community composition of the lower GI tract was clearly delineated. Notably, the Abert's squirrel lower GI community was more stable in composition and enriched for microbes that play a role in the degradation of plant fiber. In contrast, overall microbial diversity was higher in fox squirrels. We hypothesize that these disparities reflect differences in diet quality and diet breadth between the species.

Biofilm Improves Isopod Growth Independent of the Dietary Cellulose Content.

Cellulose is an abundant source of carbon, accounting for more than 50% of foliage and 90% of woody tissues of plants. Despite the diversity of species that include living or dead plant tissue in their diets, the ability to digest cellulose through self-produced enzymatic machinery is considered rare in the animal kingdom. The majority of animals studied to date rely on the cellulolytic activity of symbiotic microorganisms in their digestive tract, with some evidence for a complementary action of endogenous cellulases. Terrestrial isopods have evolved a lifestyle including feeding on a lignocellulose diet. Whether isopods utilize both external and internal cellulases for digestion of a diet is still not understood. We experimentally manipulated the content of cellulose (30%, 60%, or 90%) and the amount of biofilm (small or large) in the offered food source and quantified growth and cellulolytic activity in the gut of the isopod Porcellio scaber. The presence of a visible biofilm significantly promoted isopod growth, regardless of the cellulose content in the diet. The activity of gut cellulases was not significantly affected by the amount of biofilm or the cellulose content. Our results do not support a significant contribution of either ingested or host enzymes to cellulose utilization in P. scaber. Cellulose might not represent a key nutrient for isopods and does not seem to affect the nutritional value of the diet-associated biofilm. We propose that it is the biofilm community that determines the quality of plant diet in terrestrial isopods and potentially also in other detrital plant feeders.

The Fecal Metagenomics of Malayan Pangolins Identifies an Extensive Adaptation to Myrmecophagy.

The characteristics of flora in the intestine of an animal, including the number and abundance of different microbial species and their functions, are closely related to the diets of the animal and affect the physical condition of the host. The Malayan pangolin (Manis javanica) is an endangered species that specializes in myrmecophagy. Analyzing the microbiome in the intestine of the pangolin is imperative to protect this species. By sequencing the metagenomes of the feces of four pangolins, we constructed a non-redundant catalog of 211,868 genes representing 1,811 metagenomic species. Taxonomic annotation revealed that Bacteroidetes (49.9%), Proteobacteria (32.2%), and Firmicutes (12.6%) are the three main phyla. The annotation of gene functions identified 5,044 genes from 88 different glycoside hydrolase (GH) families in the Carbohydrate-Active enZYmes database and 114 gene modules related to chitin-degrading enzymes, corresponding to the catalytic domains of GH18 family enzymes, containing chitinase genes of classes III and V in the dataset. Fourteen gene modules corresponded to the catalytic domains of GH19 family enzymes, containing chitinase genes of classes I, II, and IV. These genes were found in 37 species belonging to four phyla: Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria. Moreover, when the metabolic pathways of these genes were summarized, 41,711 genes were associated with 147 unique KEGG metabolic pathways, and these genes were assigned to two Gene Ontology terms: metabolic process and catalytic activity. We also found several species that likely play roles in the digestion of cellulose and may be able to degrade chitin, including Enterobacter cloacae, Lactococcus lactis, Chitinimonas koreensis, and Chitinophaga pinensis. In addition, we identified some intestinal microflora and genes related to diseases in pangolins. Twenty-seven species were identified by STAMP analysis as differentially abundant in healthy and diseased animals: 20 species, including Cellulosilyticum lentocellum and Lactobacillus reuteri, were more abundant in healthy pangolins, while seven species, including Odoribacter splanchnicus, Marinilabilia salmonicolor, Xanthomonas citri, Xanthomonas vasicola, Oxalobacter formigenes, Prolixibacter bellariivorans, and Clostridium bolteae, were more abundant in diseased pangolins. These results will support the efforts to conserve pangolins.

Effects of Oligosaccharides Isolated From Pinewood Hot Water Pre-hydrolyzates on Recombinant Cellulases.

Loblolly pine residues have enormous potential to be the raw material for advanced biofuel production due to extensive sources and high cellulose content. Hot water (HW) pretreatment, while being a relatively economical and clean technology for the deconstruction of lignocellulosic biomass, could also inhibit the ensuing enzymatic hydrolysis process because of the production of inhibitors. In this study, we investigated the effect of oligosaccharide fractions purified from HW pre-hydrolyzate of pinewood using centrifugal partition chromatography (CPC) on three recombinant cellulolytic enzymes (E1, CBHI and CBHII), which were expressed in the transgenic corn grain system. The efficiency of recombinant enzymes was measured using either a 4-methylumbelliferyl-ß-D-cellobioside (MUC) or a cellulose-dinitrosalicylic acid (DNS) assay system. The results showed that HW pre-hydrolyzate CPC fractions contain phenolics, furans, and monomeric and oligomeric sugars. Among CPC fractions, oligomers composed of xylan, galactan, and mannan were inhibitory to the three recombinant enzymes and to the commercial cellulase cocktail, reducing the enzymatic efficiency to as low as 10%.

The transformation of Saperda calcarata (Coleoptera: Cerambycidae) into a cellulose digester through the inclusion of fungal enzymes in its diet.

The larvae of the aspen borer, Saperda calcarata, which feed on the inner bark and sapwood of living aspen stems, are unable to digest cellulose. However, they can be transformed into cellulose digesters by adding the active cellulase complex of the fungus, Penicillium funiculosum to their diet. S. calcarata larvae are preadapted to exploit the digestive potential of ingested microbial enzymes. We argue that ingested fungal enzymes may be responsible for cellulose digestion in many, perhaps most or even all, cellulose digesting cerambycid beetles.

Gene expression and molecular phylogenetic analyses of beta-glucosidase in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae).

Beta-glucosidase (BG) is known as a multifunctional enzyme for social maintenance in terms of both cellulose digestion and social communication in termites. However, the expression profiles of each BG gene and their evolutionary history are not well understood. First, we cloned two types of BG homologs (RsBGI and RsBGII) from the termite Reticulitermes speratus (Kolbe). Gene expression analyses showed that RsBGI expression levels of primary queens and kings from 30 to 100 days after colony foundation were high, but those of reproductives dropped after day 400. Extremely low gene expression levels of RsBGI were observed in eggs, whereas workers had significantly higher expression levels than those of soldiers and other colony members. Consequently, RsBGI gene expression levels changed among each developmental stage, and RsBGI was shown to be involved in cellulose digestion. On the other hand, the RsBGII gene was consistently expressed in all castes and developmental stages examined, and notable expression changes were not observed among them, including in eggs. It was indicated that RsBGII is a main component involved in social communication, for example, the egg-recognition pheromone shown in this species previously. Finally, we obtained partial gene homologs from other termite and cockroach species, including the woodroach (genus Cryptocercus), which is the sister group to termites, and performed molecular phylogenetic analyses. The results showed that the origin of the BG gene homologs preceded the divergence of termites and cockroaches, suggesting that the acquisition of multifunctionality of the BG gene also occurred in cockroach lineages.