Simultaneous saccharification and fermentation of cellulose to lactic acid

Recent interest in the industrial manufacture of ethanol and other organic chemicals from biomass has led to the utilization of surplus grain and cane juice as a fermentation feedstock. Since those starting materials are also foods, they are expensive. As an alternative, cellulosic substances-the most abundant renewable resources on earth(1)-have long been considered for conversion to readily utilizable hydrolyzates.(2, 3)For the production of ethanol from cellulose, we have proposed the simultaneous saccharification and fermentation (SSF) process.(4) In SSF, enzymatic cellulose hydrolysis and glucose fermentation to ethanol by yeast proceed simultaneously within one vessel. The process advantages-reduced reactor volume and faster saccharification rates-have been confirmed by many researchers.(5-8) During SSF, the faster saccharification rates result because the glucose product is immediately removed, considerably diminishing its inhibitory effect on the cellulase system.(9)To effectively apply the SSF method to produce substances fermented from glucose, several conditions should be satisfied. One is coincident enzymatic hydrolysis and fermentation conditions, such as pH and temperature. The other is that cellulase inhibition by the final product is less than that by glucose and/or cellobiose. One of us has reported that acetic acid, citric acid, itaconic acid, alpha-ketoglutaric acid, lactic acid, and succinic acid scarcely inhibit cellulase.(10) This suggests that if the microorganisms which produce these organic acids were compatible with cellulase reaction conditions, the organic acids could be produced efficiently from cellulosic substrates by SSF.In this article, the successful application of SSF to lactic acid production from cellulose is reported. Though there have been several reports of direct cellulose conversion to organic acids by anaerobes such as Clostridium, only trace amounts of lactic acid were detected in the fermentation medium among the low-molecular-weight fatty acid components.(11-13) Lactic acid is one of the most important organic acids and has a wide range of food-related and industrial applications.     

Ingestion of Salmonella enterica serotype Poona by a free-living mematode,Caenorhabditis elegans,and protection against inactivation by produce sanitizers

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables. Caenorhabditis elegans was selected as a model to study the effectiveness of sanitizers in killing Salmonella enterica serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on S. enterica serotype Poona were treated with produce sanitizers. Treatment with 20 microg of free chlorine/ml significantly (alpha = 0.05) reduced the population of S. enterica serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of S. enterica serotype Poona cells surviving treatments with 20 to 500 microg of chlorine/ml, suggesting that reductions caused by treatment with 20 microg of chlorine/ml resulted from inactivation of S. enterica serotype Poona on the surface of C. elegans but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 microg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log(10) CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 microg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log(10) CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing S. enterica serotype Poona and lactic acid was the most effective. Treatment with up to 500 microg of chlorine/ml, 1% hydrogen peroxide, 2,550 microg of Sanova/ml, 40 microg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of C. elegans. Treatments were also applied to cantaloupe rind and lettuce inoculated with S. enterica serotype Poona or C. elegans that had ingested S. enterica serotype Poona. Protection of ingested S. enterica serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that S. enterica serotype Poona ingested by C. elegans may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.     

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.     

Cellulose Fermentation: Effect of Substrate Pretreatment on Microbial Growth

The effects of chemical, physical, and enzymatic treatments of rice straw and sugarcane bagasse on the microbial digestibility of cellulose have been investigated. Treatment with 4% NaOH for 15 min at 100 C increased the digestibility of cellulose from 29.4 to 73%. Treatment with 5.2% NH₃ could increase digestibility to 57.0% Treatments with sulfuric acid and crude cellulase preparation solubilized cellulose but did not increase the digestibility. Grinding or high-pressure cooking of the substrate had little effect on increasing the digestibility of cellulosic substrates by the Cellulomonas species.     

Qualitative and quantitative composition of acids formed by bifidobacteria

The dynamics of acid production in 18 strains of bifidobacteria, belonging to 5 different species, has been studied Bifidobacteria have been found to produce 3 acids, lactic, acetic and formic, in the process of their metabolism. The lactic acid/acetic acid quantitative ratio varies, depending on the culture medium on the average, 1:2 in Blaurock medium, 1:5 in milk hydrolysate medium. Various strains have also been found to differ in the dynamics of acid production with respect to the amounts of lactic and acetic acids. The study has shown that, despite the active production of acids for a period of up to 72 hours, a decrease in the pH of the medium is observed for not more than 48 hours. The existence of a specific mechanism permitting bifidobacteria to regulate the acidity of their environment is supposed.     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.     

