Dr. Miranda Maki

Development of bacterial systems for large-scale production of cellulase and bioethanol

SUPERVISOR: Dr. Wensheng Qin  (Biology)    
ABSTRACT: The wide varieties of extant bacterial species are often resistant to various environmental stresses. This demonstrates their frequent ability to adapt to and thrive in challenging environments. One such adaptation in wood-degrading species that may be exploited to produce a product of high value to humans is a more efficient cellulase activity, which may help to overcome current challenges in biofuel production. In this study, 18 efficient cellulase-producing bacteria were isolated from organic fertilizers and paper mill sludges and characterized for consideration in large scale biorefining. All cellulase positive isolates were further characterized to identify those with the greatest cellulase activities for potential industrial application. Six of these isolates produced greater cellulase activity on soluble cellulose in 48 h than the positive control (Cellulomonas xylanilytica). Phylogenetic analysis of a portion of the 16S rDNA gene revealed genera belonging to two major phyla of Gram positive bacteria: Firmicutes and Actinobacteria. Additionally, isolates E2 and E4 (Paenibacillus species) displayed qualitative cellulase activities towards filter paper under limited oxygen condition. When total cellulase activities of E2 and E4 were examined, it was shown that 1% (w/v) carboxymethyl cellulose (CMC) could induce total cellulase activities of 1652 ± 62 and 1457 ± 31 nM of glucose equivalents that were 8- and 5.6-fold greater than total cellulase activities induced by filter paper for E2 and E4, respectively. The genus Paenibacillus includes many highly-expressing cellulase producing strains, and E2 and E4 represent excellent candidates for further cellulase activity analysis and characterization. Cellulose hydrolysis is only one of the rate-limiting steps in the industrial production of biofuels which can be improved by isolation and characterization of novel enzymes. In addition, pretreatment of lignocellulosic biomass is a costly hurdle which can be improved by the application of bacteria capable of producing a greater variety of enzymes. The potential use of lignocellulosic biomass for biofuel production has been hampered due to the complexity of its composition and the lack of microorganisms capable of modifying or decomposing the different components. Thus, CMC-containing agar was used to isolate and characterize 20 cellulase-producing bacterial strains from peat and municipal wastes that belonged to four major phyla: Firmicutes, Actinobacteria, Proteobacteria and Bacteriodetes. Seven of the cellulase positive isolates also exhibited filter paper activities, while 13 exhibited activities towards xylan. Moreover, 10 of the isolates were capable of surviving 21 days incubation with 1% black liquor. Five strains increased the absorbance of black liquor by greater than 10-fold. Similarly, these five strains could also increase the absorbance of lignin at 280 nm when grown with 0.1% pure lignin. Additionally, although FTIR analysis of 1% barley straw treated for 21 days with these 5 strains showed a preference for consumption of hemicelluloses over lignin, a change in lignin was observed. Two isolates, 55S5 and AS1, a Bacillus sp. and Pseudomonas sp., respectively, have the highest lignocellulase activity, that is activities towards cellulose, hemicellulose and lignin, and possess the greatest potential for industrial use because of their concomitantly high cellulase activities, including filter paper activity and in addition, xylanase activity. The anaerobic, thermophilic and ethanogenic bacterium Clostridium thermocellum has great potential for use in consolidated bioprocessing for a more cost effective production of biofuels. However, its application