Cofermentation

Toon, S.T., et al. Enhanced Cofermentation of Glucose and Xylose by Recombinant Saccharomyces Yeast Strains in Batch and Continuous Operating Modes , Appl Biochem. Bbtechnoi, 63-65, 243 -255 (1997). 

The use of recombinant microorganisms for cofermentation is one of the most promising approaches in the field of bioethanol production, though their use for large-scale industrial processes still requires fine-tuning of the reliability of the entire process (2). The technical hurdles of cofermentation increase when real biomass hydrolysates have to be fermented. In fact, whatever the biomass pretreatment, the formation of degradation byproducts that could inhibit the fermentation usually requires the addition of a further detoxification step. Therefore, the production of ethanol from hydrolysates should be considered in its entirety, from the optimal pretreatment to the choice of the proper fermentation process. 

Figure 4 presents data on the cofermentation of glucose and xylose. The production of ethanol still appeared to be at the expense of glucose consumption. Note that systems E and F did not show significant differences whereas the amount of ethanol in system D was roughly 25% higher. This could imply that a threshold cell concentration is necessary. 

Fig. 4. Cofermentation of model solutions of glucose and xylose. Systems D, E, and F contained beads of S. cerevisiae and P. stipitis coimmobilized with a loading ratio of P. stipitis/ S. cerevisiae of 4 g/g. The gel fractions were as follows system D, 0.10 system E, 0.05 and system F, 0.025 g/g. The total cells concentrations were as follows system D, 4.80 x 10u system E, 2.40 x 10u and system F, 1.20 x 1011 cells/L.

Fig. 5. Cofermentation of model solutions of glucose and xylose in FBR. The total initial cell concentration was 6.57 x 10ucells/L and the gel fraction was 0.1 g/g. The ration of P. stipitis/S. cerevisiae was 4 g/g. The number of beads inside the column was 3286. The airflow in the column and inside the reservoir was 0.030 wm.

Fig. 6. Effect of aeration (0.030 vvm) through Teflon-made diffusers (system 1) and acetone addition (50 mM) (system 2) on cofermentation of synthetic solutions of glucose and xylose in STR containing marbles. In both cases, the beads were prepared with an internal ratio of P. stipitis/S. cerevisiae of 4 g/g, and the gel fraction was almost 0.1 g/g. The initial cell concentration in system 1 was 8.6 x 1011 cells/L while that in system 2 was 8.42 x 10u cells/L.

Fig. 7. Cofermentation of model solutions of glucose and xylose with P. st ip it is and S. cerevisiae separately immobilized (system G) and coimmobilized (system H) in Ca-alginate beads. The gel fraction in system G was made of 0.20 g/g of beads containing P. stipitis and 0.05 g/g of beads containing S. cerevisiae. The initial concentrations of P. stipitis and S. cerevisiae cells were 5.64 x 1012 and 1.89 x 10u cells/L, respectively. The gel fraction in system H was made of 0.25 g/g of beads containing P. stipitis and S. cerevisiae coimmobilized with a loading ratio of P. stipitis/S. cerevisiae of 4 g/g of dry cells. The total cells concentration was 6.01 x 1012 cells/L.

Fig. 8. Cofermentation of detoxified hydrolysates in shake flasks (A and B) and in STR modified with perforated cylinder. For the experimental conditions of graph A, refer to graph G in Fig. 7, and for those of graphs B and C refer to graph H in Fig. 7.

Considering the results obtained in the test runs of synthetic solutions, the cofermentation of detoxified hydrolysates was carried out at flask scale using the same experimental conditions as described in Fig. 7 the results are presented in Fig. 8. The ethanol yields were slightly higher than those obtained working with synthetic solutions, but they were still comparable with respect to the composition of the sugar syrup used (0.39 ge/gs). It is likely that in the absence of furfural, the residual amount of acetic acid in the detoxified hydrolysate could have enhanced ethanol production (21,22). 

