Microbial contamination of fuel ethanol fermentations

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1 Letters in Applied Microbiology ISSN UNDER THE MICROSCOPE Microbial contamination of fuel ethanol fermentations M. Beckner, M.L. Ivey and T.G. Phister Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, USA Key words bioethanol, Dekkera bruxellensis, lactobacilli. Correspondence Trevor G. Phister, Department of Food, Bioprocessing and Nutrition Sciences, 100 Schaub Hall, Campus Box 7624, North Carolina State University, Raleigh, NC , USA : received 6 January 2011, revised 6 June 2011 and accepted 28 June 2011 doi: /j x x Abstract Microbial contamination is a pervasive problem in any ethanol fermentation system. These infections can at minimum affect the efficiency of the fermentation and at their worse lead to stuck fermentations causing plants to shut down for cleaning before beginning anew. These delays can result in costly loss of time as well as lead to an increased cost of the final product. Lactic acid bacteria (LAB) are the most common bacterial contaminants found in ethanol production facilities and have been linked to decreased ethanol production during fermentation. Lactobacillus sp. generally predominant as these bacteria are well adapted for survival under high ethanol, low ph and low oxygen conditions found during fermentation. It has been generally accepted that lactobacilli cause inhibition of Saccharomyces sp. and limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients. However, a number of researchers have demonstrated that these mechanisms may not completely account for the amount of loss observed and have suggested other means by which bacteria can inhibit yeast growth and ethanol production. While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. Though many yeast species have the ability to thrive in a fermentation environment, Dekkera bruxellensis has been repeatedly targeted and cited as one of the main contaminant yeasts in ethanol production. Though widely studied for its detrimental effects on wine, the specific species species interactions between D. bruxellensis and S. cerevisiae are still poorly understood. Introduction Fuel ethanol fermentations are designed to be carried out in the presence of chronic microbial contaminations (Bayrock and Ingledew 2004). In fact, it is even expected that these infections will occur. However, the infections can affect the efficiency of the fermentation and may even lead to a stuck fermentation causing the plant to shut down for cleaning before restarting the fermentation (Narendranath 2003; Skinner and Leathers 2004). While a number of genera from Lactic acid bacteria (LAB) have been identified from contaminated ethanol fermentations, Lactobacillus sp. are generally the predominant bacterial contaminants. These bacteria are well adapted for survival under high ethanol, low ph and low oxygen conditions found during fermentations. Processors often try to control this contamination using antibiotics, which can cause waste disposal issues. Wild yeast contamination is also a pervasive problem, the most notorious species being Dekkera bruxellensis, which is even more difficult to control without impacting Saccharomyces cerevisiae. In this review, we will discuss the impact of microbial contamination on bioethanol fermentations. Bacterial contamination of fuel ethanol fermentations Skinner and Leathers (2004) monitored three corn-based fuel fermentations, one wet mill operation that did not use antibiotics and two dry grind fermentations, one of which added antibiotics only to the yeast propagation tank while the other dosed the fermentation tank every Letters in Applied Microbiology 53, ª 2011 The Society for Applied Microbiology 387

2 Bioethanol contamination M. Beckner et al. 4 h. The wet mill facility reached bacterial contamination levels of 10 6 colony-forming units per millilitre (CFU ml )1 ) with between 44 and 60% of the total population being Lactobacillus sp. predominantly Lact. delbrueckii subsp. delbrueckii. In the first dry grind facility, bacterial contamination levels reached CFU ml )1 before subsiding by the end of the fermentation. Between 37 and 39% of the bacterial isolates were Lactobacilli, predominantly Lact. delbrueckii subsp. lactis with Lact. delbrueckii subsp. delbrueckii not being found. The next most common bacterial isolates were pediococci representing about 24% of the isolates. In the second dry grind facility, which dosed the fermentation with virginiamycin every 4 h, Lactobacillus sp. accounted for 69 87% of