Hemicellulase Activities Research Articles

Construction of consolidated bio-saccharification biocatalyst and process optimization for highly efficient lignocellulose solubilization

BackgroundThe industrial conversion of biomass to high-value biofuels and biochemical is mainly restricted by lignocellulose solubilization. Consolidated bio-saccharification (CBS) is considered a promising process for lignocellulose solubilization depending on whole-cell biocatalysts that simultaneously perform effective cellulase production and hydrolysis. However, it usually takes a long time to reach a high saccharification level using the current CBS biocatalyst and process.ResultsTo promote the saccharification efficiency and reduce the cost, a Clostridium thermocellum recombinant strain ∆pyrF::KBm was constructed as a new CBS biocatalyst in this study. The key CBS factors, including the medium, inoculum size and cultivation, and substrate load, were investigated and optimized. The saccharification process was also stimulated by adding free hemicellulases, suggesting the need to further enhance hemicellulase activity of the whole-cell catalyst. Under the optimal conditions, the CBS process was shortened by 50% with pretreated wheat straw as the substrate. The sugar yield reached 0.795 g/g and the saccharification level was 89.3%.ConclusionsThis work provided a new biocatalyst and an optimized process of CBS and confirmed that CBS is a feasible strategy for cost-efficient solubilization of lignocellulose, which will greatly promote the industrial utilization of lignocellulosic biomass.

Cellulase−Hemicellulase Activities and Bacterial Community Composition of Different Soils from Algerian Ecosystems

Soil microorganisms are important mediators of carbon cycling in nature. Although cellulose- and hemicellulose-degrading bacteria have been isolated from Algerian ecosystems, the information on the composition of soil bacterial communities and thus the potential of their members to decompose plant residues is still limited. The objective of the present study was to describe and compare the bacterial community composition in Algerian soils (crop, forest, garden, and desert) and the activity of cellulose- and hemicellulose-degrading enzymes. Bacterial communities were characterized by high-throughput 16S amplicon sequencing followed by the in silico prediction of their functional potential. The highest lignocellulolytic activity was recorded in forest and garden soils whereas activities in the agricultural and desert soils were typically low. The bacterial phyla Proteobacteria (in particular classes α-proteobacteria, δ-proteobacteria, and γ-proteobacteria), Firmicutes, and Actinobacteria dominated in all soils. Forest and garden soils exhibited higher diversity than agricultural and desert soils. Endocellulase activity was elevated in forest and garden soils. In silico analysis predicted higher share of genes assigned to general metabolism in forest and garden soils compared with agricultural and arid soils, particularly in carbohydrate metabolism. The highest potential of lignocellulose decomposition was predicted for forest soils, which is in agreement with the highest activity of corresponding enzymes.

Metagenomic Mining and Functional Characterization of a Novel KG51 Bifunctional Cellulase/Hemicellulase from Black Goat Rumen.

A novel KG51 gene was isolated from a metagenomic library of Korean black goat rumen and its recombinant protein was characterized as a bifunctional enzyme (cellulase/hemicellulase). In silico sequence and domain analyses revealed that the KG51 gene encodes a novel carbohydrate-active enzyme that possesses a salad-bowl-like shaped glycosyl hydrolase family 5 (GH5) catalytic domain but, at best, 41% sequence identity with other homologous GH5 proteins. Enzymatic profiles (optimum pH values and temperatures, as well as pH and thermal stabilities) of the recombinant KG51 bifunctional enzyme were also determined. On the basis of the substrate specificity data, the KG51 enzyme exhibited relatively strong cellulase (endo-β-1,4-glucanase [EC 3.2.1.4]) and hemicellulase (mannan endo-β-1,4-mannosidase [EC 3.2.1.78] and endo-β-1,4-xylanase [EC 3.2.1.8]) activities, but no exo-β-1,4-glucanase (EC 3.2.1.74), exo-β-1,4-glucan cellobiohydrolase (EC 3.2.1.91), and exo-1,4-β-xylosidase (EC 3.2.1.37) activities. Finally, the potential industrial applicability of the KG51 enzyme was tested in the preparation of prebiotic konjac glucomannan hydrolysates.

