Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species.

IMPORTANCE: The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.

Reconstructing electron transfer components from an Fe(II) oxidizing bacterium.

Neutrophilic Fe(II) oxidizing bacteria play an important role in biogeochemical processes and have also received attention for multiple technological applications. These micro-organisms are thought to couple their metabolism with extracellular electron transfer (EET) while oxidizing Fe(II) as electron donor outside the cell. Sideroxydans lithotrophicus ES-1 is a freshwater chemolithoautotrophic Fe(II) oxidizing bacterium that is challenging to culture and not yet genetically tractable. Analysis of the S. lithotrophicus ES-1 genome predicts multiple EET pathways, which are proposed to be involved in Fe(II) oxidation, but not yet validated. Here we expressed components of two of the proposed EET pathways, including the Mto and Slit_0867-0870 PCC3 pathways, from S. lithotrophicus ES-1 into Aeromonas hydrophila, an established model EET organism. We demonstrate that combinations of putative inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways partially complemented EET activity in Aeromonas mutants lacking native components. Our results provide evidence for electron transfer functionality and interactions of inner membrane and periplasmic components from the Mto and Slit_0867-0870 PCC3 pathways. Based on these findings, we suggest that EET in S. lithotrophicus ES-1 could be more complicated than previously considered and raises questions regarding directionality of these electron transfer pathways.

Evidence for Quinol Oxidation Activity of ImoA, a Novel NapC/NirT Family Protein from the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1.

Sideroxydans species are important chemolithoautotrophic Fe(II)-oxidizing bacteria in freshwater environments and play a role in biogeochemical cycling of multiple elements. Due to difficulties in laboratory cultivation and genetic intractability, the electron transport proteins required for the growth and survival of this organism remain understudied. In Sideroxydans lithotrophicus ES-1, it is proposed that the Mto pathway transfers electrons from extracellular Fe(II) oxidation across the periplasm to an inner membrane NapC/NirT family protein encoded by Slit_2495 to reduce the quinone pool. Based on sequence similarity, Slit_2495 has been putatively called CymA, a NapC/NirT family protein which in Shewanella oneidensis MR-1 oxidizes the quinol pool during anaerobic respiration of a wide range of substrates. However, our phylogenetic analysis using the alignment of different NapC/NirT family proteins shows that Slit_2495 clusters closer to NirT sequences than to CymA. We propose the name ImoA (inner membrane oxidoreductase) for Slit_2495. Our data demonstrate that ImoA can oxidize quinol pools in the inner membrane and is able to functionally replace CymA in S. oneidensis. The ability of ImoA to oxidize quinol in vivo as opposed to its proposed function of reducing quinone raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus. IMPORTANCE Fe(II)-oxidizing bacteria play an important role in biogeochemical cycles. At circumneutral pH, these organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside the cell, potentially through a porin-cytochrome complex in the outer membrane encoded by the Mto pathway. Electrons from Fe(II) oxidation would then be transported to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. Directly demonstrating the functionality of genes in neutrophilic iron oxidizers is challenging due to the absence of robust genetic methods. Here, we heterologously expressed a NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to reduce the quinone pool. ImoA functionally replaced the inner membrane c-type cytochrome CymA in the Fe(III)-reducing bacterium Shewanella oneidensis. We suggest that ImoA may function primarily to oxidize quinol in S. lithotrophicus.

PioABC-Dependent Fe(II) Oxidation during Photoheterotrophic Growth on an Oxidized Carbon Substrate Increases Growth Yield.

Microorganisms that carry out Fe(II) oxidation play a major role in biogeochemical cycling of iron in environments with low oxygen. Fe(II) oxidation has been largely studied in the context of autotrophy. Here, we show that the anoxygenic phototroph, Rhodopseudomonas palustris CGA010, carries out Fe(II) oxidation during photoheterotrophic growth with an oxidized carbon source, malate, leading to an increase in cell yield and allowing more carbon to be directed to cell biomass. We probed the regulatory basis for this by transcriptome sequencing (RNA-seq) and found that the expression levels of the known pioABC Fe(II) oxidation genes in R. palustris depended on the redox-sensing two-component system, RegSR, and the oxidation state of the carbon source provided to cells. This provides the first mechanistic demonstration of mixotrophic growth involving reducing power generated from both Fe(II) oxidation and carbon assimilation. IMPORTANCE The simultaneous use of carbon and reduced metals such as Fe(II) by bacteria is thought to be widespread in aquatic environments, and a mechanistic description of this process could improve our understanding of biogeochemical cycles. Anoxygenic phototrophic bacteria like Rhodopseudomonas palustris typically use light for energy and organic compounds as both a carbon and an electron source. They can also use CO2 for carbon by carbon dioxide fixation when electron-rich compounds like H2, thiosulfate, and Fe(II) are provided as electron donors. Here, we show that Fe(II) oxidation can be used in another context to promote higher growth yields of R. palustris when the oxidized carbon compound malate is provided. We further established the regulatory mechanism underpinning this observation.