the total bacteria isolated with Lact. delbrueckii subsp. delbrueckii the most prevalent. A study of an industrial ethanol plant in Korea which used tapioca as a substrate found that lactobacilli were the only bacterial contaminates to survive from the processing of tapioca through the end of the fermentation where they reached levels of CFU ml )1 (Chang et al. 1995). In further laboratory experiments, Lactobacillus fermentum was found to be the most detrimental lactobacilli to the fermentation causing a 10% reduction in ethanol production (Chang et al. 1995). (Table 1 presents an overview of the various bacterial and yeast contaminants found in fermentations.) In Brazil, the second largest bioethanol producer, 360 active distilleries use sugarcane juice and or sugar molasses as a fermentation substrate. Four of these distilleries were sampled between 2007 and 2008, and a total of 489 LAB isolates were obtained. The abundance of LAB in the fermentation tanks varied between 6Æ and 8Æ CFU ml )1. rrna operon enzyme restriction profiles indicated that most LAB isolates belonged to the genus Lactobacillus with the majority of the species belonging to Lact. fermentum and Lact. vini. These species were found in medium containing up to 10% ethanol. This suggests the selection of ethanol tolerant bacteria over time throughout the process (Lucena et al. 2010). Effects of lactobacilli contamination on biofuel fermentations The effects of lactobacilli contamination have been extensively studied in the laboratory. It has been generally Table 1 Bacterial and yeast contaminants found in Fuel Ethanol Fermentations Organism Reference Bacteroides forsythus Skinner and Leathers (2004) Bifidobacterium sp., Bif. adolescentis, Bif. angulatum, unidentified Bifidobacterium sp. Skinner and Leathers (2004) Clostridium sp., Cl. aerotolerans, Cl. clostridiiforme Skinner and Leathers (2004) Eubacterium biforme Skinner and Leathers (2004) Fusobacterium nucleatum Skinner and Leathers (2004) Lactobacillus sp., Lact. acidophilus, Lact. amylovorus, Lact. brevis, Lact. buchneri, Lact. casei, Lact. crispatus, Lact. delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. diolivorans-like, Lact. ferintoshensis, Lact. fermentum, Lact. gasseri, Lact. helveticus, Lact. hilgardii, Lact. lindneri, Lact. manihotivorans, Lact. mucosae, Lact. nagelii, Lact. paracasei subsp. paracasei, Lact. pentosus, Lact. plantarum, Lact. reuteri, Lact. rhamnosus, Lact. salivarius, Lact. vini, unidentified Lactobacillus sp. Skinner and Leathers (2004); Lucena et al. (2010); Chang et al. (1995) Lactococcus sp., L. lactis subsp. lactis, L. raffinolactis Skinner and Leathers (2004) Leuconostoc sp., Leuc. carnosum, Leuc. citreum, Leuc. lactis subsp. lactis, Leuc. mesenteroides subsp. cremoris Skinner and Leathers (2004) Oenoccocuss kitaharae-like Lucena et al. (2010) Pediococcus sp., Ped. acidilactici, Ped. damnosus, Ped. parvulus, Ped. pentosaceus, unidentified Skinner and Leathers (2004) Pediococcus sp. Propionibacterium granulosum Skinner and Leathers (2004) Weisella sp., W. confusa, W. paramesenteroides, W. viridescens Skinner and Leathers (2004); Lucena et al. (2010) Candida sp., C. intermedia, C. lusitaniae, C. pararugosa, C. parapsilosis, C. tropicalis, C. xylopsoci Basilio et al. (2008) Dekkera bruxellensis Basilio et al. (2008) Exophiala dermatitides Basilio et al. (2008) Hanseniaspora guilliermondii Basilio et al. (2008) Issatchenkia orientalis Basilio et al. (2008) Pichia anomala, P. caribbica, P. fabianii, P. galeiformis, P. guilliermondii, P. ohmeri Basilio et al. (2008) Pseudozyma hubeiensis Basilio et al. (2008) Saccharomycodes ludwigii Basilio et al. (2008) Williopsis sp. da Silva-Filho et al. (2005) Zygoascus hellenicus Basilio et al. (2008) Zygosaccharomyces fermentati Basilio et al. (2008) 388 Letters in Applied Microbiology 53, ª 2011 The Society for Applied Microbiology

3 M. Beckner et al. Bioethanol contamination believed that lactobacilli cause inhibition of Saccharomyces or limit ethanol production through two basic methods; either production of lactic and acetic acids or through competition for nutrients (Narendranath et al. 1997; Narendranath 2003). However, a number of researchers have demonstrated that these mechanisms may not completely account for this inhibition. Thomas et al. (2001) found that growth of Lactobacillus fermentum for 24 h prior to inoculation with Saccharomyces caused a 22% loss in ethanol production. Unlike previous work suggesting loss of ethanol yield, in part because of utilization of carbohydrates, in this study, all available