Correction to: 'The effect of alkali-soluble lignin on purified core cellulase and hemicellulase activities during hydrolysis of extractive ammonia-pretreated lignocellulosic biomass'.

[This corrects the article DOI: 10.1098/rsos.171529.].

The effect of alkali-soluble lignin on purified core cellulase and hemicellulase activities during hydrolysis of extractive ammonia-pretreated lignocellulosic biomass.

Removing alkali-soluble lignin using extractive ammonia (EA) pretreatment of corn stover (CS) is known to improve biomass conversion efficiency during enzymatic hydrolysis. In this study, we investigated the effect of alkali-soluble lignin on six purified core glycosyl hydrolases and their enzyme synergies, adopting 31 enzyme combinations derived by a five-component simplex centroid model, during EA-CS hydrolysis. Hydrolysis experiment was carried out using EA-CS(−) (approx. 40% lignin removed during EA pretreatment) and EA-CS(+) (where no lignin was extracted). Enzymatic hydrolysis experiments were done at three different enzyme mass loadings (7.5, 15 and 30 mg protein g−1 glucan), using a previously developed high-throughput microplate-based protocol, and the sugar yields of glucose and xylose were detected. The optimal enzyme combinations (based on % protein mass loading) of six core glycosyl hydrolases for EA-CS(−) and EA-CS(+) were determined that gave high sugar conversion. The inhibition of lignin on optimal enzyme ratios was studied, by adding fixed amount of alkali-soluble lignin fractions to EA-CS(−), and pure Avicel, beechwood xylan and evaluating their sugar conversion. The optimal enzyme ratios that gave higher sugar conversion for EA-CS(−) were CBH I: 27.2–28.2%, CBH II: 18.2–22.2%, EG I: 29.2–34.3%, EX: 9.0–14.1%, βX: 7.2–10.2%, βG: 1.0–5.0% (at 7.5–30 mg g−1 protein mass loading). Endoglucanase was inhibited to a greater extent than other core cellulases and xylanases by lignin during enzyme hydrolysis. We also found that alkali-soluble lignin inhibits cellulase more strongly than hemicellulase during the course of enzyme hydrolysis.

Use of enzyme preparations for improvement of the flour baking properties

The main reason for the wheat flour poor quality is a deviation in the enzymatic complex and the biopolymers state due to poor agricultural and technical conditions, pest damage to grain stocks, unfavorable storage conditions. The use of various bakery improvements allows to make the technological process and the quality of bread better.The article presents research on the stabilization of the quality of bakery flour with low amylolytic activity (PE - 426 s) and strong gluten (the quality of gluten on the DIG (deformation index of gluten) device is 40 conditional units. Enzyme preparations with amylase and hemicellulase activity and sulfur-containing amino acid cysteine were used as improvers. The effect of each improver on the baking properties of the flour was determined by the results of the laboratory baking test. Using a complex of these baking improvers makes it possible to increase the efficiency of each component due to the synergy of their action. The complex of improvers in medium dosages showed best results of bread quality, organoleptic and physical parameters of bread improved, the specific volume of bread having increased from 2.4 to 4.3 cm3/g, i.е. in 1.8 times.

Deactivation and activation of lignocellulose degrading enzymes in the presence of laccase.

Cellulase and hemicellulase activities in a 1:1 ratio of enzymes extracted from Chrysoporthe cubensis and Penicillium pinophilum were evaluated in the presence of known monocomponent phenolic inhibitors and also with phenol mixtures derived from alkali pretreated sugarcane bagasse. The cellulolytic activities from C. cubensis:P. pinophilum displayed a much higher tolerance to phenolic inhibitors than equivalent enzyme activities obtained from Trichoderma reesei and Aspergillus niger. Enzymes from T. reesei and A. niger were deactivated at 0.3 and 1.5mg phenols/mg protein, respectively, as reported previously, while enzymes from C. cubensis:P. pinophilum resisted deactivation at 35mg phenols/mg protein. However, tolerance of xylanase with respect to phenols required the presence of laccase. Removal of laccase (enzyme) activity using sodium azide resulted in a 2x higher xylanase deactivation (from 40% to 80%). This paper identifies enzymes that are phenol tolerant, and whose adoption for lignocellulose hydrolysis could contribute to reductions in enzyme loading needed to hydrolyze alkali pretreated lignocellulosic substrates in the presence of lignin derived phenols.