Enzyme enhanced solid-state fermentation of kenaf core fiber for storage and pretreatment

Kenaf is an annual fiber crop adaptable to a wide range of climates and soil types. This study investigated the use of kenaf core fiber as a feedstock for enzyme-enhanced fermentation. Triplicate kenaf core fiber samples were treated with enzymes having cellulase:hemicellulase activity ratios of 0:1, 0.015:1, 0.45:1, and 2.54:1 at a rate of 5010 IU/kg dry matter hemicellulase activity, vacuum-sealed, and incubated at 37 degrees C for 21 d. Samples were analyzed for pH, water soluble carbohydrates, organic acids, and hemicellulose and cellulose concentrations. All treatments produced a pH less than 4.0, which is sufficient for stable storage. Treatments with 2.54:1 and 0.45:1 produced the highest water soluble carbohydrate and lactic acid concentrations. Enzymes with no or low cellulase activity produced results similar to the control. Utilizing enzyme mixtures with high cellulase activity is an effective pretreatment method for ensiled kenaf core fiber.     

Isolation and characterisation of cellulose obtained by a two-stage treatment with organosolv and cyanamide activated hydrogen peroxide from wheat straw

Seven wheat straw cellulose preparations were isolated by a two-stage acidic organosolv treatment followed by cyanamide activated hydrogen peroxide bleaching. The effects of concentration of acetic and formic acids on the yield of cellulose and degradation of lignin and non-cellulose polysaccharides were investigated. Organic acids were more effective than alcohols on the degradation of lignin and hemicelluloses. Formic acid/acetic acid/water (30/60/10, v/v/v) system was found to be the most effective in delignification and removal of non-cellulose polysaccharides from the straw and did not have any undesirable effects on cellulose properties such as its intrinsic viscosity. In this case, the treatment removed 94.1% of the original lignin and 76.5% of the original hemicelluloses using 0.1% HCl as a catalyst at 85 °C for 4 h. Cyanamide activated hydrogen peroxide bleaching degraded substantial amounts of residual hemicelluloses and lignin, produced the cellulose samples having a relatively high purity. Under a best condition, a cellulose relatively free of lignin (0.7%) and with intrinsic viscosity of 393 ml g⁻¹ and favourable molar mass (213,940 g mol⁻¹) was obtained. Both unbleached and bleached cellulose preparations were further characterised by FT-IR and CP/MAS ¹³C NMR spectroscopy, and thermal stability.     

Ingestion of Salmonella enterica Serotype Poona by a Free-Living Nematode, Caenorhabditis elegans, and Protection against Inactivation by Produce Sanitizers

Free-living nematodes are known to ingest food-borne pathogens and may serve as vectors to contaminate preharvest fruits and vegetables. Caenorhabditis elegans was selected as a model to study the effectiveness of sanitizers in killing Salmonella enterica serotype Poona ingested by free-living nematodes. Aqueous suspensions of adult worms that had fed on S. enterica serotype Poona were treated with produce sanitizers. Treatment with 20 μg of free chlorine/ml significantly (α = 0.05) reduced the population of S. enterica serotype Poona compared to results for treating worms with water (control). However, there was no significant difference in the number of S. enterica serotype Poona cells surviving treatments with 20 to 500 μg of chlorine/ml, suggesting that reductions caused by treatment with 20 μg of chlorine/ml resulted from inactivation of S. enterica serotype Poona on the surface of C. elegans but not cells protected by the worm cuticle after ingestion. Treatment with Sanova (850 or 1,200 μg/ml), an acidified sodium chlorite sanitizer, caused reductions of 5.74 and 6.34 log₁₀ CFU/worm, respectively, compared to reductions from treating worms with water. Treatment with 20 or 40 μg of Tsunami 200/ml, a peroxyacetic acid-based sanitizer, resulted in reductions of 4.83 and 5.34 log₁₀ CFU/worm, respectively, compared to numbers detected on or in worms treated with water. Among the organic acids evaluated at a concentration of 2%, acetic acid was the least effective in killing S. enterica serotype Poona and lactic acid was the most effective. Treatment with up to 500 μg of chlorine/ml, 1% hydrogen peroxide, 2,550 μg of Sanova/ml, 40 μg of Tsunami 200/ml, or 2% acetic, citric, or lactic acid had no effect on the viability or reproductive behavior of C. elegans. Treatments were also applied to cantaloupe rind and lettuce inoculated with S. enterica serotype Poona or C. elegans that had ingested S. enterica serotype Poona. Protection of ingested S. enterica serotype Poona against sanitizers applied to cantaloupe was not evident; however, ingestion afforded protection of the pathogen on lettuce. These results indicate that S. enterica serotype Poona ingested by C. elegans may be protected against treatment with chlorine and other sanitizers, although the basis for this protection remains unclear.     

