Kinetics and Metabolism of Cellulose Degradation at High Substrate Concentrations in Steady-State Continuous Cultures of Clostridium cellulolyticum on a Chemically Defined Medium

The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate. At a dilution rate (D) of 0.048 h⁻¹, biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose liter⁻¹ the cell density at steady state leveled off. The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose liter⁻¹, respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed. The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis. A kinetics study of C. cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose liter⁻¹. Compared to cellulose limitation (M. Desvaux, E. Guedon, and H. Petitdemange, J. Bacteriol. 183:119–130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD⁺, and q_{NADH produced}/q_{NADH used} ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y) (19.4 g of biomass per mol of ATP) were lowered. C. cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions. However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients. The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E. Guedon, S. Payot, M. Desvaux, and H. Petitdemange, Biotechnol. Bioeng. 67:327–335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells⁻¹ h⁻¹ could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells⁻¹ h⁻¹; (ii) while the NADH/NAD⁺ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions. Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.     

Combined inactivation of the <Emphasis Type="Italic">Clostridium cellulolyticum</Emphasis> lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

Background  

The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H₂ and CO₂, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.     

Comparison of batch and semicontinuous cultures for production of protein from mesquite wood by Brevibacterium sp. JM98A

The production of protein by a Brevibacterium sp. JM98A usingmesquite wood as the substrate was compared in batch and semicontinuous cultures. A 14 liter glass fermentor with automatic pH, temperature, and foam control was used for the study. A pH range of 6.6 to 7.2 was optimum for the growth of JM98A. The batch and semicontinuous cultures were compared on the basis of viable cell counts, protein production, CMC-Ase (β-1,4-glucanase) activity, and filter paper cellulase (β-1,4-glucan cellobiohydrolyase) activity. Total hexose, cellulose, and reducing sugar consumption were measured. The semicontinuous process yielded 2.97 times as much protein in 72 hr as the batch cultures. Most of the biomass resulted from the utilization of soluble sugars rather than from the degradation of cellulose during the semicontinuous process.     

Bioconversion of hemicelluloses into fungal protein

Summary The utilization of cellulose from one ton of lignocellulose for ethanol production would yield 150–250 kg of hemicelluloses. The total soluble solids in the hemicellulose fraction (HF) obtained with the Université de Sherbrooke (UdeS) process contained about 56% carbohydrates. These carbohydrates were present in the form of oligomers of various sugars, predominantly xylose. All the test fungi,Chaetomium cellulolyticum, C. cellulolyticum (asporogenous mutant) andPleurotus sajor-caju, were capable of utilizing all the carbohydrates present in HF.C. cellulolyticum gave the highest amount of protein (7 g/l) from 19 g carbohydrates/l. The yield of protein was higher than expected, indicating that carbon compounds other than reducing sugars present in HF might have been consumed for fungal growth. The inhibitory effect of toxic compounds on protein production increased with an increase in concentration of soluble solids in HF. The inhibitory effect was overcome by increasing the pH of the medium to 6.0 or 7.0. Fungal protein production from hemicelluloses will give extra revenue in our integrated approach for ethanol production from lignocelluloses.NRCC publication No. 26277.     

Flux Analysis of the Metabolism of Clostridium cellulolyticum Grown in Cellulose-Fed Continuous Culture on a Chemically Defined Medium under Ammonium-Limited Conditions

An investigation of cellulose degradation by the nonruminal, cellulolytic, mesophilic bacterium Clostridium cellulolyticum was performed in cellulose-fed chemostat cultures with ammonium as the growth-limiting nutrient. At any dilution rate (D), acetate was always the main product of the catabolism, with a yield of product from substrate ranging between 37.7 and 51.5 g per mol of hexose equivalent fermented and an acetate/ethanol ratio always higher than 1. As D rose, the acetyl coenzyme A was rerouted in favor of ethanol pathways, and ethanol production could represent up to 17.7% of the carbon consumed. Lactate was significantly produced, but with increasing D, the specific lactate production rate declined, as did the specific rate of production of extracellular pyruvate. The proportion of the original carbon directed towards phosphoglucomutase remained constant, and the carbon surplus was balanced mainly by exopolysaccharide and glycogen biosyntheses at high D values, while cellodextrin excretion occurred mainly at lower ones. With increasing D, the specific rate of carbon flowing down catabolites increased as well, but when expressed as a percentage of carbon it declined, while the percentage of carbon directed through biosynthesis pathways was enhanced. The maximum growth and energetic yields were lower than those obtained in cellulose-limited chemostats and were related to an uncoupling between catabolism and anabolism leading to an excess of energy. Compared to growth on cellobiose in ammonium-limited chemostats (E. Guedon, M. Desvaux, and H. Petitdemange, J. Bacteriol. 182:2010–2017, 2000), (i) a specific consumption rate of carbon of as high as 26.72 mmol of hexose equivalent g of cells⁻¹ h⁻¹ could not be reached and (ii) the proportions of carbon directed towards cellodextrin, glycogen, and exopolysaccharide pathways were not as high as first determined on cellobiose. While the use of cellobiose allows highlighting of metabolic limitation and regulation of C. cellulolyticum under ammonium-limited conditions, some of these events should then rather be interpreted as distortions of the metabolism. Growth of cellulolytic bacteria on easily available carbon and nitrogen sources represents conditions far different from those of the natural lignocellulosic compounds.     

