Impact of chemical components of organic wastes on L(+)-lactic acid production

Lactic acid production from several organic wastes that had different chemical compositions was examined, and the factors strongly impacting yield were determined. The bioconversion of sugars to lactic acid was affected by the ratio of total sugars to total nitrogen content (the TS/N ratio), and was improved by nitrogen supplementation to adjust the TS/N ratio > or =10. Lactic acid yield was also affected by the fermentable sugars contents, i.e. various oligosaccharides constituted of mainly C6-sugars. The estimation of the fermentable sugars was determined by the total sugars content in starchy materials, such as kitchen wastes, but in lignocellulosic materials, the estimation was affected by the hemicellulose contents. The estimation model of the fermentable sugars was proposed by multivariate analysis using organic components as variables.     

Variation of proteolytic activity in ewe during in vivo digestion of two pea-based diets differing by their fermentability characteristics.

In order to study the effects of a small difference in starch and nitrogen availability on proteolysis, two different diets were supplied to four ewes fitted with rumen fistulae. They differed in the ratio of fermentable nitrogen over fermentable energy. with 144 g of fermentable nitrogen (FN) per kg of fermentable energy (FE) for diet I and 126 g FN x kg(-1) FE for diet II. The diets were constituted of 700 g hay grass, 200 g ground pea and either 100 g ground wheat (diet I) or 100 g corn starch (diet II). After two weeks of an adapting period to the diets, rumen content was sampled after feeding over time. The rate of disappearance of soluble proteins was 2.5 times higher with diet II and ammonia concentrations were significantly lower (from -28 to -43%) with diet II. Total proteolytic activity, by considering all the bacterial compartments, was significantly higher with diet II (+40 EU/mL x h(-1)): changes in the total proteolytic activity in the particulate and the liquid phases of the rumen could explain the difference observed between the two diets. Moreover, with diet II, exopeptidase activities increased more in the liquid phase, especially leucine aminopeptidase and Dipeptidyl peptidase I (DPP-I), and the diversity of endopeptidase activities increased in the particulate phase. These two facts could account for the higher total proteolytic activity in the rumen content with diet II.     

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.     

Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Optimization of processing conditions for the pretreatment of wheat straw using aqueous ionic liquid

Pretreatment of wheat straw with the aqueous ionic liquid, 1-ethyl-3-methylimidazolium acetate, was optimized to maximize fermentable sugars recovery. The optimization process employed a central composite design, where the investigated variables were temperature (130-170 °C), time (0.5-5.5 h) and ionic liquid concentration (0-100%). All the tested variables were identified to have significant effects (p < 0.05) on fermentable sugars recovery. The optimum pretreatment conditions were 158 °C, an ionic liquid concentration of 49.5% (w/w), and a duration of 3.6 h. Cellulose and xylan digestibility generally increased with increasing temperature, time and ionic liquid concentration; but, the carbohydrates recovered in the washed solids following pretreatment decreased. Thus, the final optimum conditions for maximizing fermentable sugars from the starting biomass were a compromise between greater digestibility and minimal carbohydrates loss during pretreatment.     

Designing the deconstruction of plant cell walls

Cell wall architecture plays a key role in the regulation of plant cell growth and differentiation into specific cell types. Gaining genetic control of the amount, composition, and structure of cell walls in different cell types will impact both the quantity and yield of fermentable sugars from biomass for biofuels production. The recalcitrance of plant biomass to degradation is a function of how polymers crosslink and aggregate within walls. Novel imaging technologies provide an opportunity to probe these higher order structures in their native state. If cell walls are to be efficiently deconstructed enzymatically to release fermentable sugars, then we require a detailed understanding of their structural organization in future bioenergy crops.     

Levels of coenzyme A--glutathione mixed disulfide in Escherichia coli.

The pool of coenzyme A--glutathione mixed disulfide (CoASSG) rapidly increased 2.0 times in response to oxygen starvation and 1.5 times in response to glucose starvation but did not change following ammonia starvation. The increase in the CoASSG pool resulted from an increase in the CoASSG fraction of the CoA pool from 42 to 66--93%. Fluoride, cyanide, chloramphenicol, and rifampicin all caused similar increases. Aerobic growth on fermentable sugars resulted in CoASSG making up 40--55% of the CoA pool while growth on nonfermentable carbon sources or anaerobic fermentation resulted in CoASSG replacing acetyl CoA and free CoA to make up 85--95% of the CoA pool. The CoASSG:ATP ratio varied inversely with the growth rate in two groupings of carbon sources made up of either fermentable or nonfermentable molecules. Cultures grown aerobically on fermentable sugars exhibited a lower CoASSG:ATP ratio reflecting the lower proportion of CoASSG in the CoA pool.     