Verotoxic Escherichia coli (STEC) from beef and its products

In the present investigation, out of 27 (24.10%) strains of Escherichia coli isolated from 112 beef samples comprising raw meat (45), kabab (36) and kofta (31), 9 (33.33%) belonging to 7 different serotypes were verotoxic as tested by vero cell cytotoxic assay. Serotype O145 was the predominant STEC in raw meat. Interestingly, one STEC-O157 strain was also detected. All the STEC strains were positive for Stx genes by polymerase chain reaction showing stx2 (77.78%) to be most predominant followed by stx1 (22.22%). Phenotypic enterohaemolysin production on washed sheep blood agar supplemented with CaCl2 revealed 6 (66.67%) STEC strains to be positive. Presence of STEC in cooked beef products, viz., kabab and kofta appeared to be a matter of concern and potential threat to public health.     

Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.     

Evaluations of cellulose accessibilities of lignocelluloses by solute exclusion and protein adsorption techniques

Cellulose accessibilities of a set of hornified lignocellulosic substrates derived by drying the never dried pretreated sample and a set of differently pretreated lodgepople pine substrates, were evaluated using solute exclusion and protein adsorption methods. Direct measurements of cellulase adsorption onto cellulose surface of the set of pretreated substrates were also carried out using an in situ UV-Vis spectrophotometric technique. The cellulose accessibilities measured by the solute exclusion and a cellulose-binding module (CBM)-containing green fluorescent protein (TGC) adsorption methods correlate well for both sets of samples. The substrate enzymatic digestibilities (SEDs) of the hornified substrates are proportional to the measured cellulose accessibilities. Approximately over 90% of the SED was contributed by the accessible pore surfaces of the hornified substrates, suggesting that the substrate external surface plays a minor role contributing to cellulose accessibility and SED. The cellulose accessibilities of the pretreated substrates correlated well with the amounts of cellulase adsorbed. The SEDs of these substrates directly correlated with the amounts of adsorbed cellulase.     

In vitro antagonistic activity evaluation of Lactic Acid Bacteria (LAB) combined with cellulase enzyme against campylobacter jejuni growth in co-culture

The antibacterial effects of nine lactic acid bacteria (LAB) against Campylobacter jejuni were investigated by using agar gel diffusion and co-culture assays. Some differences were recorded between the inhibition effects measured with these two methods. Only two LAB, Lb. pentosus CWBI B78 and E. faecium THT, exhibited a clear anti- Campylobacter activity in co-culture assay with dehydrated poultry excreta mixed with ground straw (DPE/GS) as the only growth substrate source. It was observed that the supplementation of such medium with a cellulase A complex (Beldem S.A.) enhanced the antimicrobial effect of both LAB strains. The co-culture medium acidification and the C. jejuni were positively correlated with the cellulase A concentration. The antibacterial effect was characterized by the lactic acid production from the homofermentative E. faecium THT and the lactic and acetic acids production from the heterofermentative Lb. pentosus CWBI B78. The antagonistic properties of LAB strains and enzyme combination could be used in strategies aiming at the reduction of Campylobacter prevalence in the poultry production chain and consequently the risk of human infection.     

Production of cellobiose by enzymatic hydrolysis: removal of beta-glucosidase from cellulase by affinity precipitation using chitosan

Removal of beta-glucosidase (BG) from cellulase is essential to the enzymatic production of cellobiose from cellulose because of the high reactivity of BG with cellobiose to form glucose. Chitosan is a reversibly soluble-insoluble polymer depending on pH, and it has an affinity with the other components, endo-beta-1,4-glucanase and cellobiohydrolase, or cellulase. The affinity precipitation technique using chitosan is an effective way to fractionate cellulase for the above purpose. Hydrolysis experiments of cellulose with the residual fractionated enzyme gave higher cellobiose contents in the soluble sugar products. (c) 1993 John Wiley & Sons, Inc.     

Isolation of cellulose—producing microbes from the intestine of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

The cellulase activities of bacterial strains in the intestine of grass carp were analyzed, using filter paper and absorbent cotton as substrates and measuring the concentration of glucose by calorimetry. Six strains were isolated and determined high cellulase activity in all grass carp. Strains showed different abilities to produce cellulase, which suggests that they interact in the grass carp intestine to digest cellulose. The presence of cellulose activity suggests that grass carp have the ability to digest cellulose in the diet. The cellulase enzymatic activity increased dramatically after 6 days of culture and reached its peak at the 7th day. Microbes are probably the main source of cellulase in grass carp diets.     