Single cell protein from various chemically pretreated wood substrates using Chaetomium cellulolyticum

The growth behavior of Chaetomium cellulolyticum, a new cellulolytic fungus, has been examined in slurry fermentation systems using various chemically pretreated sawdusts from hardwoods as substrates. Both acid- and alkali-pretreatment methods were used and the fermentation media included the spent pretreatment liquor in an attempt to concurrently maximize substrate utilization and minimize the biological oxygen demand (BOD) level in the process effluent. Diauxic growth patterns were found in the three cases studied, suggesting an initial utilization of soluble hemicellulose sugars followed by utilization of the insoluble cellulose. This behavior patterns was supported by separate growth experiments using the major sugars of hemicellulose as carbon sources. The organism was found to be a good convertor of both cellulose and hemicelluloses into single cell protein (SCP). In terms of rate and extent of protein production in the insoluble biomass product, acid pretreatment appears to be better than alkali pretreatment if the product is intended as ruminant feed.     

Metabolism of cellobiose and cellulose byBacteroides cellulosolvens

Summary The metabolism ofBacteroides cellulosolvens was studied on cellobiose and cellulose as energy and carbon sources. The growth rate was faster on cellobiose; however, growth on cellulose resulted in consumption of 55% more hexose equivalents, and in production of 49% more biomass, and 30% more metabolites (ethanol, acetate, and lactate). On each substrateB. cellulosolvens exhibited two distinct ranges of molar growth yields (Y _H g cells/mol hexose). At low substrate concentrations (less than 30 mmol) hexoseY _H values were 25.5 for cellulose and 28.5 for cellobiose, while at hexose levels greater than 30 mmolY _H values were 13.5 and 15, respectively. Shifts in metabolism towards greater lactic acid production resulted in decreased ATP production; however, this did not cause early growth cessation, as these shifts occurred after the drop inY _H.Issued as NRCC No. 27409.     

Genome-scale metabolic modeling of a clostridial co-culture for consolidated bioprocessing

An alternative consolidated bioprocessing approach is the use of a co-culture containing cellulolytic and solventogenic clostridia. 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, suggesting the presence of syntrophy between these two species. However, the metabolic interactions in the co-culture are not well understood. To understand the metabolic interactions in the co-culture, we developed a genome-scale metabolic model of C. cellulolyticum comprising of 431 genes, 621 reactions, and 603 metabolites. The C. cellulolyticum model can successfully predict the chemostat growth and byproduct secretion with cellulose as the substrate. However, a growth arrest phenomenon, which occurs in batch cultures of C. cellulolyticum at cellulose concentrations higher than 6.7 g/L, cannot be predicted by dynamic flux balance analysis due to the lack of understanding of the underlying mechanism. These genome-scale metabolic models of the pure cultures have also been integrated using a community modeling framework to develop a dynamic model of metabolic interactions in the co-culture. Co-culture simulations suggest that cellobiose inhibition cannot be the main factor that is responsible for improved cellulose utilization relative to mono-culture of C. cellulolyticum.     

Improvement of Cellulolytic Properties of Clostridium cellulolyticum by Metabolic Engineering

Cellulolytic clostridia have evolved to catabolize lignocellulosic materials at a seasonal biorhythm, so their biotechnological exploitation requires genetic improvements. As high carbon flux leads to pyruvate accumulation, which is responsible for the cessation of growth of Clostridium cellulolyticum, this accumulation is decreased by heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. In comparison with that of the wild strain, growth of the recombinant strain at the same specific rate but for 145 h instead of 80 h led to a 150% increase in cellulose consumption and a 180% increase in cell dry weight. The fermentation pattern was shifted significantly: lactate production decreased by 48%, whereas the concentrations of acetate and ethanol increased by 93 and 53%, respectively. This study demonstrates that the fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using genetically engineered C. cellulolyticum.     