Efficient conversion of sugarcane stalks into ethanol employing low temperature alkali pretreatment method

An alternative route for bio-ethanol production from sugarcane stalks (juice and bagasse) featuring a previously reported low temperature alkali pretreatment method was evaluated. Test-tube scale pretreatment-saccharification experiments were carried out to determine optimal LTA pretreatment conditions for sugarcane bagasse with regard to the efficiency of enzymatic hydrolysis of the cellulose. Free fermentable sugars and bagasse recovered from 2 kg of sugarcane stalks were jointly converted into ethanol via separate enzymatic hydrolysis and fermentation (SHF). Results showed that 98% of the cellulose present in the optimally pretreated bagasse was hydrolyzed into glucose after 72-h enzymatic saccharification using commercially available cellulase and β-glucosidase preparations at relatively low enzyme loading. The fermentable sugars in the mixture of the sugar juice and the bagasse hydrolysate were readily converted into 193.5 mL of ethanol by Saccharomyces cerevisiae within 12h, achieving 88% of the theoretical yield from the sugars and cellulose.     

Effective production of fermentable sugars from brown macroalgae biomass

Brown macroalgae are renewable and sustainable biomass resources for the production of biofuels and chemicals, owing to their high levels of carbohydrates and low levels of lignin. To increase the biological usage of brown macroalgae, it is necessary to depolymerize the polysaccharides that generate macroalgal monomeric sugars or sugar derivatives and to convert them into fermentable sugars for the production of biofuels and chemicals. In this review, we discuss the chemical and enzymatic saccharification of the major carbohydrates found in brown macroalgae and the use of the resulting constituents in the production of biofuels and chemicals, as well as high-value health-benefiting functional oligosaccharides and sugars. We also discuss recently reported experimental results, novel enzymes, and technological breakthroughs that are related to polysaccharide depolymerization, fermentable sugar production, and the biological conversion of non-favorable sugars for fermentation using industrial microorganisms. This review provides a comprehensive perspective of the efficient utilization of brown macroalgae as renewable resources for the production of biofuels and chemicals.     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Novel renewable ionic liquids as highly effective solvents for pretreatment of rice straw biomass by selective removal of lignin

Cholinium amino acids ionic liquids ([Ch][AA] ILs), a novel type of bio‐ILs that can easily be prepared from renewable biomaterials, were investigated for pretreatment of rice straw by selective extraction of lignin from this abundant lignocellulosic biomass material. Of the eight ILs examined, most were demonstrated to be excellent pretreatment solvents. Upon pretreatment using these ILs, the initial saccharification rates of rice straw residues were substantially improved as well as the extent to which polysaccharides could be digested (>90% for cellulose and >60% for xylan). Enzymatic hydrolysis of pretreated rice straw by Trichoderma reesei cellulase/xylanase furnished glucose and xylose with the yields in excess of 80% and 30%, respectively. Detailed spectroscopic characterization showed that the enhancement of polysaccharides degestibility derived mainly from delignification rather than changes in cellulose crystallinity. The yields of fermentable reducing sugars were significantly improved after individual optimization of pretreatment temperature and duration. With [Ch][Lys] as the solvent, the sugar yields of 84.0% for glucose and 42.1% for xylose were achieved after pretreatment at 90°C for 5 h. The IL [Ch][Lys] showed excellent reusability across five successive batches in pretreatment of rice straw. These bio‐ILs performed as well as or better than previously investigated non‐renewable ILs, and thus present a new and environmentally friendly way to pretreat lignocellulose for production of fermentable sugars and total utilization of the biomass. Biotechnol. Bioeng. 2012; 109: 2484–2493. © 2012 Wiley Periodicals, Inc.     

Corecovery of lipids and fermentable sugars from Rhodosporidium toruloides using ionic liquid cosolvents: Application of recycle to batch fermentation

This work evaluates the ability of an ionic liquid‐methanol cosolvent system to extract lipids and recycle fermentable sugars recovered from oil‐bearing Rhodosporidium toruloides grown in batch culture on defined media using glucose and xylose as carbon sources. Growth on the recycled mixed carbon substrate was successful with glucose consumed before xylose and overall cell mass to lipid yields (Y_P/X) between 57% and 61% (w/w relative to whole dried cell mass) achieved. Enzymatic hydrolysis of the delipified carbohydrate fraction recovered approximately 9%–11% (w/w) of the whole dried cell mass as fermentable sugars, which were successfully recycled as carbon sources without further purification. In total, up to 70% (w/w) of the whole dried cell mass was recovered as lipids and fermentable sugars and the substrate to lipid yields (Y_P/S) was increased from 0.12 to 0.16 g lipid/g carbohydrate consumed, highlighting the promise of this approach to process lipid bearing cell biomass. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1239–1242, 2014     

Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass

Room temperature ionic liquids (RTILs) are emerging as attractive and green solvents for lignocellulosic biomass pretreatment. The unique solvating properties of RTILs foster the disruption of the 3D network structure of lignin, cellulose, and hemicellulose, which allows high yields of fermentable sugars to be produced in subsequent enzymatic hydrolysis. In the current review, we summarize the physicochemical properties of RTILs that make them effective solvents for lignocellulose pretreatment including mechanisms of interaction between lignocellulosic biomass subcomponents and RTILs. We also highlight several recent strategies that exploit RTILs and generate high yields of fermentable sugars suitable for downstream biofuel production, and address new opportunities for use of lignocellulosic components, including lignin. Finally, we address some of the challenges that remain before large-scale use of RTILs may be achieved.     