sugar was consumed (Makanjuola et al. 1992; Thomas et al. 2001). Further, Thomas et al. (2001) state The loss of this magnitude could not be explained in terms of sugars being diverted for bacterial growth, production of acetic acid or lactic acid, or by increased synthesis of glycerol alone. Very few studies on the effects of organic acids on ethanol production have been actually undertaken in production medium. The few that have, usually using corn mash, all demonstrated a protective effect of the more complex medium because of increased buffering capacity (Thomas et al. 2002; Graves et al. 2006, 2007). Graves et al. (2006a) found that lactic acid had a maximum effect on ethanol production at a concentration of 4% w v in a fermentation at ph 4 (corresponding to a 2Æ36% w v undissociated acid concentration). Acetic acid was found to accelerate ethanol production at low levels ( 0Æ2%), regardless of ph value. At ph values of 5 and 5Æ5, Saccharomyces was found to be resistant to 0Æ8% w v acetic acid at, and ethanol production was reduced at lower ph values (4 and 4Æ5) with lower levels of acetic acid ( 0Æ4% v w) (Graves et al. 2006a). While the acids are known to work synergistically (Graves et al. 2007), Narendranath and Power (2005) suggested increasing the medium ph would diminish the effects of these acids as the higher ph would result in less undissociated acid. The authors also found that an increase in ph from 4 to 5Æ5 in media containing between 20 and 35% dissolved solids (dissolved solids in normal production may be up to 30% w v) ethanol production increased by c. 2%in each case (Narendranath and Power 2005). These results suggest that developing methods that allow fermentation to occur at higher less stressful phs and still limit the impact of LAB may help improve the efficiency of ethanol production and not just limit the loss of ethanol because of bacterial contamination. Work by Bayrock and Ingledew (2001, 2004) clearly demonstrates that LAB would still have an effect on these higher ph fermentations and that more is involved in the inhibition of Saccharomyces by lactobacilli than just the production of lactic acid. They first inoculated 2Æ CFU ml )1 of the rapid growing homofermentive (lactic acid production only) Lactobacillus paracasei strain into a multistage continuous culture fermentation system with a steady-state Saccharomyces population ( CFU ml )1 ) and no ph control. The lactobacillus numbers declined to a steady state of CFU ml )1, and no effect was observed on Saccharomyces growth or ethanol production. The same effect was observed even with inoculation rates up to 100 : 1 lactobacilli to Saccharomyces. This corresponded to results from other work and caused the authors to state that this low level of chronic contamination was a ticking time bomb for ethanol producers (Magnus et al. 1986; Bayrock and Ingledew 2001; Thomas et al. 2001). If various fermentation conditions were to change such as ph or temperature, it could allow the lactobacilli to outgrow the Saccharomyces and cause either a loss of final product or a stuck fermentation (Bayrock and Ingledew 2001). To demonstrate this concept, Bayrock and Ingledew (2001) initiated ph control in a steady-state fermentation raising the ph to 6Æ0, and within 3 days, the lactobacilli population increased by 4Æ4 logs and the Saccharomyces population decreased by 83%. The ethanol concentration decreased by 44% and the lactic acid levels increased from 0Æ41% w v to 2% w v. In further work examining the effects of lactic acid on Saccharomyces ethanol production in a similar multistage continuous culture fermentation (differing only slightly in dilution rate), the authors demonstrated that the amount of lactic acid produced could not completely account for the level of Saccharomyces inhibition at a ph of 6Æ0 (Bayrock and Ingledew 2004). Under these conditions, only 0Æ01% of the lactic acid would be undissociated while at least 3Æ4% undissociated lactic acid was required to achieve a 50% reduction in yeast numbers (Bayrock and Ingledew 2004). This corresponds to 644% w v lactic acid needed at the controlled ph conditions to achieve a 50% inhibition of Saccharomyces. Therefore, the 2% lactic acid produced in the previous experiment could not possibly account for the much larger reduction of Saccharomyces demonstrated in their earlier work (Bayrock and Ingledew 2001, 2004). The authors concluded that the competition for trace nutrients such as thiamine played a key role in the competition between Saccharomyces and lactobacilli (Bayrock and Ingledew 2004). However, while they suggested that the addition of trace nutrients could lessen the impact of lactobacilli they never performed an experiment to demonstrate these benefits (Bayrock and Ingledew 2004). Ngang et al. (1990) also addressed the concern over competition for nutrients in their study. They observed inhibition of ethanol production by Lactobacillus contamination at levels over 1Æ CFU ml )1 in a beet molasses fermentation. They further demonstrated that 10 g l )1 Letters in Applied Microbiology 53, ª 2011 The Society for Applied Microbiology 389