is still hindered by such obstacles as end-product inhibition, i.e. feedback inhibition to cellulase activity by cellobiose. To increase cellulase activity and ethanol production, the copy number of β-glucosidase A (bglA) in C. thermocellum 27405 was increased using shuttle vector pIBglA to lower the end-product inhibition of cellulase. Using a modified electrotransformation protocol, C. thermocellum transformant (+MCbglA) harbouring pIBglA was successfully produced. The β-glucosidase activity of +MCbglA was 2.3- and 1.6-fold greater than wild-type (WT) during late log and stationary phases of growth, respectively. Similarly, total cellulase activity of +MCbglA was shown to be 1.7-, 2.3- and 1.6-fold greater than WT during, log, late log and stationary phases of growth. However, there was no significant correlation found between increased cellulase production and increased ethanol titres for +MCbglA compared to the WT, perhaps due to the accumulation of toxic end-products (i.e. ethanol). We successfully increased total cellulase activity by increased expression of bglA and thereby increased the productivity of C. thermocellum during the hydrolysis stage in consolidated bioprocessing. Our work also provides insight into the complex metabolism of C. thermocellum for future further improvement of this strain. The co-culture of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum has great potential in the production of biofuels because it will consolidate the hydrolysis and fermentation steps and potentially increase bioethanol titres. However, there is little knowledge of the industrial application of this kind of co-culture such as substrate conditions and the number of generations for stable co-culture in addition to the effect of ethanol titres. The goal of this study was to develop a stable co-culture of C. thermocellum 27405 and T. saccharolyticum 31097 which can produce greater ethanol titres than mono-cultures in batch fermentation. Comparison of C. thermocellum and T. saccharolyticum growth in reducing sugar (1% (w/v) cellobiose and 0.5% (w/v) xylose) and polysaccharide (1% (w/v) Avicel and 0.5% (w/v) cellobiose) media, showed that T. saccharolyticum could grow 2-fold faster in reducing sugar medium compared to C. thermocellum, while C. thermocellum grew to 2.3-fold greater turbidity in polysaccharide medium in mono-cultures. Subsequent co-culture batch cultures revealed that both strains could only co-exist for complete cell culture in reducing sugar medium, as confirmed by biomarker genes (bglA and xylB, respectively) detected by PCR, while in the subsequent subcultures only T. saccharolyticum was detected. In polysaccharide medium, both strains were detected continuously for 4 generations in batch culture trials, using the same biomarker genes. After the fourth continuous subculture, the co-culture required re-establishing or further media optimization due to growth inhibition of strains. Additionally, the ethanol titres also increased by 2.01-fold in the first and second subcultures compared to the mono-cultures. However, third and fourth subcultures did not have significantly different ethanol titres. Nonetheless, C. thermocellum and T. saccharolyticum co-culture has potential application if added during the hydrolysis stage of complex polysaccharides but not if added to simple sugars such as short poly- and oligo-saccharides produced during the fermentation stage. All of the work presented here in this thesis, focuses on the potential exploitation of bacteria to improve the economic feasibility of biofuels from stages of pretreatment, to hydolysis and fermentation. Due to the large variety and extreme environmental resistance, as well as genetic advances in prokaryotic systems the potential to improve existing bacterial systems or isolate new strains for industrial application is immense.