The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

Automated operative workflow analysis of endoscopic pituitary surgery using machine learning: development and preclinical evaluation (IDEAL stage 0).

OBJECTIVE: Surgical workflow analysis involves systematically breaking down operations into key phases and steps. Automatic analysis of this workflow has potential uses for surgical training, preoperative planning, and outcome prediction. Recent advances in machine learning (ML) and computer vision have allowed accurate automated workflow analysis of operative videos. In this Idea, Development, Exploration, Assessment, Long-term study (IDEAL) stage 0 study, the authors sought to use Touch Surgery for the development and validation of an ML-powered analysis of phases and steps in the endoscopic transsphenoidal approach (eTSA) for pituitary adenoma resection, a first for neurosurgery. METHODS: The surgical phases and steps of 50 anonymized eTSA operative videos were labeled by expert surgeons. Forty videos were used to train a combined convolutional and recurrent neural network model by Touch Surgery. Ten videos were used for model evaluation (accuracy, F1 score), comparing the phase and step recognition of surgeons to the automatic detection of the ML model. RESULTS: The longest phase was the sellar phase (median 28 minutes), followed by the nasal phase (median 22 minutes) and the closure phase (median 14 minutes). The longest steps were step 5 (tumor identification and excision, median 17 minutes); step 3 (posterior septectomy and removal of sphenoid septations, median 14 minutes); and step 4 (anterior sellar wall removal, median 10 minutes). There were substantial variations within the recorded procedures in terms of video appearances, step duration, and step order, with only 50% of videos containing all 7 steps performed sequentially in numerical order. Despite this, the model was able to output accurate recognition of surgical phases (91% accuracy, 90% F1 score) and steps (76% accuracy, 75% F1 score). CONCLUSIONS: In this IDEAL stage 0 study, ML techniques have been developed to automatically analyze operative videos of eTSA pituitary surgery. This technology has previously been shown to be acceptable to neurosurgical teams and patients. ML-based surgical workflow analysis has numerous potential uses-such as education (e.g., automatic indexing of contemporary operative videos for teaching), improved operative efficiency (e.g., orchestrating the entire surgical team to a common workflow), and improved patient outcomes (e.g., comparison of surgical techniques or early detection of adverse events). Future directions include the real-time integration of Touch Surgery into the live operative environment as an IDEAL stage 1 (first-in-human) study, and further development of underpinning ML models using larger data sets.

Oligo-heterotrophic Activity of Marinobacter subterrani Creates an Indirect Fe(II) Oxidation Phenotype in Gradient Tubes.

Autotrophic bacteria utilizing Fe(II) as their energy and electron sources for growth affect multiple biogeochemical cycles. Some chemoheterotrophic bacteria have also been considered to exhibit an Fe(II) oxidation phenotype. For example, several Marinobacter strains have been reported to oxidize Fe(II) based on formation of oxidized iron bands in semi-solid gradient tubes that produce opposing concentration gradients of Fe(II) and oxygen. While gradient tubes are a simple and visually compelling method to test for Fe(II) oxidation, this method alone cannot confirm if, and to what extent, Fe(II) oxidation is linked to metabolism in chemoheterotrophic bacteria. Here we probe the possibility of protein-mediated and metabolic by-product-mediated Fe(II) oxidation in Marinobacter subterrani JG233, a chemoheterotroph previously proposed to oxidize Fe(II). Results from conditional and mutant studies, along with measurements of Fe(II) oxidation rates, suggest M. subterrani is unlikely to facilitate Fe(II) oxidation under microaerobic conditions. We conclude that the Fe(II) oxidation phenotype observed in gradient tubes inoculated with M. subterrani JG233 is a result of oligo-heterotrophic activity, shifting the location where oxygen dependent chemical Fe(II) oxidation occurs, rather than a biologically mediated process. IMPORTANCE Gradient tubes are the most commonly used method to isolate and identify neutrophilic Fe(II)-oxidizing bacteria. The formation of oxidized iron bands in gradient tubes provides a compelling assay to ascribe the ability to oxidize Fe(II) to autotrophic bacteria whose growth is dependent on Fe(II) oxidation. However, the physiological significance of Fe(II) oxidation in chemoheterotrophic bacteria is less well understood. Our work suggests that oligo-heterotrophic activity of certain bacteria may create a false-positive phenotype in gradient tubes by altering the location of the abiotic, oxygen-mediated oxidized iron band. Based on the results and analysis presented here, we caution against utilizing gradient tubes as the sole evidence for the capability of a strain to oxidize Fe(II) and that additional experiments are necessary to ascribe this phenotype to new isolates.