4 Bioethanol contamination M. Beckner et al. of lactic acid was needed to achieve a 50% inhibition of Saccharomyces growth while the Lactobacillus casei sp. used in the study only produced 2Æ5 gl )1. The possibility that Saccharomyces growth was suppressed through competition for nutrients was dismissed, as growth was still suppressed even upon the addition of glutamic acid or yeast extract to the mixed fermentation. Using an extract of spent medium of a pure Lact. casei culture, they demonstrated inhibition of ethanol production and yeast growth but were unable to identify the inhibitory compound. Inhibitory mechanisms of lactic acid bacteria Currently, there are numerous theories to account for the interaction of LAB and yeast. Accumulation of both lactic and acetic acids are major inhibitory end-products produced by LAB. While these acids lower the ph of any fermentation, their true inhibitory effect is seen in the undissociated form of the acid, as it is capable of diffusing through the cell membrane. Once inside the cell, it can then dissociate, releasing H + ions that acidify the cytoplasm of the cell (Schnürer and Magnusson 2005). There are numerous other compounds produced by LAB that are known to contribute to the inhibition of ethanol production such as diacetyl, reuterin and fatty acids (Fig. 1). Additionally, competition for trace nutrients is thought to contribute to the effect of LAB on yeast, but it is not possible to clearly separate the inhibition because of the depletion of nutrients verses the effects of end-products produced by the contaminant. Therefore, as of now, there is little to no evidence available in the literature to suggest that competition for nutrients, rather than inhibition by metabolic end-products, is the major reason for the loss of ethanol and viability in contaminated industrial ethanol fermentations (Bayrock and Ingledew 2004). Numerous metabolites have been associated with the inhibitory effects of both LAB and yeasts. Diacetyl (2,3- butandione) produces the characteristic aroma of butter and is produced by strains within all genera of LAB (Lindgren and Dobrogosz 1990). In general, yeasts and Gram-negative bacteria are more sensitive to this compound than non-lab and Gram-positive bacteria. Jay (1982) found that Candida lipolytica, Debaryomyces cantarellii and Rhodototula tubra were all significantly inhibited at more than 200 lg ml )1 diacetyl at a ph <7Æ7 using several types of substrates. During metabolism of glycerol, some LAB produce an intermediate known as reuterin (3-hydroxypropionaldehyde) (Schnürer and Magnusson 2005). Species known to produce this compound are Lact. reuteri, Lact. brevis, Lact. buchneri, Lact. coolinoides and Lact. coryniformis (Claisse and Lonvaud-Funel 2000; Magnusson et al. 2003). Reuterin is a broad-spectrum antibiotic, active against different micro-organisms including Gram-positive and Gram-negative bacteria, yeast and fungi, specifically species of the genera: Candida, Torulopsis, Saccharomyces, Aspergillus and Fusarium (Chung et al. 1989; Schnürer and Magnusson 2005). There has been some work that suggests proteinaceous compounds can also affect yeasts. Okkers et al. (1999) found a medium length peptide from Lactobacillus pentosus that had fungistatic effects against C. albicans while Magnusson and Schnürer (2001) found a small, heat stable peptide from Lactobacillus coryniformis subsp. coryniformis that has antifungal effects against several moulds and yeasts. Sjögren et al. (2003) found that hydroxylated fatty acids produced by Lact. plantarum have a strong effect against a broad spectrum of yeasts at a range of lg ml )1. While these compounds do inhibit the growth of yeast, production of organic acids, specifically lactic and acetic acid, is still considered the major inhibitory factors produced by LAB. Cyclic dipeptides Caproic acid Carbon dioxide Lactic acid Acetic acid Diacetyl 3-hydroxy fatty acids Proteinaceous compounds Lactic acid bacteria Phenyllactic acid Reuterin Hydrogen Peroxide Figure 1 Summary of the main antifungal compounds produced by different lactic acid bacteria. Lactic and acetic acid are the main compounds produced that effect Saccharomyces cerevisiae. 390 Letters in Applied Microbiology 53, ª 2011 The Society for Applied Microbiology

