Value of cane trash in nitrogen nutrition of sugarcane

The significance of trash containing 0.3 to 0.5% N in the N nutrition of sugarcane (Saccharum hybrid sp.) was investigated in pot- and field experiments using¹⁵N-labelled trash. The data obtained from the pot study with 2 silty-clay loams (a Humic Nitosol and a Humic Acrisol) showed that surface-applied trash (10 tonnes/ha), although ground to pass a 1-mm sieve, contributed less than 10% of N removed by sugarcane. Uptake of trash N was most active during the initial 6 months of the experiment though at the end of the study period of 18 months less than 15% of trash N was altogether recovered by sugarcane. In the absence of fertilizer N in a field study on the Humic Acrisol (C/N ratio 22), unground trash (5 tonnes. ha⁻¹) depressed soil N uptake by sugarcane by immobilizing available soil N. The field study moreover confirmed that the contribution of trash N in the supply of N to sugarcane is negligible. The value of trash would reside in its capacity to increase over the long term the organic matter level in the soils.     

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several β‐glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site‐saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl β‐glucosides, methyl α‐glucoside and cellobiose. In comparison with the wild‐type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413–420. © 2010 Wiley Periodicals, Inc.     

Contribution of N from green harvest residues for sugarcane nutrition in Brazil

Brazil is recognized as a prominent renewable energy producer due to the production of ethanol from sugarcane. However, in order for this source of energy to be considered truly sustainable, conservation management practices, such as harvesting the cane green (without burning) and retaining the trash in the field, need to be adopted. This management practice affects mostly the nitrogen (N) cycle through the effect of trash on immobilization–mineralization of N by soil microorganisms. The aim of the experiments reported here was to evaluate N recovery from trash (trash‐N) by sugarcane during three ratoon crop seasons: 2007, 2008 and 2009. Two field experiments were carried out, one in Jaboticabal and the other in Pradopolis, in the state of Sao Paulo, Brazil. The experiments were set up in a randomized block design with four replications. Within each plot, microplots were installed where the original trash was replaced by trash labelled with ¹⁵N, and maintained up to the fourth crop cycle. Trash‐N recovery was higher in the Jaboticabal site, the most productive one, than in the Pradópolis site. The average trash‐N recovery across the two sites after three crop cycles was 7.6 kg ha^?1 (or 16.2% of the initial N content in trash), with the remaining trash‐N being incorporated into soil organic matter reserves. While these results indicate that the value of trash for sugarcane nutrition is limited in the short term, maintaining trash on the field will serve as a long‐term source of N and C for the soil.     

Crystalline index change in cellulose during aerobic and anaerobic Cellulomonas uda growth

Summary Cultures of Cellulomonas uda were monitored under both aerobic and anaerobic conditions using three commercially available celluloses with varying degrees of crystallinity. In all cases, a high level of cellulose was metabolized and the same maximum carboxymethylcellulase activity (2.6 IU/mg of cellular protein) was observed. Measurement of the crystalline index of celluloses during cellulose growth revealed that the amorphous and crystalline regions were solubilized simultaneously. Investigation of the solubilization rate showed that a decline occurred when a considerable amount of cellulose still remained in the medium. Hypotheses were suggested to explain the biphasic pattern of the kinetics obtained.     

Biohydrogen production from cellulosic hydrolysate produced via temperature-shift-enhanced bacterial cellulose hydrolysis

A “temperature-shift” strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 °C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 °C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H₂ via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H₂/g reducing sugar.     

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

Molecular cloning of cellulolytic enzyme genes from Cellulomonas flavigena in E. coli

A genomic bank of Cellulomonas flavigena was constructed in E. coli using the pUC18 vector, and over 14000 clones screened for cellulolytic activity. Three different cellulolytic enzyme genes were cloned, one coding for an endo-β-glucanase (pJS10, CMC activity) and two coding for β-glucosidases, each with a distinct substrate specificity (pJS3, X-glu, and pJS4, X-glu and MUC activities). These three inserts have different restriction patterns to each other and the previously isolated cellulolytic enzyme genes from C. fimi and C. uda.     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998     

Cellulase genes of Cellulomonas uda CB4. II. Cloning and expression of a CM-cellulose hydrolyzing enzyme (endoglucanase) gene in Escherichia coli

A CM-cellulose hydrolyzing enzyme (endoglucanase, CMCase) gene in Cellulomonas uda CB4 was cloned in E. coli with pAT325 constructed from pAT153 and pBR325. Plasmid pCM41 was isolated from the transformant producing CMCase, and the CMCase gene cloned was a 4.8 kb BamHI fragment. About 70% of the CMCase activity was observed in the periplasmic space of E. coli carrying pCM41. The optimal pH and temperature for the CMCase thus cloned were pH 6–8 and 55–60°C, respectively, as was the case for the donor.     