Surgical Management of Giant Thoracic Paraspinal Schwannomas.

BACKGROUND: Giant paraspinal thoracic schwannomas (GPTSs) are benign, slow-growing, encapsulated lesions. They can be intracanalicular, span more than 2 vertebral bodies, and/or have a foraminal component with extraspinal extension >2.5 cm. They pose surgical challenges because of the often unfamiliar complex regional anatomy. We report the largest series of GPTSs and discuss regional surgical strategies for tumors in the thoracic spine. METHODS: We conducted a retrospective review of GPTSs operated at a national spinal referral center between December 2008 and October 2019. Inclusion criteria included World Health Organization grade 1 GPTS. Patient demographics, clinical features, radiology, and histopathology were assessed. RESULTS: Seventeen patients (12 females, 5 males) had a mean age of 48.1 years (range 21-65 years). Five GPTS (29%) were located at T1-T3, 6 (35%) at T4-6, and 6 (35%) below T6. The mean maximum diameter was 58.5 ± 19.1 mm (range 30-91 mm). Mean volume was 90.9 cm3 (range 19.1-350.6 cm3). Twelve (70%) had a fluorodeoxyglucose positron emission tomography scan showing low (25%) or moderate to high (75%) uptake. Six patients (35%) had preoperative computed tomography-guided biopsy. Surgical approaches included 1) manubriotomy and variations (4/17); 2) high lateral thoracotomy (4/17); 3) posterior parascapular (1/17); 4) standard lateral thoracotomy (3/16); 5) posterior/posterolateral (2/17); and 6) combined posterior and thoracotomy (3/17). All patients had gross total resection and were grade 1 cellular schwannomas. No recurrence at final follow-up (mean 36.1 months, range 8-130 months). CONCLUSIONS: A number of approaches are available to resect GPST in specific locations in the thoracic spine. Total resection is achievable despite complex regional anatomy, location, and tumor extension but often requires anterior or combined approaches.

Engineering lithoheterotrophy in an obligate chemolithoautotrophic Fe(II) oxidizing bacterium.

Neutrophilic Fe(II) oxidizing bacteria like Mariprofundus ferrooxydans are obligate chemolithoautotrophic bacteria that play an important role in the biogeochemical cycling of iron and other elements in multiple environments. These bacteria generally exhibit a singular metabolic mode of growth which prohibits comparative "omics" studies. Furthermore, these bacteria are considered non-amenable to classical genetic methods due to low cell densities, the inability to form colonies on solid medium, and production of copious amounts of insoluble iron oxyhydroxides as their metabolic byproduct. Consequently, the molecular and biochemical understanding of these bacteria remains speculative despite the availability of substantial genomic information. Here we develop the first genetic system in neutrophilic Fe(II) oxidizing bacterium and use it to engineer lithoheterotrophy in M. ferrooxydans, a metabolism that has been speculated but not experimentally validated. This synthetic biology approach could be extended to gain physiological understanding and domesticate other bacteria that grow using a single metabolic mode.

Evidence for auxiliary anaerobic metabolism in obligately aerobic Zetaproteobacteria.