5 M. Beckner et al. Bioethanol contamination Yeast contamination of bioethanol fermentations While LAB are the primary contaminates of concern in industrial ethanol fermentations, wild yeast may also affect the productivity of these fermentations. The most prominent yeast contaminants of biofuel fermentations include the following: Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis and Candida sp. For reasons not fully understood, all strains are associated with decreased productivity in bioethanol production with D. bruxellensis being the most troublesome of the yeasts (Basílio et al. 2008). Production losses because of wild yeasts Wild yeast are a persistent problem in all types of fermentations, including biofuel (Muthaiyan et al. 2011). Contamination by wild yeast can cause losses in ethanol yield. A loss of even 1% ethanol production has a large financial impact (Muthaiyan et al. 2011). However, controlling wild yeast presents different challenges from controlling bacterial contamination. The best way to control wild yeast is through monitoring by molecular and microbiological techniques (da Silva-Filho et al. 2005; de Souza Liberal et al. 2005). Once a wild yeast contamination has been identified, the treatment is a complete change of the yeast population within the fermentors (de Souza Liberal et al. 2007). This also adds an additional cost to the affects of wild yeast contamination. Dekkera bruxellensis contamination Dekkera bruxellensis has been repeatedly cited as one of the main contaminant yeasts in bioethanol production (Tavares 1995; Abbott et al. 2004; Abbott and Ingledew 2005; de Souza Liberal et al. 2005, 2007; Basílio et al. 2008; Elsztein et al. 2008; Muthaiyan and Ricke 2010;). Though other strains have been routinely identified as potentially detrimental to fuel ethanol fermentations, none have been so widely studied as D. bruxellensis. For all of the publications on D. bruxellensis, the means by which this yeast is able to inhibit S. cerevisiae growth are still poorly understood. Several publications have indicated that D. bruxellensis is able to produce more acetic acid than S. cerevisiae and propose this as the cause for decreased ethanol yield (Abbott et al. 2005; de Souza Liberal et al. 2007). While it is true that acetic acid formation is associated with aerobic growth of D. bruxellensis (Narendranath et al. 1997; Abbott and Ingledew 2005; Blomqvist et al. 2010), there is not enough produced to completely explain the drop in ethanol production (Abbott and Ingledew 2005). Additionally, D. bruxellensis grows more slowly and therefore does not present itself as a contaminant until later in the fermentation (Abbott and Ingledew 2005). It has been reported that industrial strains of D. bruxellensis displayed ethanol yields equivalent to or higher than two industrial strains of S. cerevisiae, as well as higher biomass yields and lower glycerol yields and growth rates, making it a more robust yeast under fermentation conditions (Blomqvist et al. 2010). Unfortunately, D. bruxellensis is able to utilize ethanol as a carbon source and will readily do so when other carbon sources are in short supply (Dias et al. 2003). This may contribute to overall decreased ethanol yield and allow D. bruxellensis to thrive where other yeast may not be able to. Not surprisingly, D. bruxellensis exhibits a remarkable ethanol tolerance, which lends itself to its ability to out compete S. cerevisiae in the latter stages of fermentations (Dias et al. 2003). Work by Passoth et al. (2007) demonstrated that D. bruxellensis and Lactobacillus vini formed a stable consortium, which supplanted the Saccharomyces species in a continuous-ethanol fermentation. While the contaminates came to dominate the fermentation ethanol, productivity did not decrease, suggesting that Dekkera sp. may not only be investigated as contaminants but also as possible ethanol production organisms (Blomqvist et al. 2010). Current detection methods The economical damage imposed by yeast contamination can be overcome if the presence of contaminating yeasts is detected early. Therefore, development of early detection methods is key to the financial profitability of the bioethanol industry and has been extensively studied. Current research shows that it is possible to use the ribosomal