Biochemical analyses of multiple endoxylanases from the rumen bacterium Ruminococcus albus 8 and their synergistic activities with accessory hemicellulose-degrading enzymes

Ruminococcus albus 8 is a ruminal bacterium capable of metabolizing hemicellulose and cellulose, the major components of the plant cell wall. The enzymes that allow this bacterium to capture energy from the two polysaccharides, therefore, have potential application in plant cell wall depolymerization, a process critical to biofuel production. For this purpose, a partial genome sequence of R. albus 8 was generated. The genomic data depicted a bacterium endowed with multiple forms of plant cell wall-degrading enzymes. The endoxylanases of R. albus 8 exhibited diverse modular architectures, including incorporation of a catalytic module, a carbohydrate binding module, and a carbohydrate esterase module in a single polypeptide. The accessory enzymes of xylan degradation were a β-xylosidase, an α-l-arabinofuranosidase, and an α-glucuronidase. We hypothesized that due to the chemical complexity of the hemicellulose encountered in the rumen, the bacterium uses multiple endoxylanases, with subtle differences in substrate specificities, to attack the substrate, while the accessory enzymes hydrolyze the products to simple sugars for metabolism. To test this hypothesis, the genes encoding the predicted endoxylanases were expressed, and the proteins were biochemically characterized either alone or in combination with accessory enzymes. The different endoxylanase families exhibited different patterns of product release, with the family 11 endoxylanases releasing more products in synergy with the accessory enzymes from the more complex substrates. Aside from the insights into hemicellulose degradation by R. albus 8, this report should enhance our knowledge on designing effective enzyme cocktails for release of fermentable sugars in the biofuel industry.     

Microbiological production of enzymes and their industrial applications

Bacteria and other fungi are industrially cultivated in a variety of ways for the commercial production of some 25 enzymes utilized in many industries ranging from the conversion of starch to fermentable sugars, through chill-proofing of beer to bating of hides.     

Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae

The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since inactivation did not occur during carbon starvation, when a fermentable sugar was added to starved cells, inactivation began, when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.     

Anaerobic fermentation: Microbes from ruminants

Fed-batch fermentation of biomass could provide a route for direct conversion of renewable resources to commercially significant chemicals. The ecosystem in the forestomach (rumen) of ruminants provides a highly reduced environment (oxidation-reduction potential of ?250 to ?450 mV) in which anaerobic bacteria directly utilize cellulose, hemicellulose, and other fermentable biomass constituents to produce acetate, butyrate, propionate, methane and carbon dioxide at pH 5.7 to 7.3. The cellulose fermentation in the rumen is impacted by the physically and chemically heterogeneous character of the insoluble substrate, as well as the properties of the mixed culture responsible for fibre hydrolysis and carbohydrate utilization. The rumen system provides an interesting case study in the context of possible process concepts for direct fermentation of biomass to commercially important chemicals such as acetate, propionate, succinate, lactate and ethanol. The role of the chemical and physical characteristics of the substrate, the microbes in the rumen system and the metabolic pathways of soluble carbohydrates are discussed in the context of cellulose and hemicellulose fermentation.     

Commercial cellulosic ethanol: The role of plant-expressed enzymes

The use and production of biofuels has risen dramatically in recent yr. Bioethanol comprises 85% of total global biofuels production, with benefits including reduction of greenhouse gas emissions and promotion of energy independence and rural economic development. Ethanol is primarily made from corn grain in the USA and sugarcane juice in Brazil. However, ethanol production using current technologies will ultimately be limited by land availability, government policy, and alternative uses for these agricultural products. Biomass feedstocks are an enormous and renewable source of fermentable sugars that could potentially provide a significant proportion of transport fuels globally. A major technical challenge in making cellulosic ethanol economically viable is the need to lower the costs of the enzymes needed to convert biomass to fermentable sugars. The expression of cellulases and hemicellulases in crop plants and their integration with existing ethanol production systems are key technologies under development that will significantly improve the process economics of cellulosic ethanol production.     

Alpha-L-arabinofuranosidases: biochemistry, molecular biology and application in biotechnology

Interest in the alpha-L-arabinofuranosidases has increased in recent years because of their application in the conversion of various hemicellulosic substrates to fermentable sugars for subsequent production of fuel alcohol. Xylanases, in conjunction with alpha-L-arabinofuranosidases and other accessory enzymes, act synergistically to degrade xylan to component sugars. The induction of alpha-L-arabinofuranosidase production, physico-chemical characteristics, substrate specificity, and molecular biology of the enzyme are described. The current state of research and development of the arabinofuranosidases and their role in biotechnology are presented.