Marinda Viljoen-Bloom

Microbiology of a biological contactor for winery wastewater treatment

Winery wastewaters are characterised by large seasonal fluctuations in volume and composition and are often discarded with little or no treatment. A rotating biological contactor (RBC) was used to investigate microorganisms associated with the biological treatment of winery wastewater. Extensive biofilms developed on the RBC discs and contained a number of yeast and bacterial species that displayed a dynamic population shift during the evaluation period. This suggested that the naturally occurring microorganisms were able to form a stable biofilm and also reduce the chemical oxygen demand (COD) of winery wastewater (on average 43% with a retention time of 1h). One of the yeast isolates, MEA 5, was able to reduce the COD of synthetic wastewater by 95% and 46% within 24h under aerated and non-aerated conditions, respectively. The yeast isolates could therefore play an important role in the degradation of organic compounds under aerobic conditions, such as those associated with an RBC.

Comparative secretome analysis of Trichoderma asperellum S4F8 and Trichoderma reesei Rut C30 during solid-state fermentation on sugarcane bagasse

BackgroundThe lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF).ResultsComparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Liquid chromatography tandem mass spectrometry analysis of the two fungal secretomes enabled the detection of 815 proteins in total, with 418 and 397 proteins being specific for S4F8 and Rut C30, respectively, and 174 proteins being common to both strains. In-depth analysis of the associated biological functions and the representation of glycoside hydrolase family members within the two secretomes indicated that the S4F8 secretome contained a higher diversity of main and side chain hemicellulases and β-glucosidases, and an increased abundance of some of these proteins compared with the Rut C30 secretome.ConclusionsIn SCB SSF, T. asperellum S4F8 produced a more complex lignocellulolytic cocktail, with enhanced hemicellulose and cellobiose hydrolysis potential, compared with T. reesei Rut C30. This bodes well for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail from T. asperellum for lignocellulosic feedstock hydrolysis.

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance

In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and β-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.

The Biochemistry of Malic Acid Metabolism by Wine Yeasts – A Review

L-Malic acid is an essential intermediate of cell metabolism and the D,L-racemic mixture is used as an acidulant in a variety of foods and beverages. In the wine industry, it plays an important role during grape must fermentation, contributing to the “fixed acidity” that is important. The latter is important in defining the quality of wine. Genetic and biochemical characterisation of the L-malate utilising pathways in several yeast species has indicated that the physiological role and regulation of L-malate metabolism differ significantly between the K(-) and K(+) yeasts. A variety of factors influence the ability of a yeast species to effectively degrade L-malate, including the conditions associated with wine fermentation and the yeast’s intrinsic ability to transport and effectively metabolise L-malate inside the cell. This paper reviews the ability of different yeast species associated with grapes and wine to degrade extracellular L-malate, and the underlying mechanisms in the differential utilisation of L-malate by different yeast species.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases

The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch‐based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon‐optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α‐amylase (SFA1) genes were δ‐integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1‐SFA1] and MEL2[TLG1‐SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch‐to‐ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre‐treatment or commercial enzyme addition) using industrial yeast strains co‐secreting both a glucoamylase and α‐amylase. Biotechnol. Bioeng. 2015;112: 1751–1760.

Utilising grape pomace for ethanol production.

Chemical analyses of grape pomace revealed the presence of significant amounts of fermentable sugars that are retained in the pomace after pressing of the grapes. Furthermore, treatment of the pomace with purified hydrolases indicated that the enzymatic biodegradation of the pomace could release additional fermentable sugars. We isolated and evaluated yeast strains associated with grape pomace for their ability to hydrolyse the complex polysaccharides found in grape pomace and to utilise the fermentable sugars for the production of ethanol. Two Pichia rhodanensis isolates were able to partially hydrolyse the pomace polysaccharides, but fermentation of the pomace resulted only in a small increase in the amount of ethanol produced. The study revealed that significant amounts of ethanol could be obtained from the residual sugars associated with grape pomace. However, the complex structure of the pomace polysaccharides apparently renders it unsusceptible to efficient hydrolysis under fermentative conditions.

Consolidated bioprocessing of raw starch with Saccharomyces cerevisiae strains expressing fungal alpha-amylase and glucoamylase combinations

&NA; Cost‐effective consolidated bioprocessing (CBP) of raw starch for biofuel production requires recombinant Saccharomyces cerevisiae strains expressing &agr;‐amylases and glucoamylases. Native Aureobasidium pullulans apuA, Aspergillus terreus ateA, Cryptococcus sp. S‐2 cryA and Saccharomycopsis fibuligera sfiA genes encoding raw‐starch &agr;‐amylases were cloned and expressed in the S. cerevisiae Y294 laboratory strain. Recombinant S. cerevisiae Y294[ApuA] and Y294[AteA] strains produced the highest extracellular &agr;‐amylase activities (2.17 U mL‐1 and 2.98 U mL‐1, respectively). Both the ApuA and AteA &agr;‐amylases displayed a preference for pH 4 to 5 and retained more than 75% activity after 5 days at 30°C. When ateA was co‐expressed with the previously reported Aspergillus. tubingensis glucoamylase gene (glaA), the amylolytic S. cerevisiae Y294[AteA‐GlaA] strain produced 45.77 g L‐1 ethanol after 6 days. Ethanol production by this strain was improved with the addition of either 2.83 &mgr;L STARGEN 002 (54.54 g L‐1 ethanol and 70.44% carbon conversion) or 20 &mgr;L commercial glucoamylase from Sigma‐Aldrich (73.80 g L‐1 ethanol and 90.19% carbon conversion). This is the first report of an engineered yeast strain that can replace up to 90% of the enzymes required for raw starch hydrolysis, and thus contributes to the realisation of a CBP yeast for starch‐based biofuel production.