Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes subsp. succinogenes

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R. flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic inhibitors, and lack of Na⁺. In contrast, the attachment was affected by the removal of divalent cations (Mg²⁺ and Ca²⁺), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine. Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at 100°C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations (Mg²⁺ and Ca²⁺), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large glycocalyx and the divalent cations Ca²⁺ and Mg²⁺. Hydrophobic bonds and enzymes may also be involved.     

Microbial Colonization and Competition on the Marine Alga Ulva australis

Pseudalteromonas tunicata and Roseobacter gallaeciensis are biofilm-forming marine bacteria that are often found in association with the surface of the green alga Ulva australis. They are thought to benefit the plant host by producing inhibitory compounds that are active against common fouling organisms. We investigated factors that influence the ability of P. tunicata and R. gallaeciensis to attach to and colonize the plant surface and also the competitive interactions that occur between these organisms and other isolates from U. australis during biofilm formation on the plant surface. A surprisingly high number of P. tunicata cells, at least 10⁸ cells ml⁻¹, were required for colonization and establishment of a population of cells that persists on axenic surfaces of U. australis. Factors that enhanced colonization of P. tunicata included inoculation in the dark and pregrowth of inocula in medium containing cellobiose as the sole carbon source (cellulose is a major surface polymer of U. australis). It was also found that P. tunicata requires the presence of a mixed microbial community to colonize effectively. In contrast, R. gallaeciensis effectively colonized the plant surface under all conditions tested. Studies of competitive interactions on the plant surface revealed that P. tunicata was numerically dominant compared with all other bacterial isolates tested (except R. gallaeciensis), and this dominance was linked to production of the antibacterial protein AlpP. Generally, P. tunicata was able to coexist with competing strains, and each strain existed as microcolonies in spatially segregated regions of the plant. R. gallaeciensis was numerically dominant compared with all strains tested and was able to invade and disperse preestablished biofilms. This study highlighted the fact that microbial colonization of U. australis surfaces is a dynamic process and demonstrated the differences in colonization strategies exhibited by the epiphytic bacteria P. tunicata and R. gallaeciensis.     

Immobilization of Delftia tsuruhatensis in macro-porous cellulose and biodegradation of phenolic compounds in repeated batch process

Delftia tsuruhatensis BM90, previously isolated from Tyrrhenian Sea and selected for its ability to degrade a wide array of phenolic compounds, was immobilized in chemically modified macro porous cellulose. The development of bacterial adhesion on the selected carrier was monitored by scanning electron microscopy. Evident colonization started already after 8 h of incubation. After 72 h, almost all the carrier surface was covered by the bacterial cells. Extracellular bacterial structures, such as pili or fimbriae, contributed to carrier colonization and cell attachment. Immobilized cells of D. tsuruhatensis were tested for their ability to biodegrade a pool of 20 phenols in repeated batch process. During the first activation batch (72 h), 90% of phenols degradation was obtained already in 48 h. In the subsequent batches (up to 360 h), same degradation was obtained after 24 h only. By contrast, free cells were slower: to obtain almost same degradation, 48 h were needed. Thus, process productivity, achieved by the immobilized cells, was double than that of free cells. Specific activity was also higher suggesting that the use of immobilized D. tsuruhatensis BM90 could be considered very promising in order to obtain an efficient reusable biocatalyst for long-term treatment of phenols containing effluents.     

Functionalized microchannels as xylem-mimicking environment: Quantifying X. fastidiosa cell adhesion

Microchannels can be used to simulate xylem vessels and investigate phytopathogen colonization under controlled conditions. In this work, we explore surface functionalization strategies for polydimethylsiloxane and glass microchannels to study microenvironment colonization by Xylella fastidiosa subsp. pauca cells. We closely monitored cell initial adhesion, growth, and motility inside microfluidic channels as a function of chemical environments that mimic those found in xylem vessels. Carboxymethylcellulose (CMC), a synthetic cellulose, and an adhesin that is overexpressed during early stages of X. fastidiosa biofilm formation, XadA1 protein, were immobilized on the device’s internal surfaces. This latter protocol increased bacterial density as compared with CMC. We quantitatively evaluated the different X. fastidiosa attachment affinities to each type of microchannel surface using a mathematical model and experimental observations acquired under constant flow of culture medium. We thus estimate that bacterial cells present ～4 and 82% better adhesion rates in CMC- and XadA1-functionalized channels, respectively. Furthermore, variable flow experiments show that bacterial adhesion forces against shear stresses approximately doubled in value for the XadA1-functionalized microchannel as compared with the polydimethylsiloxane and glass pristine channels. These results show the viability of functionalized microchannels to mimic xylem vessels and corroborate the important role of chemical environments, and particularly XadA1 adhesin, for early stages of X. fastidiosa biofilm formation, as well as adhesivity modulation along the pathogen life cycle.     