Zetaproteobacteria are obligate chemolithoautotrophs that oxidize Fe(II) as an electron and energy source, and play significant roles in nutrient cycling and primary production in the marine biosphere. Zetaproteobacteria thrive under microoxic conditions near oxic-anoxic interfaces, where they catalyze Fe(II) oxidation faster than the abiotic reaction with oxygen. Neutrophilic Fe(II) oxidizing bacteria produce copious amounts of insoluble iron oxyhydroxides as a by-product of their metabolism. Oxygen consumption by aerobic respiration and formation of iron oxyhydroxides at oxic-anoxic interfaces can result in periods of oxygen limitation for bacterial cells. Under laboratory conditions, all Zetaproteobacteria isolates have been shown to strictly require oxygen as an electron acceptor for growth, and anaerobic metabolism has not been observed. However, genomic analyses indicate a range of potential anaerobic pathways present in Zetaproteobacteria. Heterologous expression of proteins from Mariprofundus ferrooxydans PV-1, including pyruvate formate lyase and acetate kinase, further support a capacity for anaerobic metabolism. Here we define auxiliary anaerobic metabolism as a mechanism to provide maintenance energy to cells and suggest that it provides a survival advantage to Zetaproteobacteria in environments with fluctuating oxygen availability.

Informed Consent for Patients Undergoing Transsphenoidal Excision of Pituitary Adenoma: Development and Evaluation of a Procedure-Specific Online Educational Resource.

OBJECTIVE: The results of recent high-profile delict and medical negligence cases now require doctors to take "reasonable care to ensure that the patient is aware of any material risks involved in any recommended treatment, and of any reasonable alternative or variant treatments." Thus, we report the development and evaluation of a procedure-specific online educational resource to support the informed consent process for patients undergoing transsphenoidal excision of pituitary adenomas. METHODS: An interactive educational multimedia website was developed using a combination of text, images, and videos. A telephone questionnaire was devised to evaluate patients' understanding that included 15 true/false questions. The questionnaire was administered to separate cohorts of patients on the waiting list for transsphenoidal excision of a pituitary adenoma before and after introduction of the website. The patients were also asked to rate the extent to which they found the website easy to understand and useful on a 10-point Likert scale. Data were compared using the χ2 and Mann-Whitney U test, with P < 0.05 considered to indicate statistical significance. RESULTS: Ten consecutive patients completed the questionnaire before the introduction of the website and nine afterward. The median questionnaire scores were significantly greater after the introduction of the website (14 of 15 vs. 12 of 15; P = 0.002), and all patients subjectively found the website easy to understand and useful (10 of 10 in both groups). CONCLUSIONS: An interactive educational multimedia website appears to be a helpful adjunct to the informed consent process for patients undergoing transsphenoidal excision of a pituitary adenoma.

Tracking Electron Uptake from a Cathode into Shewanella Cells: Implications for Energy Acquisition from Solid-Substrate Electron Donors.

While typically investigated as a microorganism capable of extracellular electron transfer to minerals or anodes, Shewanella oneidensis MR-1 can also facilitate electron flow from a cathode to terminal electron acceptors, such as fumarate or oxygen, thereby providing a model system for a process that has significant environmental and technological implications. This work demonstrates that cathodic electrons enter the electron transport chain of S. oneidensis when oxygen is used as the terminal electron acceptor. The effect of electron transport chain inhibitors suggested that a proton gradient is generated during cathode oxidation, consistent with the higher cellular ATP levels measured in cathode-respiring cells than in controls. Cathode oxidation also correlated with an increase in the cellular redox (NADH/FMNH2) pool determined with a bioluminescence assay, a proton uncoupler, and a mutant of proton-pumping NADH oxidase complex I. This work suggested that the generation of NADH/FMNH2 under cathodic conditions was linked to reverse electron flow mediated by complex I. A decrease in cathodic electron uptake was observed in various mutant strains, including those lacking the extracellular electron transfer components necessary for anodic-current generation. While no cell growth was observed under these conditions, here we show that cathode oxidation is linked to cellular energy acquisition, resulting in a quantifiable reduction in the cellular decay rate. This work highlights a potential mechanism for cell survival and/or persistence on cathodes, which might extend to environments where growth and division are severely limited.IMPORTANCE The majority of our knowledge of the physiology of extracellular electron transfer derives from studies of electrons moving to the exterior of the cell. The physiological mechanisms and/or consequences of the reverse processes are largely uncharacterized. This report demonstrates that when coupled to oxygen reduction, electrode oxidation can result in cellular energy acquisition. This respiratory process has potentially important implications for how microorganisms persist in energy-limited environments, such as reduced sediments under changing redox conditions. From an applied perspective, this work has important implications for microbially catalyzed processes on electrodes, particularly with regard to understanding models of cellular conversion of electrons from cathodes to microbially synthesized products.