Improved chromatic and sensory characteristics of Plavac Mali wines - efficiency of maceration enzymes

Two commercial enzyme preparations were used in the production of wine from the Croatian autochthonous red grape variety Plavac Mali in order to improve the extraction of polyphenolic components from grapes, chromatic parameters, and sensory quality. During two vintages, the conventional maceration without enzymes was compared with the maceration using products with different characteristics: pectinase with additional cellulase and hemicellulase activity and pectinase with inactive yeast cells. Both products affected polyphenolic extraction and colour parameters: intensity and hue, and ratio between the yellow, red, and blue colour in young wines (2 months after fermentation) and at the moment of bottling (9 months after fermentation). The correlation between anthocyanins and colour intensity was very strong. The expected reduction of quantitative chromatic parameters during aging was confirmed. Significantly better results were observed in wines produced with pectinase, in relation to all analysed physical and chemical parameters. The sensory analysis showed that wines produced with pure pectolytic enzymes were significantly better than those produced without the enzymes. A product of the combination of pectolytic enzymes and inactive yeast cells had a partial influence on the improvement of the phenolic and sensory quality. The overall quality was significantly more expressed in wines produced with pectolytic enzymes, especially in young wines.

Effects of Brevibacillus brevis FJAT-1501-BPA on growth performance, faecal microflora, faecal enzyme activities and blood parameters of weaned piglets.

A feeding expriment was performed to investigate the effects of dietary supplementation with Brevibacillus brevis FJAT-1501-BPA fermentation on the growth performance, faecal microflora, faecal enzyme activities and blood parameters of weaned piglets. A total of 150 weaned piglets were randomly assigned to different treatments groups, which were fed the same basic diet supplemented with 10, 1, 0.1, 0.01 and 0% B. brevis FJAT-1501-BPA fermentation. The results showed that a diet supplemented with 10% B. brevis FJAT-1501-BPA fermentation could significantly increase the final body weight (P<0.05) and decrease feed to gain ratio, which was 37.1% lower than that of the control group. The addition of B. brevis FJAT-1501-BPAexhibited a trend of reducing the contents of the Escherichia coli, Lactobacillus and Salmonella compared with the control. During the 35day experimental period, cellulase and protease activities were significantly increased by the dietary inclusion of the B. brevis FJAT-1501-BPA fermentation (P<0.05). The cellulase activity for piglets fed diet containing 1% B. brevis FJAT-1501-BPA fermentation, 21.8U/g, was highest among the different treatments. The protease activity for piglets fed diet containing 10% B. brevis FJAT-1501-BPA fermentation, 50.4 U/g, was highest among the different treatments. The amylase and hemicellulase activities for piglets fed diet containing 10% B. brevis FJAT-1501-BPA fermentation were significantly higher than those on the control diet and other treatments (P<0.05). Moreover, usage of feed dietary supplementation with B. brevis FJAT-1501-BPA had positive effects on levels of enzymes and minerals in blood. The alkaline phosphatase, alanine aminotransferase, Fe and Mg concentrations for weaned piglets fed diet containing B. brevis FJAT-1501-BPA fermentation were significantly higher than for those on the control diet (P<0.05). Furthermore, concentration of IgG in serum was higher in weaned piglets fed diet containing 1% B. brevis FJAT-1501-BPA fermentation compared to other treatments. These results indicated that feeding with B. brevis FJAT-1501-BPA has the potential to improve growth performance, faecal microflora, faecal enzyme activities and blood parameters of weaned piglets.

The use of T-DNA insertional mutagenesis to improve cellulase production by the thermophilic fungus Humicola insolens Y1.

