Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol

The effects of d-limonene concentration, enzyme loading, and pH on ethanol production from simultaneous saccharification and fermentation (SSF) of citrus peel waste by Saccharomyces cerevisiae were studied at 37 °C. Prior to SSF, citrus peel waste underwent a steam explosion process to remove more than 90% of the initial d-limonene present in the peel waste. d-Limonene is known to inhibit yeast growth and experiments were performed where d-limonene was added back to peel to determine threshold inhibition amounts. Ethanol concentrations after 24 h were reduced in fermentations with initial d-limonene concentrations greater than or equal to 0.33% (v/v) and final (24 h) d-limonene concentrations greater than or equal to 0.14% (v/v). Ethanol production was reduced when enzyme loadings were (IU or FPU/g peel dry solids) less than 25, pectinase; 0.02, cellulase; and 13, beta-glucosidase. Ethanol production was greatest when the initial pH of the peel waste was adjusted to 6.0.     

Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

The degradation and utilization of wheat-straw cell-wall monosaccharide components by defined ruminal cellulolytic bacteria

Cell walls (CW) of untreated wheat straw and sulphur-dioxide (SO₂)-treated wheat straw were used as model substrates for the hydrolysis and utilization of CW carbohydrates by pure cultures or pair-combinations of defined rumen bacterial strains. Fibrobacter succinogenes S85 and BL2 strains and their co-cultures with D1 were the best degraders of CW among ruminal cultures, solubilizing 37.2–39.6% of CW carbohydrates of untreated straw and 62.2–74.5% of SO₂-treated straw. Complementary action between Butyrivibrio fibrisolvens D1 and the F. succinogenes strains was identified with respect to co-culture growth and carbohydrate utilization. However, the extent of CW solubilization was determined mainly by the F. succinogenes strains. In both substrates, utilization of solubilized cellulose by F. succinogenes S85 and BL2 monocultures was higher than that of xylan and hemicellulose: 96.5–98.3%, 34.4–40.5% and 33.5–36.2%, respectively. Under scanning electron microscopy visualization, S85 and BL2 cells of the co-cultures comprised the most dense layer of bacterial cell mass attached to and colonized on straw stems and leaves, whereas D1 cells were always nearby. Stems and leaves of the untreated straw were less crowded by attached bacteria than that of the SO₂-treated straw. In both materials, the cell surface topography of S85 and BL2 bacteria attached to CW particles was specified by a coat of characteristic protuberant structures, polycellulosome complexes.     

Kinetic evaluation of products inhibition to succinic acid producers <Emphasis Type="Italic">Escherichia coli</Emphasis> NZN111, AFP111, BL21, and <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> 130Z<Superscript>T</Superscript>

Succinic acid is one of the platform compounds and its production via natural feedstocks has drawn worldwide concerns. To evaluate the inhibitory effects of fermentation products on the growth of Actinobacillus succinogenes 130Z^T and Escherichia coli NZN111, AFP111, BL21, fermentations with addition of individual products in medium were carried out. The cell growth was inhibited when the concentrations of formate, acetate, lactate, and succinate were at range of 8.8–17.6 g/L, 10–40 g/L, 9–18 g/L, and 10–80 g/L, respectively. For these two species of bacteria, E. coli was more resistant to acid products than A. succinogenes, while both endured succinate rather than by-products. As a result of end product inhibition, succinate production yield by A. succinogenes decreased from 1.11 to 0.49 g/g glucose. Logistic and Monod mathematical models were presented to simulate the inhibition kinetics. The Logistic model was found more suitable for describing the overall synergistic inhibitory effects.     

Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

Consolidated Bioprocessing of Lignocellulosic Feedstocks for Ethanol Fuel Production

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.     

