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A Multispecies Fungal Biofilm Approach to Enhance the Celluloyltic Efficiency of Membrane Reactors for Consolidated Bioprocessing of Plant Biomass.

ABSTRACT: The constraints and advantages in cellulolytic enzymes production by fungal biofilms for a consolidated bioconversion process were investigated during this study. The biofilm cultivations were carried out in reactors designed for consolidated bioprocessing Multispecies Biofilm Membrane reactors, (MBM) where an aerobic fungal biofilm produces the lignocellulolytic enzymes while a fermenting microorganism forms the fermentation product at anaerobic conditions. It was shown that although mycelial growth was limited in the MBM reactors compared to submerged cultivations, the secretion of cellulolytic enzymes per cell dry weight was higher. When Trichoderma reesei was used as the sole enzyme producer, cellobiose accumulated in the liquid medium as the result of the deficiency of ?-glucosidase in the fungal secretome. To enhance ?-glucosidase activity, T. reesei was co-cultivated with A. phoenicis which is a ?-glucosidase overproducer. The two fungi formed a multispecies biofilm which produced a balanced cellulolytic cocktail for the saccharification of plant biomass. The mixed biofilm reached a 2.5 fold increase in ?-glucosidase production, compared to the single T. reesei biofilm. The enzymatic systems of single and mixed biofilms were evaluated regarding their efficiency on cellulosic substrates degradation. Washed solids from steam pretreated beechwood, as well as microcrystalline cellulose were used as the substrates. The enzymatic system of the multispecies biofilm released four times more glucose than the enzymatic system of T. reesei alone from both substrates and hydrolyzed 78 and 60% of the cellulose content of washed solids from beechwood and microcrystalline cellulose, respectively.

SUBMITTER: Xiros C

PROVIDER: S-EPMC5641325 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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A Multispecies Fungal Biofilm Approach to Enhance the Celluloyltic Efficiency of Membrane Reactors for Consolidated Bioprocessing of Plant Biomass.

Xiros Charilaos C Studer Michael H MH

Frontiers in microbiology 20171010

The constraints and advantages in cellulolytic enzymes production by fungal biofilms for a consolidated bioconversion process were investigated during this study. The biofilm cultivations were carried out in reactors designed for consolidated bioprocessing Multispecies Biofilm Membrane reactors, (MBM) where an aerobic fungal biofilm produces the lignocellulolytic enzymes while a fermenting microorganism forms the fermentation product at anaerobic conditions. It was shown that although mycelial g ...[more]

PMID: 29067006

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Project description:BackgroundAlthough the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics.ResultsThe screening of five different cellulase producers with the freeze assay identified Trichoderma reesei and Penicillium verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P. verruculosum but a similar cellulase production rate afterwards, compared to T. reesei. The freeze assay measurement depicted that P. verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the different initial enzyme/cellulose ratios as well as a discrepancy in cellulose digestibility are the main differences between the two approaches.ConclusionsWith two complementary methods to quantify cellulase activity and the dynamics of cellulase production for CBP applications, T. reesei and P. verruculosum were identified as compatible candidates for the chosen model process. The presented methods can easily be adapted to screen for suitable cellulose degrading organisms for various other applications.

Project description:BACKGROUND: The recalcitrant nature of hemicellulosic materials and the high cost in depolymerization are the primary obstacles preventing the use of xylan as feedstock for fuel and chemical production. Consolidated bioprocessing, incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single microorganism, could potentially avoid the cost of the dedicated enzyme generation in the process of xylan utilization. In this study, we engineered Escherichia coli strains capable of exporting three hemicellulases to the broth for the succinate production directly from beechwood xylan. RESULTS: Xylanases were extracellular environment-directed by fusing with OsmY. Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinations were characterized and tested. The combination of XynC-A from Fibrobacter succinogenes S85 and XyloA from Fusarium graminearum which appeared to have optimal enzymatic properties was identified as the best choice for xylan hydrolysis (0.18 ± 0.01 g/l protein in the broth with endoxylanase activity of 12.14 ± 0.34 U/mg protein and xylosidase activity of 92 ± 3 mU/mg protein at 8 h after induction). Further improvements of hemicellulases secretion were investigated by lpp deletion, dsbA overexpression and expression level optimization. With co-expression of ?-arabinofuranosidase, the engineered E. coli could hydrolyze beechwood xylan to pentose monosaccharides. The hemicellulolytic capacity was further integrated with a succinate-producing strain to demonstrate the production of succinate directly from xylan without externally supplied hydrolases and any other organic nutrient. The resulting E. coli Z6373 was able to produce 0.37 g/g succinate from xylan anaerobically equivalent to 76% of that from xylan acid hydrolysates. CONCLUSIONS: This report represents a promising step towards the goal of hemicellulosic chemical production. This engineered E. coli expressing and secreting three hemicellulases demonstrated a considerable succinate production on the released monosaccharides from xylan. The ability to use lower-cost crude feedstock will make biological succinate production more economically attractive.

Project description:BackgroundClostridial co-culture containing cellulolytic and solventogenic species is a potential consolidated bioprocessing (CBP) approach for producing biochemicals and biofuels from cellulosic biomass. It has been demonstrated that the rate of cellulose utilization in the co-culture of Clostridium acetobutylicum and Clostridium cellulolyticum is improved compared to the mono-culture of C. cellulolyticum (BL 5:119-124, 1983). However, the metabolic interactions in this co-culture are not well understood. To investigate the metabolic interactions in this co-culture we dynamically characterized the physiology and microbial composition using qPCR.ResultsThe qPCR data suggested a higher growth rate of C. cellulolyticum in the co-culture compared to its mono-culture. Our results also showed that in contrast to the mono-culture of C. cellulolyticum, which did not show any cellulolytic activity under conditions similar to those of co-culture, the co-culture did show cellulolytic activity even superior to the C. cellulolyticum mono-culture at its optimal pH of 7.2. Moreover, experiments indicated that the co-culture cellulolytic activity depends on the concentration of C. acetobutylicum in the co-culture, as no cellulolytic activity was observed at low concentration of C. acetobutylicum, and thus confirming the essential role of C. acetobutylicum in improving C. cellulolyticum growth in the co-culture. Furthermore, butanol concentration of 350 mg/L was detected in the co-culture batch experiments.ConclusionThese results suggest the presence of synergism between these two species, while C. acetobutylicum metabolic activity significantly improves the cellulolytic activity in the co-culture, and allows C. cellulolyticum to survive under harsh co-culture conditions, which do not allow C. cellulolyticum to grow and metabolize cellulose independently. It is likely that C. acetobutylicum improves the cellulolytic activity of C. cellulolyticum in the co-culture through exchange of metabolites such as pyruvate, enabling it to grow and metabolize cellulose under harsh co-culture conditions.

Project description:BackgroundTerrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed.ResultsThe total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I.ConclusionsThe significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum.

Dataset Information

A Multispecies Fungal Biofilm Approach to Enhance the Celluloyltic Efficiency of Membrane Reactors for Consolidated Bioprocessing of Plant Biomass.

Publications

A Multispecies Fungal Biofilm Approach to Enhance the Celluloyltic Efficiency of Membrane Reactors for Consolidated Bioprocessing of Plant Biomass.

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