Humicola insolens is an excellent producer of pH-neutral active, thermostable cellulases that find many industrial applications. In the present study, we developed an efficient Agrobacterium tumefaciens-mediated transformation system for H. insolens. We transformed plasmids carrying the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of H. insolens driving the transcription of genes encoding neomycin phosphotransferase, hygromycin B phosphotransferase, and enhanced green fluorescent protein. We optimized transformation efficiency to obtain over 300 transformants/106 conidia. T-DNA insertional mutagenesis was employed to generate an H. insolens mutant library, and we isolated a transformant termed T4 with enhanced cellulase and hemicellulase activities. The FPase, endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase activities of T4, measured at the end of fermentation, were 60%, 440%, 320%, 41%, and 81% higher than those of the wild-type strain, respectively. We isolated the sequences flanking the T-DNA insertions and thus identified new genes potentially involved in cellulase and hemicellulase production. Our results show that it is feasible to use T-DNA insertional mutagenesis to identify novel candidate genes involved in cellulase production. This will be valuable when genetic improvement programs seeking to enhance cellulase production are planned, and will also allow us to gain a better understanding of the genetics of the thermophilic fungus H. insolens.

Effects of microbial enzymes on starch and hemicellulose degradation in total mixed ration silages.

ObjectiveThis study investigated the association of enzyme-producing microbes and their enzymes with starch and hemicellulose degradation during fermentation of total mixed ration (TMR) silage.MethodsThe TMRs were prepared with soybean curd residue, alfalfa hay (ATMR) or Leymus chinensis hay (LTMR), corn meal, soybean meal, vitamin-mineral supplements, and salt at a ratio of 25:40:30:4:0.5:0.5 on a dry matter basis. Laboratory-scale bag silos were randomly opened after 1, 3, 7, 14, 28, and 56 days of ensiling and subjected to analyses of fermentation quality, carbohydrates loss, microbial amylase and hemicellulase activities, succession of dominant amylolytic or hemicellulolytic microbes, and their microbial and enzymatic properties.ResultsBoth ATMR and LTMR silages were well preserved, with low pH and high lactic acid concentrations. In addition to the substantial loss of water soluble carbohydrates, loss of starch and hemicellulose was also observed in both TMR silages with prolonged ensiling. The microbial amylase activity remained detectable throughout the ensiling in both TMR silages, whereas the microbial hemicellulase activity progressively decreased until it was inactive at day 14 post-ensiling in both TMR silages. During the early stage of fermentation, the main amylase-producing microbes were Bacillus amyloliquefaciens (B. amyloliquefaciens), B. cereus, B. licheniformis, and B. subtilis in ATMR silage and B. flexus, B. licheniformis, and Paenibacillus xylanexedens (P. xylanexedens) in LTMR silage, whereas Enterococcus faecium was closely associated with starch hydrolysis at the later stage of fermentation in both TMR silages. B. amyloliquefaciens, B. licheniformis, and B. subtilis and B. licheniformis, B. pumilus, and P. xylanexedens were the main source of microbial hemicellulase during the early stage of fermentation in ATMR and LTMR silages, respectively.ConclusionThe microbial amylase contributes to starch hydrolysis during the ensiling process in both TMR silages, whereas the microbial hemicellulase participates in the hemicellulose degradation only at the early stage of ensiling.

A method for rapid purification and evaluation of catalytically distinct lignocellulolytic glycosyl hydrolases from thermotolerant fungus Acrophialophora sp.

A novel thermotolerant fungal strain P2 identified as Acrophialophora sp. was found to be a good source of cellulases and hemicellulases. A rapid approach was devised to purify different lignocellulolytic glycosyl hydrolases from secretome of Acrophialophora sp. employing separation of proteins by SDS-PAGE followed by renaturation, slicing of gel and elution of proteins in tandem with microtitre plate based method for assay of cellulases and hemicellulase activities. This approach resulted in purification of an array of glycosyl hydrolases i.e., CBH I (42.0 kDa), EG I (64.0 kDa), EG II (32.0 kDa), EG III (22.0 kDa), EG IV (20.0 kDa), Xyl I (28.0 kDa), Xyl II (20.6 kDa), β-xylosidase (66.7 kDa) and α-arabinofuranosidase (66.0 kDa). The purified enzymes were analysed for physicochemical characters as well as substrate specificity. The enzyme extract from Acrophialophora sp. was evaluated for saccharification of alkali treated rice straw that primarily resulted in release of xylobiose, glucose, xylose, cellobiose and arabinose as the major hydrolysis products. Furthermore the proteome analysis by LTQ-Velos-Orbitrap mass spectrometry unveiled the identity of proteins involved in carbohydrate catabolism, stress as well as lipid and protein biosynthetic pathway.