Pentose metabolism byBacteroides ruminicola subsp.brevis strain B14

The fermentation ofd-arabinose byBacteroides ruminicola strain B₁4 occurs in a manner similar to or identical with that shown previously forl-arabinose metabolism by the organism, a combination of hexose resynthesis and the Embden-Meyerhof sequence. The use ofd-arabinose by strain B₁4 was repressed by prior growth in medium containingd-glucose and induced by prior growth in the presence ofl-arabinose ord-xylose. The use ofd-ribose andd-xylose by strain B₁4 is different from that ford-arabinose. During growth in the presence of 1-14C-d-arabinose, labeled acetate, propionate, and succinate were formed, whereas during 1-14C-d-ribose growth only labeled acetate and propionate were obtained. Under the conditions used,d-xylose growth failed to allow formation of acetate, propionate, or succinate. Strain B₁4 incorporates label from 1- or 2-labeled glycine into acetate, propionate, and succinate by a mechanism involving the cleavage of glycine and equilibration of glycine carbons 1 and 2 with different metabolic pools.     

Further studies on Egyptian soil fungi: Succession of sugar and osmophilic fungi in soil amended with five organic substrates

The sugar and osmophilic fungal composition of soils amended with five organic substrates (newspaper, orange peel, bromegrass leaves, wheat straw and wood sawdust) was estimated after 2, 4, 6, 8 and 10 weeks using the dilution plate method on glucose and 50% sucrose Czapek's agar media. Wheat straw was the best substrate for total counts of both sugar and osmophilic fungi followed by newspaper, bromegrass leaves, wood sawdust and orange peel. Wood sawdust supported the highest average counts of total sugar fungi, Fusarium, Mucor, Scopulariopsis, Trichoderma and Trimmatostroma spp.; Newspaper, of Aspergillus (8 spp.), Penicillium (4 spp.) and Chaetomium sp. bromegrass leaves of Cladosporium sp., Humicola sp. and Sporotrichum sp.; orange peel, ofAlternaria sp., Circinella sp. and Stachybotrys sp.; and wheat straw, of Botryotrichum sp. and Myrothecium sp. Bromegrass leaves and orange peel supported the highest average counts of total osmophilic fungi, Aspergillus (10 spp.), Cladosporium sp. Paecillomyces sp. and Rhizopus sp.; and of Stemphylium sp., Trichoderma sp., Humicola sp. and Circinella sp. respectively; wheat straw, of Epicoccum sp., Scopulariopsis sp. and Trichothecium sp.; newspaper, of Penicillium (4 spp.) and Alternaria sp.; and wood sawdust of Curvularia sp. and Fusarium (3 spp.). The best colonizers throughout the experimental periods were Aspergilus and Penicillium spp.     

Particulate bioprocessing: A novel process strategy for biorefineries

A novel process strategy based on particulate bioprocessing has been developed for the production of value-added chemicals and biofuels. The process, which involves two main steps, fungal fermentation and discontinuous extraction, leads to the production of generic fermentation feedstocks from cereals. Partially pearled whole wheat grains were used as substrate for the growth of Aspergillus awamori in a packed bed bioreactor. Water was trickled through the bed of particles intermittently every 6 h to extract glucose and other nutrients and to maintain moisture and temperature levels. The feedstocks obtained through this system have been used for subsequent fermentations by Wautersia eutropha to produce the biodegradable plastic PHB (polyhydroxybutyrate) and by Saccharomyces cerevisiae for ethanol production. These preliminary results demonstrate the potential suitability of the novel concept of particulate bioprocessing in the development of biorefineries.     

Effects of fumarate,l-malate,and anAspergillus oryzae fermentation extract ond-lactate Utilization by the ruminal bacteriumSelenomonas ruminantium

Growth ofSelenomonas ruminantium HD4 in medium that contained 21mm d-lactate was stimulated to varying degrees by 10mm l-malate, 10mm fumarate, and 2% (v/v)Aspergillus oryzae fermentation extract (Amaferm). Amaferm treatment caused the greatest growth stimulation. Initial uptake rates (30s) and long-term uptake rates (30 min) ofd-lactate by whole cells ofS. ruminantium were increased in the presence of 10mm l-malate. Amaferm (25 l/ml) also stimulated long-term uptake rates ofd-lactate, whereas fumarate had no effect. Initial uptake ofd-lactate was depressed in the presence of fumarate or Amaferm. When eitherl-malate, fumarate, or Amaferm was included in thed-lactate growth medium, a homosuccinate fermentation resulted and an inverse relationship was observed between growth (protein synthesis) and succinate production. Recent research demonstrated that Amaferm containsl-malate, and this dicarboxylic acid may be involved in stimulatingd-lactate utilization byS. ruminantium.     

Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO₂-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates. Received: 2 September 1996 / Accepted: 10 January 1997     

Protective effect of Premna tomentosa extract (L. verbanacae) on acetaminophen-induced mitochondrial dysfunction in rats

Allurement of herbs as health beneficial foods (physiologically functional foods) and as a source material for the development of new drugs, has led to greater furtherance in the study of herbal medicines during recent years. Plant extracts are being utilized to treat a wide variety of diseases like hepatotoxicity. Premna tomentosa is one such medicinal plant used widely in Indian ayurvedic medicine for the treatment of liver disorders. This study appraised the effectiveness of P. tomentosa leaf extract in protecting the liver against mitochondrial damage induced by acetaminophen, since mitochondrial injury has been investigated as a potential initiator of hepatotoxicity. Normal Wistar strain rats were pre-treated with P. tomentosa extract (750 mg/kg, orally) for 15 days and then intoxicated with acetaminophen (640 mg/kg, orally). Mitochondria were isolated from liver of experimental animals and assessed for the levels of lipid peroxide products, GSH and mitochondrial enzymes (isocitrate dehydrogenase, -keto glutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase and cytochrome-C-oxidase). The levels of Lipid peroxidation products were increased and the levels of the other assessed parameters were significantly decreased in hepatotoxicity induced animals. Whereas, the levels were brought back to normal in P. tomentosa pre-treated rats, which shows the protective effect of the extract against mitochondrial damage. Presence of anti-oxidant compound d-limonene (58%) in P. tomentosa leaves, which is known to enhance conjugation of toxic metabolites by maintaining liver GSH concentrations may explain the hepatoprotective property of the extract.     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

d-Pentose metabolism byBacteroides vulgatus strain 8482

Bacteroides vulgatus strain 8482 metabolizedd-arabinose by a mechanism involving a 32 (top to bottom) cleavage of the arabinose carbon skeleton. During growth in the presence of 1-¹⁴C-d-arabinose, acetate, propionate, and succinate were labeled, but during growth in the presence of 5-labeledd-arabinose, only labeled acetate and succinate were formed. The metabolism ofd-ribose by strain 8482 differed from that ford-arabinose. Strain 8482 converted glycolic acid and glycine to acetate and succinate, but not propionate, by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons and equilibration of carbons 1 and 2 of glycolic acid and glycine with nonequivalent metabolic pools. The metabolism ofd-arabinose,d-ribose,d-glycine, andd-glycolic acid by strain 8482 was similar, in some respects, to that ofBacteroides fragilis strain 2044, but differed substantially from the metabolism of the same substances byBacteroides ruminicola strain B₁4.     

Propionate Formation from Cellulose and Soluble Sugars by Combined Cultures of Bacteroides succinogenes and Selenomonas ruminantium

Succinate is formed as an intermediate but not as a normal end product of the bovine rumen fermentation. However, numerous rumen bacteria are present, e.g., Bacteroides succinogenes, which produce succinate as a major product of carbohydrate fermentation. Selenomonas ruminantium, another rumen species, produces propionate via the succinate or randomizing pathway. These two organisms were co-cultured to determine if S. ruminantium could decarboxylate succinate produced by B. succinogenes. When energy sources used competitively by both species, i.e. glucose or cellobiose, were employed, no succinate was found in combined cultures, although a significant amount was expected from the numbers of Bacteroides present. The propionate production per S. ruminantium was significantly greater in combined than in single S. ruminantium cultures, which indicated that S. ruminantium was decarboxylating the succinate produced by B. succinogenes. S. ruminantium, which does not use cellulose, grew on cellulose when co-cultured with B. succinogenes. Succinate, but not propionate, was produced from cellulose by B. succinogenes alone. Propionate, but no succinate, accumulated when the combined cultures were grown on cellulose. These interspecies interactions are models for the rumen ecosystem interactions involved in the production of succinate by one species and its decarboxylation to propionate by a second species.     

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

Degradation of Wheat Straw by Fibrobacter succinogenes S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with ¹³C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of ¹³C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.