Plasticizers Increase Adhesion of the Deteriogenic Fungus Aureobasidium pullulans to Polyvinyl Chloride

Initial adhesion of fungi to plasticized polyvinyl chloride (pPVC) may determine subsequent colonization and biodeterioration processes. The deteriogenic fungus Aureobasidium pullulans was used to investigate the physicochemical nature of adhesion to both unplasticized PVC (uPVC) and pPVC containing the plasticizers dioctyl phthalate (DOP) and dioctyl adipate (DOA). A quantitative adhesion assay using image analysis identified fundamental differences in the mechanism of adhesion of A. pullulans blastospores to these substrata. Adhesion to pPVC was greater than that to uPVC by a maximum of 280% after a 4-h incubation with 10⁸ blastospores ml⁻¹. That plasticizers enhance adhesion to PVC was confirmed by incorporating a dispersion of both DOA and DOP into the blastospore suspension. Adhesion to uPVC was increased by up to 308% in the presence of the dispersed plasticizers. Hydrophobic interactions were found to dominate adhesion to uPVC because (i) a strong positive correlation was observed between substratum hydrophobicity (measured by using a dynamic contact angle analyzer) and adhesion to a range of unplasticized polymers including uPVC, and (ii) neither the pH nor the electrolyte concentration of the suspension buffer, both of which influence electrostatic interactions, affected adhesion to uPVC. In contrast, adhesion to pPVC is principally controlled by electrostatic interactions. Enhanced adhesion to pPVC occurred despite a relative reduction of 13° in the water contact angle of pPVC compared to that of uPVC. Furthermore, adhesion to pPVC was strongly dependent on both the pH and electrolyte concentration of the suspension medium, reaching maximum levels at pH 8 and with an electrolyte concentration of 10 mM NaCl. Plasticization with DOP and DOA therefore increases adhesion of A. pullulans blastospores to pPVC through an interaction mediated by electrostatic forces.     

Continuous Cellulosic Bioethanol Fermentation by Cyclic Fed-Batch Cocultivation

Cocultivation of cellulolytic and saccharolytic microbial populations is a promising strategy to improve bioethanol production from the fermentation of recalcitrant cellulosic materials. Earlier studies have demonstrated the effectiveness of cocultivation in enhancing ethanolic fermentation of cellulose in batch fermentation. To further enhance process efficiency, a semicontinuous cyclic fed-batch fermentor configuration was evaluated for its potential in enhancing the efficiency of cellulose fermentation using cocultivation. Cocultures of cellulolytic Clostridium thermocellum LQRI and saccharolytic Thermoanaerobacter pseudethanolicus strain X514 were tested in the semicontinuous fermentor as a model system. Initial cellulose concentration and pH were identified as the key process parameters controlling cellulose fermentation performance in the fixed-volume cyclic fed-batch coculture system. At an initial cellulose concentration of 40 g liter⁻¹, the concentration of ethanol produced with pH control was 4.5-fold higher than that without pH control. It was also found that efficient cellulosic bioethanol production by cocultivation was sustained in the semicontinuous configuration, with bioethanol production reaching 474 mM in 96 h with an initial cellulose concentration of 80 g liter⁻¹ and pH controlled at 6.5 to 6.8. These results suggested the advantages of the cyclic fed-batch process for cellulosic bioethanol fermentation by the cocultures.     

Are Cellulosome Scaffolding Protein CipC and CBM3-Containing Protein HycP,Involved in Adherence of Clostridium cellulolyticum to Cellulose?