Monitoring the fibrolytic potential of microbial ecosystems from domestic and wild ruminants browsing tanniferous forages.

Although the rumen microbiome has been reported to synthesize a rich source of symbiotic enzymes (exocellulase, endocellulase, hemicellulase and cellobiase), the digestion of tropical C4 grasses and browses by ruminants is still limited. Therefore, this study aimed to unveil potential fibrolytic microbial ecosystems from giraffe, kudu, impala and consortia (A1 [giraffe + kudu], A2 [giraffe + impala], A3 [kudu + impala], and A4 [giraffe + kudu + impala]) browsing tanniferous plants, which can be used to improve forage utilization in domesticated goat. Crude protein enzyme extracts (CPZ) from fresh faecal samples were precipitated by 60% ammonium sulphate and assayed for exocellulase, endocellulase and hemicellulase by incubating with crystalline cellulose, carboxymethyl cellulose and xylan at 38 °C with optimum pH of 5.5 to 6.5 for 1, 2, and 48 h, respectively. Enzyme specific activities were defined as μg of reducing sugar/mg CPZ. In vitro fermentation study was done by transferring 33 mL of fresh faecal inoculum into 67 mL of salivary buffer containing 1 g Acacia sieberiana and incubating for 72 h at 38 °C. Apparent degradability (APDeg), true degradability (TD), neutral detergent fibre degradability (NDFdeg), acid detergent fibre degradability (ADFdeg), microbial yield (MY), metabolizable energy (ME) and total gas emitted (Gas) were measured. Exocellulase activities were higher (P < 0.05) in all wild animals and consortia than those in goat except for A4. Minimal differences in hemicellulase activities (P < 0.05) were observed among goat and wild animals and consortia, while endocellulase activity was generally higher (P < 0.05) in goat than that in the rest of the systems. Apart from A3, TDeg, NDFdeg and ADFdeg were higher (P < 0.05) in all microbial ecosystems from wild animals and consortia than those in goat. Apparent degradability, MY and ME also varied (P < 0.05) among these systems. Giraffe, Kudu and A3 produced lower (P < 0.05) gas than the goat system. This study showed that microbial ecosystems from wild browsers (especially impala) and consortia possess a higher potential to digest tanniferous forage with less enteric gas production compared with domesticated goat, hence those microbiome could be exploited as microbial feed additives for improving digestibility and reducing enteric gas production in domesticated goat. Improvements of goat's digestibility will depend on the survival and establishment of microbial species in the rumen as well as their fibrolytic and symbiotic potential including tannin tolerance.

Novel enzyme preparations with high pectinase and hemicellulase activity based on Penicillium canescens strains

Recombinant strains of Penicillium canescens producing homologous pectin lyase A and heterologous endo- 1,5-α-arabinase A and endo- 1,4-α-polygalacturonase, as well as enzymes of the host strain (α-L-arabinofuranosidases, xylanases, and others), were obtained by genetic engineering. The enzyme preparations (EPs) obtained from the cultural medium of recombinant P. canescens strains efficiently hydrolyzed raw plant material with a high content of pectin compounds. It was shown that the yield of reducing sugars and arabinose increased 16 and 22% in comparison with the control EP based on the host strain when one of the obtained EPs was used for beet pulp hydrolysis. It was established that the most active EP consisted of pectin lyase (10%), endo-1,5-arabinase (26%), α-L-arabinofuranosidase and arabinoxylan-arabinofuranohydrolase (12%), and xylanase (10%). The activities of pectin lyase, polygalacturonase, and arabinase of the EP in reactions with various substrates were determined. The specificity, pH and T-optima, and thermal stability of the homogenous recombinant endo- 1,5-α-arabinase were investigated. The kinetic parameters (K(m), K(cat)) of the linear arabinan hydrolysis were determined.

