Elsevier

Journal of Biotechnology

Volume 96, Issue 2, 26 June 2002, Pages 155-168
Journal of Biotechnology

Evaluation of a recombinant Klebsiella oxytoca strain for ethanol production from cellulose by simultaneous saccharification and fermentation: comparison with native cellobiose-utilising yeast strains and performance in co-culture with thermotolerant yeast and Zymomonas mobilis

https://doi.org/10.1016/S0168-1656(02)00026-3Get rights and content

Abstract

In the simultaneous saccharification and fermentation to ethanol of 100 g l−1 microcrystalline cellulose, the cellobiose-fermenting recombinant Klebsiella oxytoca P2 outperformed a range of cellobiose-fermenting yeasts used in earlier work, despite producing less ethanol than reported earlier for this organism under similar conditions. The time taken by K. oxytoca P2 to produce up to about 33 g l−1 ethanol was much less than for any other organism investigated, including ethanol-tolerant strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Zymomonas mobilis. Ultimately, it produced slightly less ethanol (maximum 36 g l−1) than these organisms, reflecting its lower ethanol tolerance. Significant advantages were obtained by co-culturing K. oxytoca P2 with S. pastorianus, K. marxianus or Z. mobilis, either isothermally, or in conjunction with temperature-profiling to raise the cellulase activity. Co-cultures produced significantly more ethanol, more rapidly, than either of the constituent strains in pure culture at the same inoculum density. K. oxytoca P2 dominated the early stages of the co-cultures, with ethanol production in the later stages due principally to the more ethanol tolerant strain. The usefulness of K. oxytoca P2 in cellulose simultaneous saccharification and fermentation should be improved by mutation of the strain to increase its ethanol tolerance.

Introduction

Bioethanol has significant environmental advantages over petroleum as a liquid fuel (Duff and Murray, 1996, Philippidis and Smith, 1995) and has the potential to compete with it economically if produced from cheap, renewable lignocellulosic feedstocks such as wood. During the last 30 years substantial progress has been made in the development of technologies for the efficient production of ethanol from lignocellulose, and pioneer commercial plants using genetically-modified organisms are at an advanced stage of planning in the USA (Wyman, 2001).

To prepare lignocellulose for ethanol production, the substrate is normally either hydrolyzed completely to the component sugars using mineral acids (Wright, 1988, Dale and Moreira, 1982) or given a milder pretreatment to solubilise the hemicellulose and lignin, leaving the residual cellulose to be saccharified enzymatically (Philippidis and Hatzis, 1997, Dale, 1987). The enzymatic route has the advantages of reduced sugar loss through side-reactions and is less corrosive of process equipment. Cellulases from the fungus Trichoderma reesei are normally employed, with ethanol production either following the hydrolysis as a separate step or occurring concurrently in a process known as simultaneous saccharification and fermentation (SSF). In a variant of the SSF process, simultaneous saccharification and co-fermentation (SSCF), the hydrolysed hemicellulose and the solid cellulose are not separated after pretreatment, allowing the hemicellulose sugars to be converted to ethanol concurrently with SSF of the cellulose (McMillan et al., 1999, Teixeira et al., 2000).

SSF and SSCF have the advantage that glucose resulting from the hydrolysis is removed by the microorganisms, reducing product inhibition of the enzyme system and allowing an overall faster rate of conversion (Takagi et al., 1977). However product inhibition is not eliminated unless the glucose dimer, cellobiose, is also consumed or hydrolysed by β-glucosidase from the enzyme preparation. As conventional Saccharomyces cerevisiae strains do not metabolise cellobiose, and cellulase preparations with sufficient β-glucosidase activity are expensive to produce (Spindler et al., 1989a), considerable research has been directed towards the use of native cellobiose-utilising yeast strains for SSF, either as an alternative to Saccharomyces, or in co-culture with it (Wyman et al., 1986, Spindler et al., 1992).

A recent alternative approach has been the incorporation of ethanol-producing genes into native cellobiose-utilising bacteria using recombinant DNA techniques. Wood and Ingram (1992) produced a strain (P2) of Klebsiella oxytoca containing chromosomally-integrated pyruvate decarboxylase and alcohol dehydrogenase genes from the ethanol-producing bacterium Zymomonas mobilis. This organism, which was designed specifically for use in SSF processes, produces ethanol rapidly and at high yield from both glucose and cellobiose (Wood and Ingram, 1992) and has been tested in SSF processes with both purified and native cellulosic substrates (Wood and Ingram, 1992, Doran et al., 1994, Moniruzzaman et al., 1997).

