Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates.

A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.

Insights into Clostridium phytofermentans biofilm formation: aggregation, microcolony development and the role of extracellular DNA.

Biofilm formation is a critical component to the lifestyle of many naturally occurring cellulose-degrading microbes. In this work, cellular aggregation and biofilm formation of Clostridium phytofermentans, a cellulolytic anaerobic bacterium, was investigated using a combination of microscopy and analytical techniques. Aggregates included thread-like linkages and a DNA/protein-rich extracellular matrix when grown on soluble cellobiose. Similar dense biofilms formed on the surface of the model cellulosic substrate Whatman no. 1 filter paper. Following initially dispersed attachment, microcolonies of ~500 µm diameter formed on the filter paper after 6 days. Enzymic treatment of both the biofilm and cellular aggregates with DNase and proteinase resulted in significant loss of rigidity, pointing to the key role of extracellular DNA and proteins in the biofilm structure. A high-throughput biofilm assay was adapted for studying potential regulators of biofilm formation. Various media manipulations were shown to greatly impact biofilm formation, including repression in the presence of glucose but not the ß(1→4)-linked disaccharide cellobiose, implicating a balance of hydrolytic activity and assimilation to maintain biofilm integrity. Using the microtitre plate biofilm assay, DNase and proteinase dispersed ~60 and 30 % of mature biofilms, respectively, whilst RNase had no impact. This work suggests that Clostridium phytofermentans has evolved a DNA/protein-rich biofilm matrix complementing its cellulolytic nature. These insights add to our current understanding of natural ecosystems as well as strategies for efficient bioprocess design.

Reversible control of biofilm formation by Cellulomonas spp. in response to nitrogen availability.

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments.

Genome and Transcriptome of Clostridium phytofermentans, Catalyst for the Direct Conversion of Plant Feedstocks to Fuels.

Clostridium phytofermentans was isolated from forest soil and is distinguished by its capacity to directly ferment plant cell wall polysaccharides into ethanol as the primary product, suggesting that it possesses unusual catabolic pathways. The objective of the present study was to understand the molecular mechanisms of biomass conversion to ethanol in a single organism, Clostridium phytofermentans, by analyzing its complete genome and transcriptome during growth on plant carbohydrates. The saccharolytic versatility of C. phytofermentans is reflected in a diversity of genes encoding ATP-binding cassette sugar transporters and glycoside hydrolases, many of which may have been acquired through horizontal gene transfer. These genes are frequently organized as operons that may be controlled individually by the many transcriptional regulators identified in the genome. Preferential ethanol production may be due to high levels of expression of multiple ethanol dehydrogenases and additional pathways maximizing ethanol yield. The genome also encodes three different proteinaceous bacterial microcompartments with the capacity to compartmentalize pathways that divert fermentation intermediates to various products. These characteristics make C. phytofermentans an attractive resource for improving the efficiency and speed of biomass conversion to biofuels.

The complete genome sequence of Clostridium indolis DSM 755(T.).

Clostridium indolis DSM 755(T) is a bacterium commonly found in soils and the feces of birds and mammals. Despite its prevalence, little is known about the ecology or physiology of this species. However, close relatives, C. saccharolyticum and C. hathewayi, have demonstrated interesting metabolic potentials related to plant degradation and human health. The genome of C. indolis DSM 755(T) reveals an abundance of genes in functional groups associated with the transport and utilization of carbohydrates, as well as citrate, lactate, and aromatics. Ecologically relevant gene clusters related to nitrogen fixation and a unique type of bacterial microcompartment, the CoAT BMC, are also detected. Our genome analysis suggests hypotheses to be tested in future culture based work to better understand the physiology of this poorly described species.

Involvement of a bacterial microcompartment in the metabolism of fucose and rhamnose by Clostridium phytofermentans.

BACKGROUND: Clostridium phytofermentans, an anaerobic soil bacterium, can directly convert plant biomass into biofuels. The genome of C. phytofermentans contains three loci with genes encoding shell proteins of bacterial microcompartments (BMC), organelles composed entirely of proteins. METHODOLOGY AND PRINCIPAL FINDINGS: One of the BMC loci has homology to a BMC-encoding locus implicated in the conversion of fucose to propanol and propionate in a human gut commensal, Roseburia inulinivorans. We hypothesized that it had a similar role in C. phytofermentans. When C. phytofermentans was grown on fucose, the major products identified were ethanol, propanol and propionate. Transmission electron microscopy of fucose- and rhamnose-grown cultures revealed polyhedral structures, presumably BMCs. Microarray analysis indicated that during growth on fucose, operons coding for the BMC locus, fucose dissimilatory enzymes, and an ATP-binding cassette transporter became the dominant transcripts. These data are consistent with fucose fermentation producing a 1,2-propanediol intermediate that is further metabolized in the microcompartment encoded in the BMC locus. Growth on another deoxyhexose sugar, rhamnose, resulted in the expression of the same BMC locus and similar fermentation products. However, a different set of dissimilatory enzymes and transport system genes were induced. Quite surprisingly, growth on fucose or rhamnose also led to the expression of a diverse array of complex plant polysaccharide-degrading enzymes. CONCLUSIONS/SIGNIFICANCE: Based on physiological, genomic, and microarray analyses, we propose a model for the fermentation of fucose and rhamnose in C. phytofermentans that includes enzymes encoded in the same BMC locus. Comparative genomic analysis suggests that this BMC may be present in other clostridial species.

A high-throughput biological conversion assay for determining lignocellulosic quality.

Lignocellulosic biomass is a source of low cost polysaccharides that some microbes can deconstruct and convert into liquid transportation fuel. Feedstocks vary in their ease of use depending on their source and handing. Estimating conversion amenability is useful to determine the effects of biomass pretreatment and genetic potential for the purposes of energy crop breeding and genetics. Here we describe a small-scale high-throughput assay that measures ethanol production from a culture of plant biomass and the ethanologen Clostridium phytofermentans.

Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality.

BACKGROUND: There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. RESULTS: We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.). CONCLUSION: Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.

Biochemical and genetic characterization of ChiA, the major enzyme component for the solubilization of chitin by Cellulomonas uda.

Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity. ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5. The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa. The region upstream of chiA included a conserved -35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins. Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases. In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein.

Clostridium phytofermentans sp. nov., a cellulolytic mesophile from forest soil.

An obligately anaerobic, mesophilic, cellulolytic bacterium, strain ISDgT, was isolated from forest soil. Cells of this isolate stained Gram-negative, despite possessing a Gram-positive cell-wall ultrastructure, and were motile, straight rods that formed spherical terminal spores that swelled the sporangium. Cellulose, pectin, polygalacturonic acid, starch, xylan, arabinose, cellobiose, fructose, galactose, gentiobiose, glucose, lactose, maltose, mannose, ribose and xylose supported growth. The major end products of fermentation were ethanol, acetate, CO2 and H2; formate and lactate were minor products. The optimum temperature for growth was 35-37 degrees C. Phylogenetic analyses based on 16S rRNA sequence comparisons showed that strain ISDgT was related to a group of anaerobes that included Clostridium herbivorans, Clostridium polysaccharolyticum and Clostridium populeti. The G+C content of this strain was 35.9 mol%. On the basis of numerous genotypic and phenotypic differences between strain ISDgT and its close relatives, strain ISDgT is proposed as a novel species in the genus Clostridium, for which the name Clostridium phytofermentans sp. nov. is proposed. The type strain is ISDgT (= ATCC 700394T).