Characterization of hemicellulase and cellulase from the extremely thermophilic bacterium Caldicellulosiruptor owensensis and their potential application for bioconversion of lignocellulosic biomass without pretreatment.

BackgroundPretreatment is currently the common approach for improving the efficiency of enzymatic hydrolysis on lignocellulose. However, the pretreatment process is expensive and will produce inhibitors such as furan derivatives and phenol derivatives. If the lignocellulosic biomass can efficiently be saccharified by enzymolysis without pretreatment, the bioconversion process would be simplified. The genus Caldicellulosiruptor, an obligatory anaerobic and extreme thermophile can produce a diverse set of glycoside hydrolases (GHs) for deconstruction of lignocellulosic biomass. It gives potential opportunities for improving the efficiency of converting native lignocellulosic biomass to fermentable sugars.ResultsBoth of the extracellular (extra-) and intracellular (intra-) enzymes of C. owensensis cultivated on corncob xylan or xylose had cellulase (including endoglucanase, cellobiohydrolase and β-glucosidase) and hemicellulase (including xylanase, xylosidase, arabinofuranosidase and acetyl xylan esterase) activities. The enzymes of C. owensensis had high ability for degrading hemicellulose of native corn stover and corncob with the conversion rates of xylan 16.7 % and araban 60.0 %. Moreover, they had remarkable synergetic function with the commercial enzyme cocktail Cellic CTec2 (Novoyzmes). When the native corn stover and corncob were respectively, sequentially hydrolyzed by the extra-enzymes of C. owensensis and CTec2, the glucan conversion rates were 31.2 and 37.9 %,which were 1.7- and 1.9-fold of each control (hydrolyzed by CTec2 alone), whereas the glucan conversion rates of the steam-exploded corn stover and corncob hydrolyzed by CTec2 alone on the same loading rate were 38.2 and 39.6 %, respectively. These results show that hydrolysis by the extra-enzyme of C. owensensis made almost the same contribution as steam-exploded pretreatment on degradation of native lignocellulosic biomass. A new process for saccharification of lignocellulosic biomass by sequential hydrolysis is demonstrated in the present research, namely hyperthermal enzymolysis (70–80 °C) by enzymes of C. owensensis followed with mesothermal enzymolysis (50–55 °C) by commercial cellulase. This process has the advantages of no sugar loss, few inhibitors generation and consolidated with sterilization.ConclusionsThe enzymes of C. owensensis demonstrated an enhanced ability to degrade the hemicellulose of native lignocellulose. The pretreatment and detoxification steps may be removed from the bioconversion process of the lignocellulosic biomass by using the enzymes from C. owensensis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0313-0) contains supplementary material, which is available to authorized users.

Protoplast fusion enhances lignocellulolytic enzyme activities in Trichoderma reesei.

Protoplast fusion was used to obtain a higher production of lignocellulolytic enzymes with protoplast fusion in Trichoderma reesei. The fusant strain T. reesei JL6 was obtained from protoplast fusion from T. reesei strains QM9414, MCG77, and Rut C-30. Filter paper activity of T. reesei JL6 increased by 18% compared with that of Rut C-30. β-Glucosidase, hemicellulase and pectinase activities of T. reesei JL6 were also higher. The former activity was 0.39 Uml(-1), while those of QM9414, MCG77, and Rut C-30 were 0.13, 0.11, and 0.16 Uml(-1), respectively. Pectinase and hemicellulase activities of JL6 were 5.4 and 15.6 Uml(-1), respectively, which were slightly higher than those of the parents. The effects of corn stover and wheat bran carbon sources on the cellulase production and growth curve of T. reesei JL6 were also investigated.