Clostridium cellulolyticum, a mesophilic anaerobic bacterium, produces highly active enzymatic complexes called cellulosomes. This strain was already shown to bind to cellulose, however the molecular mechanism(s) involved is not known. In this context we focused on the gene named hycP, encoding a 250-kDa protein of unknown function, containing a Family-3 Carbohydrate Binding Module (CBM3) along with 23 hyaline repeat modules (HYR modules). In the microbial kingdom the gene hycP is only found in C. cellulolyticum and the very close strain recently sequenced Clostridium sp BNL1100. Its presence in C. cellulolyticum guided us to analyze its function and its putative role in adhesion of the cells to cellulose. The CBM3 of HycP was shown to bind to crystalline cellulose and was assigned to the CBM3b subfamily. No hydrolytic activity on cellulose was found with a mini-protein displaying representative domains of HycP. A C. cellulolyticum inactivated hycP mutant strain was constructed, and we found that HycP is neither involved in binding of the cells to cellulose nor that the protein has an obvious role in cell growth on cellulose. We also characterized the role of the cellulosome scaffolding protein CipC in adhesion of C. cellulolyticum to cellulose, since cellulosome scaffolding protein has been proposed to mediate binding of other cellulolytic bacteria to cellulose. A second mutant was constructed, where cipC was inactivated. We unexpectedly found that CipC is only partly involved in binding of C. cellulolyticum to cellulose. Other mechanisms for cellulose adhesion may therefore exist in C. cellulolyticum. In addition, no cellulosomal protuberances were observed at the cellular surface of C. cellulolyticum, what is in contrast to reports from several other cellulosomes producing strains. These findings may suggest that C. cellulolyticum has no dedicated molecular mechanism to aggregate the cellulosomes at the cellular surface.     

Key role for clumping factor B in Staphylococcus aureus nasal colonization of humans

Background

Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor B (ClfB) promotes adhesion to squamous epithelial cells in vitro and might be a physiologically relevant colonization factor.

Methods and Findings

We define the role of the staphylococcal cytokeratin-binding protein ClfB in the colonization process by artificial inoculation of human volunteers with a wild-type strain and its single locus ClfB knock-out mutant. The wild-type strain adhered to immobilized recombinant human cytokeratin 10 (CK10) in a dose-dependent manner, whereas the ClfB⁻ mutant did not. The wild-type strain, when grown to the stationary phase in a poor growth medium, adhered better to CK10, than when the same strain was grown in a nutrient-rich environment. Nasal cultures show that the mutant strain is eliminated from the nares significantly faster than the wild-type strain, with a median of 3 ± 1 d versus 7 ± 4 d (p = 0.006). Furthermore, the wild-type strain was still present in the nares of 3/16 volunteers at the end of follow-up, and the mutant strain was not.

Conclusions

The human colonization model, in combination with in vitro data, shows that the ClfB protein is a major determinant of nasal-persistent S. aureus carriage and is a candidate target molecule for decolonization strategies.     

Acetobixan,an Inhibitor of Cellulose Synthesis Identified by Microbial Bioprospecting

In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI), named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6^prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM) to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited ¹⁴C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis.     

Alternative Environmental Roles for Cellulose Produced by Acetobacter xylinum

The cellulose-producing bacterium Acetobacter xylinum has been considered a strict aerobe, and it has been suggested that the function of cellulose is to hold cells in an aerobic environment. In this study, we showed that A. xylinum is capable of growing microaerophilically. Cellulose pellicles provided significant protection to A. xylinum cells from the killing effects of UV light. In experiments measuring colonization by A. xylinum, molds, and other bacteria on pieces of apple, cellulose pellicles enhanced colonization of A. xylinum on the substrate and provided protection from competitors which use the same substrate as a source of nutrients. Cellulose pellicles produced by A. xylinum may have multiple functions in the growth and survival of the organism in nature.     

CelR,an Ortholog of the Diguanylate Cyclase PleD of Caulobacter,Regulates Cellulose Synthesis in Agrobacterium tumefaciens

Cellulose fibrils play a role in attachment of Agrobacterium tumefaciens to its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, including A. tumefaciens. In this study, we identified two putative diguanylate cyclase genes, celR (atu1297) and atu1060, that influence production of cellulose in A. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion of celR, but not atu1060, resulted in decreased cellulose biosynthesis. celR overexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis of celR and Δcel mutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing both celR and celA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lack celA use CelR, called PleD, to regulate specific processes associated with polar localization and cell division.     

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane.     

