Bagasse is the fibrous derivative of sugar cane, that is grown on a commercial scale in many tropical and sub-tropical countries, where ideal climatic conditions are experienced. The seasonality of sugar cane presents storage problems for bagasse, since this lignocellulosic material is susceptible to degradation by a diverse range of microorganisms, mainly fungi. The decay that is brought about contributes largely to the losses of fibre in a bagasse pile. The surrounding microclimate, and conditions within the pile, needs to be carefully monitored in order to understand the factors that support the fungal populations and biochemical activity. The microclimate at the surface and inside the bagasse pile at a paper mill in Stanger (South Africa) was carefully monitored over a one-year storage period. Significant changes were noted in temperature, pH and moisture content, between the surface and the inside of the pile, as the pile aged. The data were compared to established parameters for bagasse preservation, and it was found that the temperature was lower than expected, thus promoting fungal growth. The pH was much higher (promoting bacteria and actinomycetes) and the moisture content was too low to produce anaerobic conditions. The environmental conditions in the bagasse pile at Stanger, therefore, promoted the proliferation of microbes, and consequently decay. Fungi that were present in the pile, were enumerated in order to investigate the diversity and fungal succession. There was a wider variety of species and higher numbers of fungi at the surface than inside the bagasse pile and the Shannon and Berger-Parker diversity confirmed these observations. Sorensons measure also showed that the types of fungal communities at the surface and inside the pile only started becoming similar toward the latter part of storage. When compared to models for abundance of species, conditions on the surface of the pile allowed maximum niche occupation at the beginning of storage, followed by the establishment of a mature community. The inside of the pile displayed minimal niche pre-emption followed by a state where most fungal species shared the domain. This study indicated that, as the storage time increased, the microbial communities became better established. Bagasse is rich in holocellulose, the basic raw material used for paper-making. Since there were many species of holocellulolytic fungi found growing on the surface and the inside of the bagasse pile, the activity of cellulases and xylanases were determined. These enzymes were found to be active at the surface and inside the pile. However, higher activities of both enzymes were noted inside the bagasse pile than on the surface. The higher levels of activity inside the pile, despite lower fungal numbers, suggested that fungal counts were not a clear indication of biomass or biochemical activity. It appeared that the environment on the inside of the bagasse pile promoted the establishment of specific fungal populations that bring about a high degree of degradation to fibre inside the bagasse pile. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

In addition to substrate-associated cellulases, Cellulomonas fimi secretes endoglucanases ( endo-1, 4-β-D-glucan glucanohydrolases, EC 3.2.1.4. ) which are recovered from the cellulose-free culture supernatant of cells grown on microcrystalline cellulose. Two such enzymes, C3.1 and C3.2 with Mrs of 130'000 and 120'000, respectively, were purified to homogeneity. The two endoglucanases were shown to share the same N-terminal amino acid sequence and to hydrolyze carboxymethylcellulose ( CMC ) with similar efficiencies ( 236u/mg protein for C3.1 and 367u/mg protein for C3.2 ). The recombinant lambda vector L47.1-169 was identified from a C.fimi DNA-lambda library on the basis of hybridization with C3.1/2-specific oligonucleotide probes. The subclone pTZ18R-8 only moderately expressed CMCase activity. The 5'-terminus of cenC ( the gene coding for C3.1/2 ) was localized in the insert by Southern transfer experiments and nucleotide sequence analysis. Results from total C.fimi RNA-DNA hybrid protection analyses defined the boundaries of cenC in pTZ18R-8 and led to the tentative identification of -10 and -35 promoter sequences. To improve the expression of cenC, its entire coding sequence, except for the start codon GTG, was fused in frame to the ATG codon of a synthetic ribosomal binding site ( PTIS ) and placed under the transcriptional control of the lac p/o system. Induction of the resulting clone ( JM101[pTZP-cenC] ) led to impaired growth in liquid cultures because overproduction of CenC inhibited cell division'" and eventually led to cell death. Analysis of cell fractions by SDS-PAGE revealed a dominant ( >10% of total cell extract proteins ), clone-specific protein with a Mr of approximately 140'000 which