Role of macrobenthic fauna in mangrove carbon fluxes indicated by their cellulase and hemicellulase activities

Numerous studies have focused on the impact of biotic factors under the role of organic matter in the dynamics of mangrove ecosystems during the last decades, but an overall view of the role of macrobenthic fauna in organic matter processing has not yet been established. Cellulase and hemicellulase (xylanase and laminarinase) activities were measured in the extracts from eight native mangrove macrobenthic species (two gastropods, three crabs and three bivalves) of southern China with plate assay and colorimetric assay. All enzyme activities were detected in the eight species except for negligible exoglucanase activity in the crabs and the bivalves. The crabs generated the largest halo zones in the plate assays, but produced the lowest amount of reducing sugar in the colorimetric assays. The highest endoglucanase, β-1,4-glucosidase and xylanase activities were detected in Crassostrea rivularis with colorimetric assays. For each mangrove macrobenthic species, the laminarinase activity was highest among the enzyme activities that were detected. The results suggest that mangrove macrobenthos participates in the carbon cycling of the mangrove ecosystem via the consumption and conversion of mangrove primary production at different ecological niches and that they prefer algae as a major food source rather than mangrove leaf litter from September to October.

Sweet Sorghum Biorefinery for Production of Fuel Ethanol and Value-Added Co-products

<abstract> <bold><sc>Abstract.</sc></bold> An integrated process has been proposed for a sweet sorghum biorefinery in which all carbohydrate components of the feedstock were used for production of fuel ethanol and industrial chemicals. In the first step, the juice was extracted from the stalks. The resultant bagasse was then pretreated using the soaking in aqueous ammonia (SAA) process, which did not result in significant loss of cellulose and hemicellulose, to enhance subsequent enzyme hydrolysis for production of fermentable sugars. Following pretreatment, the bagasse was hydrolyzed with a commercial enzyme product containing high hemicellulase activity (Accellerase XY). The xylose-rich solution obtained after solid/liquid separation was used for production of value-added co-products using suitable microorganisms. The value-added co-products produced to demonstrate the feasibility were astaxanthin and D-ribose. The residual solids were then hydrolyzed with a commercial enzyme product containing high cellulase activity (Accellerase 1500), with the juice extracted in the first step used as make-up water. By combining the sugar in the juice with the glucose released from the residual solids by enzyme hydrolysis, high ethanol concentrations could be achieved, which resulted in lower distillation cost than if pure water were used for enzyme hydrolysis and subsequent fermentation, as normally performed in cellulosic ethanol production.

Two Major Facilitator Superfamily Sugar Transporters from Trichoderma reesei and Their Roles in Induction of Cellulase Biosynthesis

Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement.

Assimilation of terrigenous organic matter via bacterial biomass as a food source for a brackish clam, Corbicula japonica (Mollusca: Bivalva)

Corbicula japonica collected from the Kitakami River estuary, northeastern Japan, showed lower δ34S values in soft-body parts (+1.7 to +11.0‰) than the ambient seawater sulfate sulfur (+21‰), and this value gradually decreased at successive sites up to 15.8 km upstream from the river mouth. Previous study using carbon and nitrogen isotopes suggests that the bivalve nonselectively assimilates particulate organic matter of marine and terrestrial origin by filter feeding. This pattern in δ34S values may indicate a considerable contribution of a food source derived from terrigenous organic matter, which has low δ34S values close to 0‰, and the bivalve has been reported to have cellulase and hemicellulase activities. Unique fatty acids (iso 17:0 and anteiso 17:0 acids), both characteristic of sulfate-reducing bacteria, were observed in the salt-free, soft-body parts of the bivalve. The concentration of monounsaturated fatty acids, possibly derived from bacteria, was also high. Trace amounts of a polyunsaturated fatty acid (20:5ω3) specific to dinoflagellates were detected. In Corbicula habitats, reductive sandy layers with ample sulfides that were sporadically intercalated into the oxidative sandy sediment were often observed. The stable isotopic signatures of sediment sulfides (acid-volatile sulfide) and associated pore-water sulfates were −8.9 to +8.6‰ and +22.4 to +26.3‰, respectively, indicating the existence of bacterial sulfate-reducing activity and thiobios biomass. These isotopic signatures of the sediment, with the fatty acid composition of the bivalve, confirm the importance of a food source derived from bacteria belonging to the thiobios in the substrate sediments via pedal feeding, rather than direct digestion of terrigenous organic matter, in this estuarine ecosystem.