By comparing their strain with data reported in the literature for the SSF of microcrystalline cellulose using similar cellulase preparations, Doran and Ingram (1993) concluded that K. oxytoca P2 gives superior results in SSF to those obtained in earlier studies with Saccharomyces strains and cellobiose-utilising yeasts, either alone or in co-culture. Such comparisons tend to be problematic, given the inevitable inter-laboratory variations in the source and concentration of the cellulase, substrate loading, medium composition, inoculum size and cultivation conditions. In a recent study (Golias et al., 2000) we found that K. oxytoca P2, whilst outperforming a S. cerevisiae strain in SSF under comparable conditions, did not produce ethanol as rapidly or in as high a yield as obtained in Ingram's laboratory, and its performance was inferior to that reported earlier for some yeast co-culture studies (Spindler et al., 1988, Spindler et al., 1992). In view of the difficulties associated with inter-laboratory comparisons, and the general lack of data on the performance of K. oxytoca P2 outside Ingram's laboratory, we have carried out an independent laboratory comparison of the performance of this organism and some of the most promising cellobiose-fermenting yeast from previous work. The comparison uses, for each organism, an identical enzyme preparation (Genencor Spezyme CP), substrate source (Sigmacell 50), inoculum size, cultivation system (pH-controlled fleakers) and analytical procedures. We also present for the first time data obtained with co-cultures of K. oxytoca P2 and strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Z. mobilis. The aim of the co-culture studies was to combine the capacity of K. oxytoca P2 for rapid glucose and cellobiose assimilation with the greater thermal and ethanol tolerance of the latter strains.

Section snippets

Strain maintenance and preparation of inocula

K. oxytoca P2 was supplied by Professor L.O. Ingram (University of Florida), and Z. mobilis ZM4 (ATCC 31821) by Professor P. Rogers, University of New South Wales, Australia. Saccharomyces pastorianus (formerly uvarum) ATCC 26602 was obtained from the University of New South Wales culture collection, Brettanomyces custersii CBS 5512 and K. marxianus CBS 712 from the CBS Culture Collection (Delft, the Netherlands) and Brettanomyces clausenii Y-1414 and Candida lusitaniae Y-5394 from the Northern

Comparative performance in SSF of K. oxytoca P2 and cellobiose-fermenting yeasts

SSF experiments using 100 g l−1 Sigmacell 50 and 15 FPU g−1 Spezyme CP cellulase were carried out with K. oxytoca P2, Brettanomyces clausenii, B. custersii and C. lusitaniae (Fig. 1). The pH (5.2) and temperature (35 °C) for K. oxytoca P2 experiments was one of two optimum combinations identified by Doran et al. (1994), for this organism. To take advantage of their greater thermotolerance and ability to grow at lower pH, the combination of 37 °C and pH 5.0 was chosen for the yeast strains,

Discussion

K. oxytoca P2 produced ethanol much more rapidly and to a higher final concentration than pure cultures of any of the cellobiose-fermenting yeasts considered in this study. This was despite the higher thermotolerance of the latter, which permitted the use of a temperature (37 °C) more favourable to cellulase activity in the yeast SSFs. The performance of K. oxytoca P2 was however not as good as reported in the original study (Doran and Ingram, 1993) using the same substrate and Spezyme CE

Conclusions

In SSFs inoculated to a standard inoculum size, K. oxytoca P2 produced up to about 33 g l−1 ethanol far more rapidly than pure cultures of any of the other organisms investigated. The maximum concentration of ethanol achievable (ca. 36 g l−1) was about 16% lower than reported in earlier studies with the strain under comparable conditions (Doran and Ingram, 1993). Ethanol yields exceeded those achievable with cellobiose-fermenting yeasts but were marginally inferior to those obtained in

Acknowledgments

We are grateful to Professor L.O. Ingram for the donation of K. oxytoca strain P2 and for generous advice regarding cultivation procedures, and to Mr S. Lewis, Genencor International Inc., for the donation of Spezyme CP cellulase. The work was supported in part by grants from the Australian Research Council.

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