was found exclusively in the cytoplasmic fraction. Conversely, 60% of the total CMC-hydrolyzing activity was localized in the periplasmic fraction indicating that the export of CenC is required for maximal expression of endoglucanase activity. Isolation of the cellulolytic activities from an osmotic shockate led to the purification to homogeneity of two recombinant cellulases, CenC1 and CenC2, with Mr of 130'000 and 120'000, respectively. Both enzymes hydrolyzed CMC with similar efficiencies ( 278u/mg protein for CenC1 and 390u/mg protein for CenC2 ). In addition, amino acid sequence analyses showed the two enzymes to have the same N-termini as the native enzymes and proved furthermore that the CenC leader peptide was functional in Escherichia coli. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Transcriptional analysis was used to investigate the molecular mechanisms which effect cellulase gene expression in the gram-positive bacterium Cellulomonas fimi strain ATCC 484 and the gram-negative bacterium Agrobacterium sp. strain ATCC 21400. The cenA, cex and cenB genes of C. fimi encoding the extracellular β-1,4-endoglucanase, EngA (EC 3.2.1.4; Mr 48,700), the extracellular β-1, 4-exoglucanase, Exg (EC 3.2.1.91; Mr 47,300) and the extracellular β-1,4-endoglucanase EngB (EC 3.2.1.4; Mr 110,000) respectively, were characterised. By northern blot analysis, cenA mRNA was detected in C. fimi RNA prepared from glycerol- and carboxymethylcellulose (CMC)-grown cells but not in RNA from glucose-grown cells. The cex mRNA was found only in RNA from CMC-grown cells. The cenB mRNA was found in all three preparations of RNA. Therefore, the expression of these genes is subject to regulation by the carbon source provided to C. fimi. High resolution nuclease SI protection studies with unique 5'-labeled DNA probes and C. fimi RNA isolated in vivo, were used to map the 5' termini of cenA and cex mRNAs. Two cenA mRNA 5' ends, 11 bases apart, mapped 51 and 62 bases upstream of the cenA start codon, suggesting that in vivo, cenA transcription was directed from two promoters in tandem. The cex mRNA 5' end was found to map 28 bases upstream of the cex start codon. Using SI mapping with unlabeled DNA probes and C. fimi RNA which had been isolatedin vivo but which had been 5'-labeled in vitro with vaccinia virus capping enzyme confirmed that true transcription initiation sites for cenA and cex mRNA had been identified. The SI mapping revealed mRNA 3' termini 1,438, 1,449, and 1, 464 bases from the major cenA start site, and one 3' terminus 1,564 bases from the major cex mRNA start site, in good agreement with the northern blot data. High resolution SI studies were also used to show that abundant mRNA 5' ends mapped upstream of the cenB start codon in RNA prepared from CMC-grown cells, while less-abundant species mapped 52 bases closer to the ATG codon in RNA prepared from C. fimi grown on any one of the three substrates. These results seem to indicate a tandem promoter arrangement with an ATG-proximal promoter directing low-level constitutive cenB transcription and a more distal promoter directing higher levels of cenB transcription as a result of C. fimi growth on cellulosic substrate. Steady- state levels were determined for cenA, cex and cenB mRNAs with RNA prepared from glycerol-, glucose-, and CMC-grown cultures of C. fimi in slot-blot hybridisations with radiolabeled oligodeoxyribonucleotide probes. A cex-linked gene (clg) was identified by sequence inspection and SI mapping. Transcripts of the abg gene encoding the β-glucosidase (Abg, EC 3.2.2.21/ Mr 50,000) of Agrobacterium sp. strain ATCC 21400 were also characterised. Northern blot analysis of Agrobacterium RNA revealed the size of the in vivo abgmRNA was approximately 1,500 bases in length. High resolution SI mapping determined abg mRNA 5' ends 22 bases upstream of the abg ATG codon and 3' ends 71 bases downstream of the abg stop codon. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

>Magister Scientiae - MSc / Consolidated bioprocessing (CBP), in which a single organism in a single reactor is responsible for the conversion of pretreated lignocellulosic biomass to bioethanol, remains an attractive option for production of commodity products if an organism fit for this process can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second generation bioethanol. Current recombinant yeast strains engineered for this purpose must overcome the drawback of generally low secretion titres. A promising strategy for directly converting lignocellulose to ethanol is by displaying heterologous cellulolytic enzymes on the cell surface by means of the glycosylphosphatidylinositol (GPI) or similar anchoring systems. Recently, a strain producing cell-adhered enzymes in a ratio-optimized manner was created that showed significant crystalline cellulose hydrolysis.