Dr. Ling Zhang

Cystathionine gamma-lyase/hydrogen sulfide system and glucose homeostasis 

CO-SUPERVISORS: Drs. Rui Wang and Guangdong Yang  (formerly with Biology)    
ABSTRACT:   Research in the last twenty years has transformed the role of hydrogen sulfide (H2S) being perceived from a toxic gas to a gaso-transmitter with diverse physiological and pathological significance. Cystathionine gamma-lyase (CSE) is the major enzyme responsible for the endogenous production of H2S in pancreatic β cells and liver cells, which are two key types of cells to regulate glucose level. The hallmarks of type 2 diabetes mellitus consist of insulin resistance, pancreatic β-cell dysfunction, and increased endogenous glucose production by liver. Pathophysiological implications of the CSE/H2S system in both type 1 and type 2 diabetes as well as diabetic complications have been reported before. Previous studies in our laboratory have shown that endogenous H2S mainly produced from CSE inhibited insulin release via activating KATP channels and decreased H2S production was observed after exposure to elevated glucose level in pancreatic β cells. However, the effect of glucose on CSE gene expression in pancreatic β cells and the underlying mechanism have not been thoroughly explored. Liver, an important organ to control glucose level, has relatively high amount of H2S production compared to other organs and CSE has been reported to be the major enzyme responsible for it. The aim of the current study was to investigate the effect of glucose on CSE expression in INS-1E cells (insulin-secreting β cells) and the role of H2S on basal and insulin-stimulated glucose uptake, glycogen synthesis and gluconeogenesis in human hepatoma HepG2 cells as well as the underlying mechanisms. In the present study, a significant decrease of CSE mRNA and protein expression by high glucose was observed in INS-1E cells and freshly isolated rat pancreatic islets. This regulation required Sp1 (specific protein-1) and p38 MAPK phosphorylation. Glucose stimulated the phosphorylation of Sp1 via p38 MAPK activation, which led to decreased CSE promoter activity and subsequent downregulation of CSE gene expression. Diminished H2S production through altered CSE activity and expression by high glucose may be involved in the fine control of glucose stimulated insulin secretion. Besides insulin secretion, insulin actions through adipose tissues, skeletal muscle and liver are also critical for blood glucose control. In liver cells, incubation with NaHS (a H2S donor) significantly impaired basal and insulin-stimulated glucose uptake and glycogen storage in HepG2 cells. DL-propargylglycine (PPG), as an irreversible inhibitor of the H2S synthesizing enzyme CSE, significantly improved glycogen storage in HepG2 cells. These impairing effects of H2S did not appear to be due to loss of cell viability, but were mediated by AMPK inhibition. We also found that H2S increased gluconeogenesis in HepG2 cells with pyruvate and lactate as substrate in association with increased phosphoenolpyruvate carboxykinase (PEPCK) activity. Decreased glucokinase activity and enhanced glycogenolysis were also observed when the cells were treated with NaHS. Moreover, adenovirus-mediated CSE overexpression had similar effects as that of low dose of exogenous H2S (below 100 μM) on glycogen storage. These data suggest that CSE/H2S system in the liver is an important potential target for regulating insulin sensitivity and glucose metabolism. A detailed understanding of the mechanisms correlating CSE/H2S system and insulin secretion, insulin action as well as glucose homeostasis under physiological or diabetes like conditions may help develop improved treatment strategies for diabetes, considering the importance of these events within the context of pancreatic β cell function and liver function. 

Dr. Bruce Rosa

Improving novel gene discovery in high-throughput gene expression datasets

SUPERVISOR: Dr. Wensheng Qin  (Biology)    
ABSTRACT: High-throughput gene expression datasets (including RNA-seq and microarray datasets) can quantify the expression level of tens of thousands of genes in an organism, which allows for the identification of putative functions for previously unstudied genes involved in treatment/condition responses. For static (single timepoint) high-throughput gene expression experiments, the most common first analysis step to discover novel genes is to filter out genes based on their degree of differential expression and the amount of inter-replicate noise. However, this filtering step may remove genes with very high baseline expression levels, and genes with important functional annotations in the experiment being studied. Chapter 2 presents a novel knowledge-based clustering approach for novel gene discovery, in which known functionally important genes as well as genes with very high expression levels (which would typically be removed by a strict fold change filter) are saved prior to filtering. In stress-related experiments on plants (including Arabidopsis), novel gene discovery is complicated by stress-induced disruption of circadian rhythm pathways, leading to differential expression of many genes which are not involved in adaptive stress responses. Chapter 3 presents the PRIISM (Pattern Recomposition for the Isolation of Independent Signals in Microarray data) algorithm, which is a frequency-based method which is able to differentiate and isolate circadian-disruption signals, improving novel gene discovery in time-series stress-response datasets. Another major factor limiting the effectiveness of novel gene discovery in time-series datasets is the experimenter’s choice of timepoints to sample; The identification of important novel treatmentresponse genes is strongly dependent on sampling the timepoints at which the most target response genes are the most significantly differentially expressed. Although there may be several other timeseries datasets with similar treatments available, there is currently no approach in the literature for using the information in these datasets to guide timepoint selection in a new experiment. Chapter 4 3 presents a new machine-learning model called Optimal Timepoint Selection (OTS) to automatically design optimized sampling rates for microarray and RNA-seq experiments based on the expression data of known treatment-response genes in existing datasets.