Effect of undecanoic acid on germination of microconidia of wild and undecanoic acid resistant mutant of trichophyton rubrum

Effects of undecanoic acid (UDA) on germination of microconidia and elongation of germ tubes in UDA sensitive (uda _s) wild type Trichophyton rubrum and UDA resistant (uda _r) mutant derived from it, were studied. UDA inhibited conidial germination of uda _s and uda _r strains at 30 g/ml and 120 g/ml respectively which were minimum inhibitory concentrations of UDA for these two strains. When spores from both uda _s and uda _r were germinated in presence of subinhibitory concentration of UDA, germ tube growth was short. The elongation of germ tubes of spores pregerminated in absence of UDA was also inhibited by dose of UDA not sufficient to inhibit germination.     

Microbial production of testosterone and testololactone in the culture of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Two metabolites were obtained by microbial transformation of androstendione in the culture of Aspergillus terreus PTCC 5283, a fungus isolated from soil. Their structures were established as testosterone and testololactone on the basis of the spectral data including ¹H NMR, ¹³C NMR, FTIR, MS and physical constants such as melting point and optical rotation. Aspergillus terreusproduced both metabolites after 3 days incubation at 27 °C. The bioconversion reactions observed were 17-carbonyl reduction and biological Baeyer–Villiger oxidation.     

Bioethanol production from sugarcane and emissions of greenhouse gases – known and unknowns

Bioethanol production from sugarcane is discussed as an alternative energy source to reduce dependencies of regional economies on fossil fuels. Even though bioethanol production from sugarcane is considered to be a beneficial and cost‐effective greenhouse gas (GHG) mitigation strategy, it is still a matter of controversy due to insufficient information on the total GHG balance of this system. Aside from the necessity to account for the impact of land use change (LUC), soil N₂O emissions during sugarcane production and emissions of GHG due to preharvest burning may significantly impact the GHG balance. Based on a thorough literature review, we show that direct N₂O emissions from sugarcane fields due to nitrogen (N) fertilization result in an emission factor of 3.87±1.16% which is much higher than suggested by IPCC (1%). N₂O emissions from N fertilization accounted for 40% of the total GHG emissions from ethanol–sugarcane production, with an additional 17% from trash burning. If LUC‐related GHG emissions are considered, the total GHG balance turns negative mainly due to vegetation carbon losses. Our study also shows that major gaps in knowledge still exist about GHG sources related to agricultural management during sugarcane production, e.g. effects of irrigation, vinasse and filter cake application. Therefore, more studies are needed to assess if bioethanol from sugarcane is a viable option to reduce energy‐related GHG emissions.     

Evaluation of thermophilic fungal consortium for paddy straw composting

Out of 10 thermophilic fungi isolated from wheat straw, farm yard manure, and soil, only three showed highest cellobiase, carboxymethyl cellulase, xylanase, and FPase activities. They were identified as Aspergillus nidulans (Th₄), Scytalidium thermophilum (Th₅), and Humicola sp. (Th₁₀). A fungal consortium of these three fungi was used to compost a mixture (1:1) of silica rich paddy straw and lignin rich soybean trash. The composting of paddy straw for 3 months, during summer period in North India, resulted in a product with C:N ratio 9.5:1, available phosphorus 0.042% and fungal biomass 6.512 mg of N-acetyl glucosamine/100 mg of compost. However, a C:N ratio of 10.2:1 and highest humus content of 3.3% was achieved with 1:1 mixture of paddy straw and soybean trash. The fungal consortium was effective in converting high silica paddy straw into nutritionally rich compost thereby leading to economical and environment friendly disposal of this crop residue.     

Optimization of pretreatment and fermentation conditions for production of extracellular cellulase complex using sugarcane bagasse

Sugarcane bagasse (SCB), a lignocellulosic byproduct of juice extraction from sugarcane, is rich in cellulose (40-42%). This could be used as a substrate for the production of cellulase complex. Fermentation conditions were optimized for production of cellulase complex (CMCase, Cellulobiase and FPase) by wild type Trichoderma sp. using sugarcane bagasse as sole carbon source. Alkaline treatment (2% NaOH) of bagasse (AlSCB) was found suitable for the production of reducing sugar over the acidic pretreatment method. After 5 days of incubation period, 5% substrate concentration at pH 5.0 and 400C resulted in maximum production of CMCase (0.622 U), while maximum (3.388 U) production of cellulobiase was obtained at 300C. The CMCase was precipitated and purified to the extent of 59.06 fold by affinity chromatography with 49.09% recovery. On 12% SDS-PAGE, a single band corresponding to 33 kDa was observed. The Km and Vmax for CMCase from Trichoderma was found 507.04 mg/ml and 65.32 mM/min, respectively. The enzyme exhibited maximum activity at 300C at pH-5.0 (0.363 U) and was stable over range of 20-60°C and pH 5.0-7.5.     