This master’s thesis is aimed at process of enzymatic hydrolysis of lignocellulosic material – waste paper as a source of raw material for production of liquid biofuels. In the theoretical part of this work are summarized previously used methods of hydrolysis and lignocellulosic materials used for the process of hydrolysis as a source of fermentable sugars for fermentation technology. The different types of waste paper are evaluated from the composition and usability with consideration to the papermaking process in order to select the appropriate type of waste paper for the enzymatic hydrolysis process. In the next part of this work are suggested technological premises and procedures for the preparation of raw materials and the subsequent enzymatic hydrolysis of these pre–treated materials. In the experimental part were optimized parameters of enzymatic hydrolysis using the Novozymes company enzyme package. Enzymatic degradation of cellulose to reducing sugars was observed using Somogyi – Nelson method. For the verification of hydrolysis conditions were used model materials with high cellulose content – pulp and filter paper. Conditions, which seems to be the best after testing on the model materials, were verified on specific waste paper materials – offset cardboard, recycled paper, matte MYsol paper and for comparison again on model materials – pulp and filter paper. The highest yields was achieved with the use of cardboard, which was further tested using various combinations of pretreatment to material for purpose of increase the yields of hydrolysis.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19758号 / 農博第2154号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4974(農学部図書室) / 32794 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 山下 洋, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The first part of the dissertation presented a potentially novel aerobic, thermophilic, and cellulolytic bacterium identified as Brevibacillus sp. Strain JXL which was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between the effects caused by these two simple sugars. After the end of the 24 hour period, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also observed when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and optimal pH range of 6-8. These cellulases also had high thermotolerance as demonstrated by retaining more than 50% activity after 1 h at 100°C. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose. In the second part of the dissertation, the effect of Cellulose- and Organic-Solvent based Lignocellulose Fractionation (COSLIF) (Zhang, Y.-H. P.; Ding, S.-Y.; Mielenz, J. R.; Elander, R.; Laser, M.; Himmel, M.; McMillan, J. D.; Lynd, L. R. Biotechnol. Bioeng.2007, 97 (2), 214−223) pretreatment conditions on sweet sorghum bagasse (SSB) feedstock was studied using Response Surface Methodology (RSM). Batch experimental matrix was set up based on response surface method's central composite design in two factors to determine the effects of reaction time and temperature on the yield of simple sugars after a sequential pretreatment-enzyme hydrolysis process. Accordingly, changes in delignification, total reducing sugar (TRS) yield, glucan retention, digestibility and overall sugar yields resulting from various combinations of reaction times and temperatures were determined. The results suggested that both pretreatment temperature and reaction time were significant factors, although temperature was more so than reaction time. COSLIF pretreatment conditions of 50°C and 40 min were found to be the optimum pretreatment conditions for the saccharification of SSB. At the end of pretreatment and enzymatic hydrolysis, maximum values of 51.4% delignification, 85% overall glucose yield, and 44% overall xylose yield at an ACCELERASE®1500 loading of 0.25 mL/g sweet sorghum bagasse were achieved. Optimum ACCELERASE®1500 dosage of 0.1 mL/g of sweet sorghum bagasse was identified which resulted in an overall glucose yield of 82.2%±1.05. An effort has also been made to prescribe predictive models which represented the correlation between independent variables (reaction time and temperature), and dependent variables (delignification, and overall glucose yield) using RSM. The significance of the correlations and adequacy of these models were statistically tested for the selected objective functions. The outcomes suggested very competent and statistically adequate regression models which provided quantitative information both for delignification and overall glucose yield for the batch experiments studied.

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