Dataset Information

Form and function of Clostridium thermocellum biofilms.

ABSTRACT: 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.

SUBMITTER: Dumitrache A

PROVIDER: S-EPMC3536079 | biostudies-other | 2013 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Form and function of Clostridium thermocellum biofilms.

Dumitrache Alexandru A Wolfaardt Gideon G Allen Grant G Liss Steven N SN Lynd Lee R LR

Applied and environmental microbiology 20121019 1

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 c ...[more]

PMID: 23087042

Similar Datasets

Project description:BackgroundMicrobial cellulose conversion by Clostridium thermocellum 27405 occurs predominantly through the activity of substrate-adherent bacteria organized in thin, primarily single cell-layered biofilms. The importance of cellulosic surface exposure to microbial hydrolysis has received little attention despite its implied impact on conversion kinetics.ResultsWe showed the spatial heterogeneity of fiber distribution in pure cellulosic sheets, which made direct measurements of biofilm colonization and surface penetration impossible. Therefore, we utilized on-line measurements of carbon dioxide (CO2) production in continuous-flow reactors, in conjunction with confocal imaging, to observe patterns of biofilm invasion and to indirectly estimate microbial accessibility to the substrate's surface and the resulting limitations on conversion kinetics. A strong positive correlation was found between cellulose consumption and CO2 production (R2?=?0.996) and between surface area and maximum biofilm activity (R2?=?0.981). We observed an initial biofilm development rate (0.46 h-1, 0.34 h-1 and 0.33 h-1) on Whatman sheets (#1, #598 and #3, respectively) that stabilized when the accessible surface was maximally colonized. The results suggest that cellulose conversion kinetics is initially subject to a microbial limitation period where the substrate is in excess, followed by a substrate limitation period where cellular mass, in the form of biofilms, is not limiting. Accessible surface area acts as an important determinant of the respective lengths of these two distinct periods. At end-point fermentation, all sheets were digested predominantly under substrate accessibility limitations (e.g., up to 81% of total CO2 production for Whatman #1). Integration of CO2 production rates over time showed Whatman #3 underwent the fastest conversion efficiency under microbial limitation, suggestive of best biofilm penetration, while Whatman #1 exhibited the least recalcitrance and the faster degradation during the substrate limitation period.ConclusionThe results showed that the specific biofilm development rate of cellulolytic bacteria such as C. thermocellum has a notable effect on overall reactor kinetics during the period of microbial limitation, when ca. 20% of cellulose conversion occurs. The study further demonstrated the utility of on-line CO2 measurements as a method to assess biofilm development and substrate digestibility pertaining to microbial solubilization of cellulose, which is relevant when considering feedstock pre-treatment options.

Project description:Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ?-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ?glyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ?glyR2 strain, and ?-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ?glyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

Dataset Information

Form and function of Clostridium thermocellum biofilms.

Publications

Form and function of Clostridium thermocellum biofilms.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets