PhD student, University of Cape Town


Improve the utilization of glucose and xylose for higher yields and productivities of bioethanol

Bioethanol plays a significant role in the world of biofuel. However, majority of bioethanol is produced from edible food crops such as corn which causes increase in demand of vacant lands for food production, and subsequently increase in the cost of food manufacturing. Therefore, alternative raw materials for bioethanol production are sorted, such as sugarcane bagasse, which is a waste material from the sugar industry. This research focuses on the study of the production of bioethanol from sugarcane bagasse. In particular, xylose and glucose hydrolyzed from the hemicellulose and cellulose fraction of bagasse in the pretreatment and hydrolysis steps will be fermented in this project. A high and efficient conversion of glucose and xylose is necessary for commercial viability of bioethanol production. In view of the methods presented (e.g. sequential cultures in one and two reactors, co-cultures) for co-fermentation of glucose and xylose, utilizing two different microorganisms (e.g. Zymomonas mobilis and Pichia stipitis for efficient fermentation of the glucose and xylose respectively) is an option. However, there are numerous problems associated with co-culturing. Firstly, xylose is only converted if the glucose concentration is adequately low due to catabolite repression. In order to increase xylose conversion, a low glucose concentration is required. Therefore two continuous reactors in series will be tested, in which immobilized Z.mobilis is inoculated in the first reactor to convert glucose rapidly, and a second reactor with immobilized P.stipitis to convert xylose. These microorganisms will be immobilized by entrapment in calcium alginate beads. Secondly, ethanol tolerance of P.stipitis is low, but in continuous conditions, even in industrial proportion, ethanol is seldom over 35 g/l in which reactions will be inhibited in P.stipitis above the threshold. Thirdly, ethanol fermentation by Z.mobilis can only occur under anaerobic conditions while xylose conversion is optimum under microaerobic conditions. Therefore, oxygen will only be sparged into the second reactor of immobilized P.stipitis.
The conversion of glucose and xylose with suspended and immobilized cultures will be modeled from the kinetic results of literature and experimental work. Examples of parameters that will be studied are initial sugar concentration, oxygen flow rates, and dilution rate. The model will be refined with the use of MATLAB.


Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4 (pZB5)

Abstract: Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in κ-carrageenan beads having a diameter of 1.5–2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2–4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5–15.3 g/L·h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars.

Pub.: 01 Mar '00, Pinned: 31 Aug '17

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis.

Abstract: Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.

Pub.: 30 Mar '06, Pinned: 31 Aug '17

Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae: a review and perspective.

Abstract: Efficient xylose utilization is one of the most important pre-requisites for developing an economic microbial conversion process of terrestrial lignocellulosic biomass into biofuels and biochemicals. A robust ethanol producing yeast Saccharomyces cerevisiae has been engineered with heterologous xylose assimilation pathways. A two-step oxidoreductase pathway consisting of NAD(P)H-linked xylose reductase and NAD(+)-linked xylitol dehydrogenase, and one-step isomerase pathway using xylose isomerase have been employed to enable xylose assimilation in engineered S. cerevisiae. However, the resulting engineered yeast exhibited inefficient and slow xylose fermentation. In order to improve the yield and productivity of xylose fermentation, expression levels of xylose assimilation pathway enzymes and their kinetic properties have been optimized, and additional optimizations of endogenous or heterologous metabolisms have been achieved. These efforts have led to the development of engineered yeast strains ready for the commercialization of cellulosic bioethanol. Interestingly, xylose metabolism by engineered yeast was preferably respiratory rather than fermentative as in glucose metabolism, suggesting that xylose can serve as a desirable carbon source capable of bypassing metabolic barriers exerted by glucose repression. Accordingly, engineered yeasts showed superior production of valuable metabolites derived from cytosolic acetyl-CoA and pyruvate, such as 1-hexadecanol and lactic acid, when the xylose assimilation pathway and target synthetic pathways were optimized in an adequate manner. While xylose has been regarded as a sugar to be utilized because it is present in cellulosic hydrolysates, potential benefits of using xylose instead of glucose for yeast-based biotechnological processes need to be realized.

Pub.: 13 May '17, Pinned: 31 Aug '17

Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

Abstract: Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130 °C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g.L(-1) .h(-1) (first to third batch) for S. stipitis and from 0.38 to 0.81 g.L(-1) .h(-1) for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. This article is protected by copyright. All rights reserved.

Pub.: 20 Jun '17, Pinned: 31 Aug '17