Engineering of cellobiose phosphorylase for glycoside synthesis

Disaccharide phosphorylases are increasingly applied for glycoside synthesis, since they are very regiospecific and use cheap and easy to obtain donor substrates. A promising enzyme is cellobiose phosphorylase (CP), which was discovered more than 50 years ago. Many other bacterial CP enzymes have since then been characterized, cloned and applied for glycoside synthesis. However, the general application of wild-type CP for glycoside synthesis is hampered by its relatively narrow substrate specificity. Recently we have taken some successful efforts to broaden the substrate specificity of Cellulomonas uda CP by directed evolution and protein engineering. This review will give an overview of the obtained results and address the applicability of the engineered CP enzymes for glycoside synthesis. CP is the first example of an extensively engineered disaccharide phosphorylase, and may provide valuable information for protein engineering of other phosphorylase enzymes.     

Bioconversion of cellulose wastes to protein and sugar

A schematic representation of the variety of products which can be obtained by microbial conversion of cellulose is presented. Alkaline pre-treatment has been used after milling in all the experiments. Solka-floc or sugarcane bagasse was used as sources of cellulose. A cellulolytic strain of Aspergillus terreus (ATCC 30514) was cultivated in batch-, fed batch and continuous culture up to 7 liter stirred tank fermenter. The general growth characteristics were determined by growing on glucose. Results of experiments on the growth of A. terreus for production of biomass on Solka-floc or Sugarcane bagasse are given, also the ability of crude cellulases to produce sugar syrups by enzymatic hydrolysis of cellulose has been evaluated.     

Optimization of polyhydroxybutyrate production by experimental design of combined ternary mixture (glucose,xylose and arabinose) and process variables (sugar concentration,molar C:N ratio)

Conversion of lignocellulosic feedstocks to polyhydroxybutyrate (PHB) could make lignocellulosic biorefineries more profitable and sustainable. Glucose, xylose and arabinose are the main sugars derived from pretreatment and hydrolysis of herbaceous feedstocks. Burkholderia sacchari DSM 17165 is a bacterium that can convert these sugars into PHB. However, the effects of sugar ratio, sugar concentration, and molar C:N ratio on PHB production have not been studied. In this study, a seven-run mixture design for sugar ratio combined with a 3² full factorial design for process variables was performed to optimize PHB production. A polynomial model was built based on experimental data, and optimum conditions for different sugar streams were derived and validated. The highest PHB production (3.81 g/L) was achieved with arabinose at a concentration of 25.54 g/L and molar C:N ratio of 74.35. Results provide references for manipulation of sugar mixture and process control to maximize PHB production.

     

β-Methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. β-Methyl-xyloside (β-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with β-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus β-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus β-mx as substrate. To our knowledge, this is the first report on the effect of β-mx on the induction of xylanase in C. flavigena.     

Glucose uptake by Cellulomonas fimi

Glucose uptake by whole-cell suspension of the facultative anaerobe Cellulomonas fimi, which was two-fold higher under aerobic conditions than under N₂ or H₂, was inhibited by inhibitors of electron transport and ATP synthesis and, particularly, by proton and metal ion ionophores. A variety of sugars, including 2-deoxyglucose, did not inhibit glucose uptake but cellobiose was a non-competitive inhibitor. Cells grown on cellobiose medium transported glucose at one half the rate of glucose-grown cells. Cellulomonas fimi has a highly specific active system for glucose transport.     

Exploring soil microbial 16S rRNA sequence data to increase carbon yield and nitrogen efficiency of a bioenergy crop

Crop residues returned to the soil are important for the preservation of soil quality, health, and biodiversity, and they increase agriculture sustainability by recycling nutrients. Sugarcane is a bioenergy crop that produces huge amounts of straw (also known as trash) every year. In addition to straw, the ethanol industry also generates large volumes of vinasse, a liquid residue of ethanol production, which is recycled in sugarcane fields as fertilizer. However, both straw and vinasse have an impact on N₂O fluxes from the soil. Nitrous oxide is a greenhouse gas that is a primary concern in biofuel sustainability. Because bacteria and archaea are the main drivers of N redox processes in soil, in this study we propose the identification of taxa related with N₂O fluxes by combining functional responses (N₂O release) and the abundance of these microorganisms in soil. Using a large‐scale in situ experiment with ten treatments, an intensive gas monitoring approach, high‐throughput sequencing of soil microbial 16S rRNA gene and powerful statistical methods, we identified microbes related to N₂O fluxes in soil with sugarcane crops. In addition to the classical denitrifiers, we identified taxa within the phylum Firmicutes and mostly uncharacterized taxa recently described as important drivers of N₂O consumption. Treatments with straw and vinasse also allowed the identification of taxa with potential biotechnological properties that might improve the sustainability of bioethanol by increasing C yields and improving N efficiency in sugarcane fields.