Dr. Mehdi Dashtban

Engineering fungi for large-scale production of cellulase

SUPERVISOR: Dr. Wensheng Qin  (Biology)    
ABSTRACT: Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi produce large amounts of extracellular cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β- glucosidases that work efficiently on cellulolytic residues in a synergistic manner. The ascomycete Hypocrea jecorina (anamorph Trichoderma reesei), an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. However, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for enzyme induction in this organism are not completely understood. In this study, the optimum conditions for cellulase production by T. reesei strains were investigated. Three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the latter group including 4 pure polysaccharides and 3 lignocelluloses. Maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred after 120 h of incubation, while RUT-C30 had the greatest cellulase activity after around 72 h. Maximum cellulase production was 0.035, 0.42 and 0.33 μmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production with the ability to grow on microcrystalline cellulose was positively correlated in QM9414 and negatively correlated in RUT-C30. Although T. reesei is widely used as an industrial strain for cellulase production, its low yield of β-glucosidase has limited its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, inhibiting further cellulase production. In order to improve β-glucosidase production and ultimately overall cellulase activity of T. reesei, a thermostable β-glucosidase gene from the fungus Periconia sp. was engineered into the genome of the T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher β-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high cellulase activity (about 39.0 FPU/mg) at 24 h of incubation, whereas the parent strain showed almost no cellulase activity at 24 h of incubation. The recombinant β- glucosidase was thermotolerant and remained fully active after two-hour incubation at temperatures as high as 60 °C. Additionally, it maintained about 88% of its maximal activity after a four-hour incubation at 25 °C across a wide range of pH values from 3.0 to 9.0. Furthermore, an enzymatic hydrolysis assay using untreated, NaOH- or Organosolv-pretreated barley straw or microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. These features suggest that the transformants can be used for β-glucosidase production as well as improving biomass conversion using cellulases. Xylitol, a naturally occurring five-carbon sugar alcohol derived from D-xylose, is currently in high demand by industries for its sweetening and anti-microbial properties. Biotechnological methods can be used for large-scale xylitol production since its current industrial production relies on chemical methods which are costly and energy intensive. While T. reesei is capable of selectively using D-xylose for xylitol production as an intermediate metabolite, its production can be enhanced by genetic engineering of the metabolic pathway. In this study, two T. reesei mutant strains were used, including a single mutant in which the endogenous xylitol dehydrogenase gene was deleted (Δxdh1), and a double mutant in which L-arabinitol-4-dehydrogenase was additionally deleted (Δlad1Δxdh1). The widely available agricultural residue barley straw was used for xylitol production by the strains after its pretreatment using NaOH- and Organosolv-pretreatment methods. High xylitol production by both strains was achieved when barley straw (untreated or pretreated) was supplemented with 2% D-xylose, whereas the D-glucose supplementation did not increase production. The highest production of xylitol was 6.1 and 13.22 g/L obtained after 96 and 168 h of incubation, respectively, using medium supplemented with 2% Organosolv-pretreated barley straw and 2% D-xylose by single and double T. reesei mutant strains, respectively. Maximum saccharification of barley straw was observed after 120 h of incubation for NaOH-pretreated by single (692 ± 1.01 mg/g reducing sugars) and double T. reesei mutant (685 ± 11.9 mg/g reducing sugars) strains, respectively. Moreover, the significant increase of xylitol production by the T. reesei strains using medium containing Organosolv-pretreated barley straw supplemented with Dxylose suggests that the pretreatment in combination with the added sugar favored xylitol production. These results suggest that agricultural residues, such as barley straw, could be a suitable resource for bioconversion to produce value-added products such as xylitol.