Fractionating recalcitrant lignocellulose at modest reaction conditions

Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose biorefineries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (50 degrees C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ( approximately 97% in 24 h) during the hydrolysis step at the enzyme loading of 15 filter paper units of cellulase and 60 IU of beta-glucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorefinery.     

H2O2 feeding enables LPMO-assisted cellulose saccharification during simultaneous fermentative production of lactic acid

Simultaneous saccharification and fermentation (SSF) is a well-known strategy for valorization of lignocellulosic biomass. Because the fermentation process typically is anaerobic, oxidative enzymes found in modern commercial cellulase cocktails, such as lytic polysaccharide monooxygenases (LPMOs), may be inhibited, limiting the overall efficiency of the enzymatic saccharification. Recent discoveries, however, have shown that LPMOs are active under anoxic conditions if they are provided with H₂O₂ at low concentrations. In this study, we build on this concept and investigate the potential of using externally added H₂O₂ to sustain oxidative cellulose depolymerization by LPMOs during an SSF process for lactic acid production. The results of bioreactor experiments with 100 g/L cellulose clearly show that continuous addition of small amounts of H₂O₂ (at a rate of 80 µM/h) during SSF enables LPMO activity and improves lactic acid production. While further process optimization is needed, the present proof-of-concept results show that modern LPMO-containing cellulase cocktails such as Cellic CTec2 can be used in SSF setups, without sacrificing the LPMO activity in these cocktails.     

Stimulation of lactic acid production in chick embryo fibroblasts by serum and high pH in the absence of external glucose

Lactic acid production by chick embryo fibroblasts occurs in the absence of exogenous glucose. Fifteen to 50-fold less lactic acid is formed in the absence of glucose than in its presence. Nevertheless, serum and pH stimulation enhances this residual lactic acid production to the same relative extent as when glucose is present. The amount of lactic acid formed cannot be accounted for by the catabolism of residual glucose in the medium since its concentration is less than one-tenth that of the lactic acid eventually produced. Moreover, the residual glucose concentration remains constant or increases during the course of the experiment. To a large extent lactic acid accumulation in the absence of external glucose is dependent on the presence of amino acids in the medium, but amino acid transport is not affected by the stimulatory agents used in this study. The results suggest that treatments which stimulate cell multiplication also activate those enzymatic pathways which convert amino acids to pyruvic and thence to lactic acid.     

Sensitivity of Shiga toxin-producing Escherichia coli (STEC) strains for colicins under different experimental conditions

Twenty Escherichia coli strains producing well-characterised colicins were tested for their inhibitory activity against five Shiga toxin-producing E. coli (STEC) strains using different media under aerobic and anaerobic conditions. The five STEC strains used were of serotype O26, O111, O128, O145 and O157:H7 which are frequently isolated serotypes associated with disease in humans. The main route of infection for humans is through the eating of badly cooked or handled beef. The major reservoir for STEC strains in cattle is the rumen. To mimic the situation in the rumen of cattle, overlay assays were also performed under anaerobic conditions in the presence of 30% rumen fluid. Colicins E1, E4, E8-J, K and S4 are most active against STEC strains under anaerobic conditions in the absence or presence of rumen fluid. These colicins will be used in future experiments with the aim to eradicate the presence of STEC in cattle.     

Evaluation of chemical pretreatment for enzymatic solubilization of rice straw

Summary Rice straw was treated with NaOH, peracetic acid (PA), and sodium chlorite (NaClO₂). Quantitative changes in the composition of the treated straw, crystallinity of the treated straw and extracted cellulose, and susceptibility of the treated straw to Trichoderma reesei cellulase were studied. The alkali treatment resulted in a remarkable decrease in hemicellulose as well as lignin. Consequently, the recovery of residual straw after NaOH treatment was lowest among the three chemical reagents evaluated. The treatment with PA or NaCIO₂ resulted in a slight loss in hemicellulose and cellulose in the straw. The three chemical treatments caused little or no breakdown of the crystalline structure of cellulose in the straw. The treated straw was solubilized with the culture filtrate of T. reesei. The degree of enzymatic solubilization relative to the amount of residual straw was 69% after treatment with 0.25 N NaOH, 42% after treatment with 20% PA, and 50% after treatments with NaClO₂ (twice). The degree of enzymatic solubilization relative to the amount of the untreated straw, however, was 30% after treatment with 0.25 N NaOH, 32% after treatment with 20% PA, and 37% after treatments with NaClO₂ (twice).