Engineering a Native Inducible Expression System in Shewanella oneidensis to Control Extracellular Electron Transfer.

Shewanella oneidensis MR-1 is a model organism for understanding extracellular electron transport, in which cells transfer intracellular electrons to an extracellular terminal electron acceptor such as insoluble minerals or poised electrodes. Biotechnological applications exploiting the respiratory capabilities of Shewanella species have led to their proposed use in wastewater treatment, bioremediation, and remote sensors. Transcriptional regulation tools can be used to rationally engineer S. oneidensis, optimizing performance in biotechnological applications, introducing new capabilities, or investigating physiology. Engineered gene expression in S. oneidensis has primarily involved the use of foreign regulatory systems from Escherichia coli. Here we characterize a native S. oneidensis pathway that can be used to induce gene expression with trimethylamine N-oxide, then engineer strains in which extracellular electron transfer is controlled by this compound. The ability to induce this pathway was assessed by measuring iron reduction over time and by analyzing anodic current produced by cells grown in bioreactors.

Correlation analysis of enzyme activities and deconstruction of ammonia-pretreated switchgrass by bacterial-fungal communities.

The mixed microbial communities that occur naturally on lignocellulosic feedstocks can provide feedstock-specific enzyme mixtures to saccharify lignocelluloses. Bacterial-fungal communities were enriched from switchgrass bales to deconstruct ammonia-pretreated switchgrass (DSG). Correlation analysis was carried out to elucidate the relationship between microbial decomposition of DSG by these communities, enzymatic activities produced and enzymatic saccharification of DSG using these enzyme mixtures. Results of the analysis showed that ß-glucosidase and xylosidase activities limited the extent of microbial deconstruction and enzymatic saccharification of DSG. The results also underlined the importance of ligninase activity for the enzymatic saccharification of pretreated lignocellulosic feedstock. The bacterial-fungal communities developed in this research can be used to produce enzyme mixtures to deconstruct DSG, and the results from the correlation analysis can be used to optimize these enzyme mixtures for efficient saccharification of DSG to produce second-generation biofuels.

Use of biliary stent in laparoscopic common bile duct exploration.

INTRODUCTION: It is well supported in the literature that laparoscopic common bile duct exploration (LCBDE) for choledocholithiasis has equal efficacy when compared to ERCP followed by laparoscopic cholecystectomy. Decompression after supra-duodenal choledochotomy is common practice as it reduced the risk of bile leaks. We conducted a prospective non-randomized study to compare outcomes and length of stay in patients undergoing biliary stent insertion versus T-tube drainage following LCBDE via choledochotomy. METHODS AND PROCEDURES: The study involved 116 patients with choledocholithiasis who underwent LCBDE and decompression of the biliary system by either ante-grade biliary stent or T-tube insertion. A 7 French straight/duodenal curve biliary Diagmed™ stent (9-11 cm) was placed in 82 patients (Biliary Stent Group). T-tube insertion was used for 34 patients (T-tube group). The length of hospital stay and complications for the selected patients were recorded. All trans-cystic common bile duct explorations were excluded from the study. RESULTS: The mean hospital stay for patients who underwent ante-grade biliary stent or T-tube insertion after LBCDE were 1 and 3.4 days, respectively. This is a statistically significant result with a p value of less than 0.001. Of the T-tube group, two patients required laparoscopic washout due to bile leaks, one had ongoing biliary stasis and one reported ongoing pain whilst the T-tube was in situ. A complication rate of 11.2%, this was a significant finding. There were no complications or concerns reported for the Biliary Stent Group. CONCLUSION: Our results show that there is a significant reduction in length of hospital stay and morbidity for patients that have ante-grade biliary stent decompression of the CBD post laparoscopic choledochotomy when compared T-tube drainage. This implies that ante-grade biliary stent insertion is likely to reduce costs and increase overall patient satisfaction. We support the use of ante-grade biliary stent insertion during LCBDE when primary closure is not preferred.

Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans.

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.