Eugéne van Rensburg

Progress and challenges in the engineering of non-cellulolytic microorganisms for consolidated bioprocessing

Lignocellulosic biomass is an abundant, renewable feedstock for the production of fuels and chemicals, if an efficient and affordable conversion technology can be established to overcome its recalcitrance. Consolidated bioprocessing (CBP) featuring enzyme production, substrate hydrolysis and fermentation in a single step is a biologically mediated conversion approach with outstanding potential if a fit-for-purpose microorganism(s) can be developed. Progress in developing CBP-enabling microorganisms is ongoing by engineering (i) naturally cellulolytic microorganisms for improved product-related properties or (ii) non-cellulolytic organisms exhibiting high product yields to heterologously produce different combinations of cellulase enzymes. We discuss progress on developing yeast and bacteria for the latter strategy and consider further challenges that require attention to bring this technology to market.

A Kinetic Model for Simultaneous Saccharification and Fermentation of Avicel With Saccharomyces cerevisiae

This work describes a numerical model for predicting simultaneous saccharification and fermentation of Avicel, an insoluble crystalline cellulose polymer. Separate anoxic cultivations of 40 g/L glucose and 100 g/L Avicel were conducted to verify model predictions and obtain parameters to describe the reaction kinetics. Saccharification of Avicel was achieved with Trichoderma reesei cellulases from the enzyme preparation Spezyme CP with an enzyme loading of 10 FPU/g cellulose. Cultivations were supplemented with 50 IU/g cellulose of β‐glucosidase from Novozym 188 to prevent product inhibition by cellobiose. Saccharomyces cerevisiae MH‐1000 is a robust industrial strain and was used to ferment glucose to ethanol, glycerol, and carbon dioxide. The numerical model presented in this paper differs from previous models by separating the endoglucanase and exoglucanase enzyme kinetics and allowing for inhibitive site competition. Assuming all enzymes remain active and that each enzyme complex has a corresponding constant specific activity, the model is capable of predicting adsorbed enzyme concentrations with reasonable accuracy. Comparison of predicted values to experimental measurements indicated that the numerical model was capable of capturing the significant elements involved with cellulose conversion to ethanol. Biotechnol. Bioeng. 2011; 108:924–933.

Enhancing the enzymatic digestibility of sugarcane bagasse through the application of an ionic liquid in combination with an acid catalyst.

Various ionic liquids have been identified as effective pretreatment solvents that can enhance the cellulose digestibility of lignocellulose by removing lignin, one of the main factors contributing to the recalcitrant nature of lignocellulose. 1‐Butyl‐3‐methylimidazolium methylsulfate ([BMiM]MeSO4) is a potential delignification reagent, hence its application as a pretreatment solvent for sugarcane bagasse (SB) was investigated. The study also evaluated the benefit of an acid catalyst (i.e., H2SO4) and the effect of pretreatment conditions, which varied within a time and temperature range of 0–240 min and 50–150°C, respectively. The use of an acid catalyst contributed to a more digestible solid and a higher degree of delignification. However, the [BMiM]MeSO4‐H2SO4 combination failed to produce a fully digestible solid, as a maximum cellulose digestibility of 77% (w/w) was obtained at the optimum pretreatment condition of 125°C for 120 min. Furthermore, up to half of the lignin content could be extracted during pretreatment, while simultaneously extensive, sometimes complete, removal of xylan, the presence of which, also hampers cellulose digestibility. Hence, [BMiM]MeSO4 has been identified an effective pretreatment solvent for SB as the application thereof both significantly improved digestibility, and simultaneously removed two of the main factors contributing to the recalcitrant nature of lignocellulose. As xylan and lignin have potential value as precursor chemicals, the existing process may in future be extended toward substrate fractionation, a biorefinery concept where value is added to all feedstock constituents.

The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture

Two recombinant strains of Saccharomyces cerevisiae Y294 producing cellulase using different expression strategies were compared to a reference strain in aerobic culture to evaluate the potential metabolic burden that cellulase expression imposed on the yeast metabolism. In a chemically defined mineral medium with glucose as carbon source, S. cerevisiae strain Y294[CEL5] with plasmid-borne cellulase genes produced endoglucanase and β-glucosidase activities of 0.038 and 0.30 U mg dry cell weight−1, respectively. Chromosomal expression of these two cellulases in strain Y294[Y118p] resulted in no detectable activity, although low levels of episomally co-expressed cellobiohydrolase (CBH) activity were detected. Whereas the biomass concentration of strain Y294[CEL5] was slightly greater than that of a reference strain, CBH expression by Y294[Y118p] resulted in a 1.4-fold lower maximum specific growth rate than that of the reference. Supplementation of the growth medium with amino acids significantly improved culture growth and enzyme production, but only partially mitigated the physiological effects and metabolic burden of cellulase expression. Glycerol production was decreased significantly, up to threefold, in amino acid-supplemented cultures, apparently due to redox balancing. Disproportionately higher levels of glycerol production by Y294[CEL5] indicated a potential correlation between the redox balance of anabolism and the physiological stress of cellulase production. With the reliance on cellulase expression in yeast for the development of consolidated bioprocesses for bioethanol production, this work demonstrates the need for development of yeasts that are physiologically robust in response to burdens imposed by heterologous enzyme production.

Evaluation of steam-treated giant bamboo for production of fermentable sugars

Giant bamboo plantations are currently being established in the Southern Africa region and can be considered as potential lignocellulosic feedstock for the production of second generation bioethanol. In this study, giant bamboo internodal material was subjected to sulphur dioxide (SO2) impregnated steam pretreatment prior to enzymatic hydrolysis. The effect of temperature, residence time, and acidity on the overall sugar recovery and byproduct formation was studied using response surface response technology according to a central composite experimental design (CCD) at a fixed SO2 concentration of 2.5% (w/w liquid) after impregnation. The results showed that pretreatment conditions with combined severity factor (CSF) values and enzyme dosages greater than 1.72 and 30 FPU/g water insoluble solid, respectively, were required to obtain an efficient glucan digestibility and a good overall glucose recovery. Up to 81.2% of the sugar in the raw material was recovered for a CSF of 2.25. However, considering overall sugar yield and byproducts concentration, the pretreated material obtained with a CSF of 1.62 can be considered as the most appropriate for SSF experiments using a xylose‐utilizing yeast. At these conditions, it could be possible to obtain up to 247 L of ethanol per dry ton of giant bamboo considering hexose and pentose sugars fermentation. This amount could be increased up to 292 L of ethanol per dry ton of giant bamboo with the maximum sugar yield obtained (CSF = 2.25) if the microorganism possesses robust fermentative characteristics as well as a high resistance to pretreatment by‐products.

Opportunities and prospects of biorefinery-based valorisation of pulp and paper sludge.

The paper and pulp industry is one of the major industries that generate large amount of solid waste with high moisture content. Numerous opportunities exist for valorisation of waste paper sludge, although this review focuses on primary sludge with high cellulose content. The most mature options for paper sludge valorisation are fermentation, anaerobic digestion and pyrolysis. In this review, biochemical and thermal processes are considered individually and also as integrated biorefinery. The objective of integrated biorefinery is to reduce or avoid paper sludge disposal by landfilling, water reclamation and value addition. Assessment of selected processes for biorefinery varies from a detailed analysis of a single process to high level optimisation and integration of the processes, which allow the initial assessment and comparison of technologies. This data can be used to provide key stakeholders with a roadmap of technologies that can generate economic benefits, and reduce carbon wastage and pollution load.

Paper sludge (PS) to bioethanol: evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentation.

Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20 FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11 FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2g/L at 66.9% and 45.5 g/L at 78.2%, respectively.

Engineering Saccharomyces cerevisiae for direct conversion of raw, uncooked or granular starch to ethanol

Abstract The production of raw starch-degrading amylases by recombinant Saccharomyces cerevisiae provides opportunities for the direct hydrolysis and fermentation of raw starch to ethanol without cooking or exogenous enzyme addition. Such a consolidated bioprocess (CBP) for raw starch fermentation will substantially reduce costs associated with energy usage and commercial granular starch hydrolyzing (GSH) enzymes. The core purpose of this review is to provide comprehensive insight into the physiological impact of recombinant amylase production on the ethanol-producing yeast. Key production parameters, based on outcomes from modifications to the yeast genome and levels of amylase production, were compared to key benchmark data. In turn, these outcomes are of significance from a process point of view to highlight shortcomings in the current state of the art of raw starch fermentation yeast compared to a set of industrial standards. Therefore, this study provides an integrated critical assessment of physiology, genetics and process aspects of recombinant raw starch fermenting yeast in relation to presently used technology. Various approaches to strain development were compared on a common basis of quantitative performance measures, including the extent of hydrolysis, fermentation-hydrolysis yield and productivity. Key findings showed that levels of α-amylase required for raw starch hydrolysis far exceeded enzyme levels for soluble starch hydrolysis, pointing to a pre-requisite for excess α-amylase compared to glucoamylase for efficient raw starch hydrolysis. However, the physiological limitations of amylase production by yeast, requiring high biomass concentrations and long cultivation periods for sufficient enzyme accumulation under anaerobic conditions, remained a substantial challenge. Accordingly, the fermentation performance of the recombinant S. cerevisiae strains reviewed in this study could not match the performance of conventional starch fermentation processes, based either on starch cooking and/or exogenous amylase enzyme addition. As an alternative strategy, the addition of exogenous GSH enzymes during early stages of raw starch fermentation may prove to be a viable approach for industrial application of recombinant S. cerevisiae, with the process still benefitting from amylase production by CBP yeast during later stages of cultivation.

Incorporating anaerobic co-digestion of steam exploded or ammonia fiber expansion pretreated sugarcane residues with manure into a sugarcane-based bioenergy-livestock nexus

The co-digestion of pretreated sugarcane lignocelluloses with dairy cow manure (DCM) as a bioenergy production and waste management strategy, for intensive livestock farms located in sugarcane regions, was investigated. Ammonia fiber expansion (AFEX) increased the nitrogen content and accelerated the biodegradability of sugarcane bagasse (SCB) and cane leaf matter (CLM) through the cleavage of lignin carbohydrate crosslinks, resulting in the highest specific methane yields (292-299 L CH4/kg VSadded), biogas methane content (57-59% v/v) and biodegradation rates, with or without co-digestion with DCM. To obtain comparable methane yields, untreated and steam exploded (StEx) SCB and CLM had to be co-digested with DCM, at mass ratios providing initial C/N ratios in the range of 18 to 35. Co-digestion with DCM improved the nutrient content of the solid digestates, providing digestates that could be used as biofertilizer to replace CLM that is removed from sugarcane fields during green harvesting.