Physiological properties of Cellulomonas fermentans,a mesophilic cellulolytic bacterium

Summary The fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h^-1, 0.040 h^-1 and 0.013 h^-1 respectively. Growth inhibition was observed, and a drop in Y_H occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h^-1 and 0.070±0.007 h^-1 respectively). Aerobic metabolism was studied using ¹⁴C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no ¹⁴CO₂ was produced. During the stationary phase, acetate was oxidized and ¹⁴CO₂ produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.     

Effect of Sugar Concentration in Jerusalem Artichoke Extract on Kluyveromyces marxianus Growth and Ethanol Production

The effect of inulin sugars concentration on the growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 was studied. A maximum ethanol concentration of 102 g/liter was obtained from 250 g of sugars per liter initial concentration. The maximum specific growth rate varied from 0.44 h⁻¹ at 50 g of sugar per liter to 0.13 h⁻¹ at 300 g of sugar per liter, whereas the ethanol yield remained almost constant at 0.45 g of ethanol per g of sugars utilized.     

Bioconversion of waste office paper to hydrogen using pretreated rumen fluid inoculum

In this study, a microbial consortium from an acid-treated rumen fluid was used to improve the yields of H₂ production from paper residues in batch reactors. The anaerobic batch reactors, which contained paper and cellulose, were operated under three conditions: (1) 0.5 g paper/L, (2) 2 g paper/L, and (3) 4 g paper/L. Cellulase was added to promote the hydrolysis of paper to soluble sugars. The H₂ yields were 5.51, 4.65, and 3.96 mmol H₂/g COD, respectively, with substrate degradation ranging from 56 to 65.4 %. Butyric acid was the primary soluble metabolite in the three reactors, but pronounced solventogenesis was detected in the reactors incubated with increased paper concentrations (2.0 and 4.0 g/L). A substantial prevalence of Clostridium acetobutylicum (99 % similarity) was observed in the acid-treated rumen fluid, which has been recognized as an efficient H₂-producing strain in addition to ethanol and n-butanol which were also detected in the reactors.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

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.     

Effect of physicochemical pre-treatment of rice straw on its digestibility by <Emphasis Type="Italic">Clostridium cellulolyticum</Emphasis>

Rice straw (RS) may serve as a low-cost biomass for the production of biofuels and biochemicals, but its native structure is resistant to enzymatic and microbial deconstruction. Therefore, an efficient pre-treatment method is required to modify crystalline cellulose to a more reactive amorphous form. This work investigated pre-treatments of rice straw involving size reduction (S) followed by either sodium hydroxide (NaOH) or diluted sulfuric acid (H₂SO₄) and liquid hot water (LHW). The shrinkage of the vascular bundles in the rice straw structure pre-treated with NaOH–LHW–S was higher than that with LHW–S and H₂SO₄–LHW–S pre-treatments. The highest levels of total fermentative products and residual sugars were obtained at the concentrations of 7.8 ± 0.2 and 2.1 ± 0.3 g/L, respectively, after fermentation by Clostridium cellulolyticum for NaOH–LHW–S pre-treated rice straw at 121 °C for 120 min. Overall, the combined physicochemical pre-treatment of RS led to improved microbial hydrolysis during cellulose degradation at the percentage of 85.5 ± 0.5.     

Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km_(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.     

Effect of glucose concentration on the rate of fructose consumption in native strains isolated from the fermentation of Agave duranguensis

Studies on hexose consumption by Saccharomyces cerevisiae show that glucose is consumed faster than fructose when both are present (9:1 fructose to glucose) in the medium during the fermentation of Agave. The objective of this work was to select strains of S. cerevisiae that consume fructose equal to or faster than glucose at high fructose concentrations by analyzing the influence of different glucose concentrations on the fructose consumption rate. The optimal growth conditions were determined by a kinetics assay using high performance liquid chromatography (HPLC) using 50?g of glucose and 50?g of fructose per liter of synthetic medium containing peptone and yeast extract. Using the same substrate concentrations, strain ITD-00185 was shown to have a higher reaction rate for fructose over glucose. At 75?g of fructose and 25?g of glucose per liter, strain ITD-00185 had a productivity of 1.02 gL^?1?h^?1 after 40?h and a fructose rate constant of 0.071?h^?1. It was observed that glucose concentration positively influences fructose consumption when present in a 3:1 ratio of fructose to glucose. Therefore, adapted strains at high fructose concentrations could be used as an alternative to traditional fermentation processes.     

Incorporation of [15N]Ammonia by the Cellulolytic Ruminal Bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using ¹⁵NH₃. At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH₃-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH₃. More cell nitrogen was formed from NH₃ during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its ¹⁵N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.