Figure 15.9 Block diagram for bioethanol from ligno-cellulosic biomass [27], CBP = consolidated biprocessing, CF = cofermetation, SSF = simultaneous saccharification and fermentation, SSCF = simultaneous saccharification and cofermentation. Components C = cellolose,...

Ho, N. W. Y., Chen, Z. D., and Brainard, A. P., Genetically engineered saccharomyces yeast capable of effective cofermentation of glucose and xylose. Applied Environmental Microbiology 1998, 64 (5), 1852-59. 

Veiga-da-Cunha, M., Foster, M.A. (1992). Sugar-glycerol cofermentations in lactobaciUi The fate of lactate. J. Bacteriaol, 174, 1013-1019. 

Howell et al. (2006) also carried out extensive metabolite profiling of the volatile metabolites produced in wines made by cofermentation with Saccharomyces strains. They found that cofermentation not only produced a metabolite profile different from wines made by monoculture fermentation, but that blends of the monoculture fermentation wine were also notably different. These various finding therefore strengthen the anecdotal observations of winemakers that cofermentation with two or more strains can potentially increase the flavour complexity of wines. These studies also suggest that cofermentation of yeasts with greater metabolic dissimilarity are likely to produce greater differences in the profile of volatile and non-volatile metabolites, as has been observed when mn-Saccharomyces yesists are cofermented with Saccharomyces (Sect. 8D.6.4). Additional studies are needed to understand better the complex metabolic interactions and to describe the major impacts on wine composition and flavour. 

Cofermentation with two or more strains has recently been exploited to enhance the aromatic profile of Sauvignon Blanc wines, in which a non-volatile 5-cysteinylated precursor (3-(hexan-1 -ol)-L-cysteine) is first hydrolysed by a carbon-sulfur lyase and subsequently esterifled by alcohol acetyltransferase (Sect. 8D.5.2). Strains of Saccharomyces cerevisiae vary in ability to carry out the two reactions (Dubourdieu et al. 2006 Swiegers and Pretorius 2007). Cofermentation with two strains, one having higher hydrolytic function (release of 3-MH) and the other higher esterification activity, substantially enhanced formation of 3-MHA, thereby increasing the passion-fruit aroma when compared to monoculture wines. The authors showed that interaction between the two strains produced more 3-MH and 3-MHA... 

A practical problem with non-Saccharomyces yeasts is that few strains are capable of completing fermentation, and in many cases only a small proportion of grape sugar will be fermented. Two strategies have evolved to enable complete fermentation these are cofermentation with a robust Saccharomyces strain and sequential fermentation, in which the non-Saccharomyces yeast and Saccharomyces strain are inoculated successively, in order to complete fermentation. Several studies have... 

Hanseniaspora uvarum (anamorph Kloeckera apiculata) is commonly the major yeast present on the grape berry and in musts and juices, but due to low tolerance to ethanol, populations decline quickly in the presence of Saccharomyces cerevisiae. Strains are typically characterised by low fermentative ability and high production of acetic acid, ethyl acetate and acetaldehyde, which render such strains more suitable to vinegar production. Nevertheless, Ciani and Maccarelli (1998) surveyed 37 isolates and found considerable variability, with some strains producing concentrations of these compounds approaching concentrations present in wines made with Saccharomyces cerevisiae (Table 8D.6). Cofermentation fermentation with Saccharomyces cerevisiae can produce wines with an acceptable balance of volatile and non-volatile compounds and sensory scores (Ciani et al. 2006 Jemec and Raspor 2005 Jolly et al. 2003b Zohre and Erten 2002). 

Hanseniaspora guilliermondii can produce more ethanol than Hanseniaspora uvarum and has a better balance of volatile and non-volatile compounds, including production of 2-phenylethyl acetate, which elicits a fruity, honey, rose-like aroma (Table 8D.6) (Rojas et al. 2003 Swiegers et al. 2005). Cofermentation with Saccha-romyces cerevisiae has potential for aroma enhancement of wine. 

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