Adhesion of Human and Animal Escherichia coli Strains in Association with Their Virulence-Associated Genes and Phylogenetic Origins

Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche, which is based on the expression of colonization factors. Escherichia coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal (exVAGs) infection. Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization. We developed new screening tools to genotypically and phenotypically characterize E. coli isolates originating in humans, domestic pigs, and 17 wild mammal and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA) and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes. inVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and the European hedgehog ( Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host, and tissue, though it was also unspecific. Occurrence of the following VAGs was associated with a higher rate of adhesion to one or more cell lines: afa-dra, daaD, tsh, vat, ibeA, fyuA, mat, sfa-foc, malX, pic, irp2, and papC. In summary, we established new screening methods which enabled us to characterize large numbers of E. coli isolates. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.     

Intra-Subtype Variation in Enteroadhesion Accounts for Differences in Epithelial Barrier Disruption and Is Associated with Metronidazole Resistance in Blastocystis Subtype-7

Blastocystis is an extracellular, enteric pathogen that induces intestinal disorders in a range of hosts including humans. Recent studies have identified potential parasite virulence factors in and host responses to this parasite; however, little is known about Blastocystis-host attachment, which is crucial for colonization and virulence of luminal stages. By utilizing 7 different strains of the parasite belonging to two clinically relevant subtypes ST-4 and ST-7, we investigated Blastocystis-enterocyte adhesion and its association with parasite-induced epithelial barrier disruption. We also suggest that drug resistance in ST-7 strains might result in fitness cost that manifested as impairment of parasite adhesion and, consequently, virulence. ST-7 parasites were generally highly adhesive to Caco-2 cells and preferred binding to intercellular junctions. These strains also induced disruption of ZO-1 and occludin tight junction proteins as well as increased dextran-FITC flux across epithelial monolayers. Interestingly, their adhesion was correlated with metronidazole (Mz) susceptibility. Mz resistant (Mz^r) strains were found to be less pathogenic, owing to compromised adhesion. Moreover, tolerance of nitrosative stress was also reduced in the Mz^r strains. In conclusion, the findings indicate that Blastocystis attaches to intestinal epithelium and leads to epithelial barrier dysfunction and that drug resistance might entail a fitness cost in parasite virulence by limiting entero-adhesiveness. This is the first study of the cellular basis for strain-to-strain variation in parasite pathogenicity. Intra- and inter-subtype variability in cytopathogenicity provides a possible explanation for the diverse clinical outcomes of Blastocystis infections.     

A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization.     

Root-to-Root Travel of the Beneficial Bacterium Azospirillum brasilense

The root-to-root travel of the beneficial bacterium Azospirillum brasilense on wheat and soybean roots in agar, sand, and light-textured soil was monitored. We used a motile wild-type (Mot⁺) strain and a motility-deficient (Mot^-) strain which was derived from the wild-type strain. The colonization levels of inoculated roots were similar for the two strains. Mot⁺ cells moved from inoculated roots (either natural or artificial roots in agar, sand, or light-textured soil) to noninoculated roots, where they formed a band-type colonization composed of bacterial aggregates encircling a limited part of the root, regardless of the plant species. The Mot^- strain did not move toward noninoculated roots of either plant species and usually stayed at the inoculation site and root tips. The effect of attractants and repellents was the primary factor governing the motility of Mot⁺ cells in the presence of adequate water. We propose that interroot travel of A. brasilense is an essential preliminary step in the root-bacterium recognition mechanism. Bacterial motility might have a general role in getting Azospirillum cells to the site where firmer attachment favors colonization of the root system. Azospirillum travel toward plants is a nonspecific active process which is not directly dependent on nutrient deficiency but is a consequence of a nonspecific bacterial chemotaxis, influenced by the balance between attractants and possibly repellents leaked by the root.     

Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

Form and Function of Clostridium thermocellum Biofilms

The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h⁻¹) 12-fold higher than the bacterium''s maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion.     

Kinetics of Insoluble Cellulose Fermentation by Continuous Cultures of Ruminococcus albus

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant (k) of 1.18 day⁻¹. The true microbial growth yield (Y) was 0.11 g g⁻¹, the maintenance coefficient (m) was 0.10 g g⁻¹ h⁻¹, and the maximum Y_ATP was 7.7 g of biomass (dry weight) mol of ATP⁻¹.