DNA locus (rdna) for early yeast detection and identification (de Souza Liberal et al. 2005). PCR amplification of this region has been used for detection of non- Saccharomyces cerevisiae yeast during spontaneous wine fermentation (Guillamon et al. 1998). This PCR method makes use of a region of rdna (the internal transcribed spacers and the 5Æ8S rrna gene) within yeast. de Souza Liberal et al. (2005) demonstrated that S. cerevisiae exhibits a very specific band size of 850 bp, while anything larger or smaller than this is representative of a contaminant yeast. That is not to say however that this yeast would be necessarily detrimental to ethanol production. Further research has been conducted to help identify specific detrimental strains using DNA sequencing, PCR-rDNA restriction analysis and PCR-fingerprinting single-primed amplification reaction with the (GTG) 5 primer (Basílio et al. 2008). This in-depth study was able to identify 22 adventitious yeasts and provided the sequence and PCRrDNA restriction banding associated with specific species. Letters in Applied Microbiology 53, ª 2011 The Society for Applied Microbiology 391

6 Bioethanol contamination M. Beckner et al. This allows for rapid identification of detrimental strains in fermentations. The identification of non-dekkera strains demonstrates how much more research is necessary to completely understand the role of contaminate yeasts and the means by which they inhibit S. cerevisiae growth and ethanol production. Current solutions Several techniques are currently being employed in an attempt to combat rampant unwanted microbial growth. Bacterial infections can be controlled by the use of antibiotics, acid treatment, ammonia (Broda and Grajek 2009) and urea hydrogen peroxide (Muthaiyan and Ricke 2010). A no antibiotic approach has obvious advantages, but can be detrimental to S. cerevisiae; therefore, there is a definite need for more research to control bacterial infections. Yeast infections, however, can cause a complete change in the yeast population within the system. In wine fermentation, sulfur dioxide (SO 2 ) is widely used to avoid contamination by spoiling yeasts (Loureiro 2003). However, given the amounts needed to maintain low contaminating yeast levels and its potentially corrosive and toxic nature, it is not a viable, long-term solution for bioethanol production facilities. Other fungicides and antimicrobial compounds such as chitosan (Escudero-Abarca et al. 2004), hydroxycinnamates, organic acids (Neves et al. 1994) and membrane active antimicrobial peptides (Bom et al. 2001) have been used with varying degrees of success. These methods, however, pose a potential biological and environmental hazard if waste in not properly disposed of (Pimentel 2008). Also, some of these treatments are also quite costly. An alternative has been proposed, however: polyhexamethyl biguanide (PHMB) that is a fungicide shown to prevent and actually kill D. bruxellensis (Elsztein et al. 2008). It is in fact the only fungicide shown to specifically kill D. bruxellensis without harming S. cerevisiae. Elsztein et al. (2008) concluded that the use of PHMB at 200 mg l )1 in combination with a high-fermenting PHMB-resistant strain may prevent the establishment of contamination in the bioethanol fermentation process. However, more research should be carried out in this area to determine the safety and cost-effectiveness of this and other such compounds. Discussion Fuel ethanol fermentations are designed to be carried out in the presence of chronic microbial contaminations. No matter the precautions taken, infections will occur and in all likelihood will be comprised of LAB and non-saccharomyces yeast species. Significant research has gone into understanding what inhibitory effects each of these contaminants has on Saccharomyces and how they might be mitigated. In the case of LAB, it has been demonstrated that neither the production of lactic and acetic acids nor the competition for nutrients completely explains the amount of ethanol loss typically observed. Of the organic acids produced by the bacteria, it is the undissociated lactic and acetic acid that pose the most risk given that they are able to cross the cell membrane and once inside can then dissociate, releasing H + ions and thus acidify the cytoplasm of the cell. It has been hypothesized however that there are yet other compounds being produced by LAB that can have a significant impact on ethanol fermentations. Diacetyl, reuterin and proteinaceous compounds have all been shown to effect yeast growth and ethanol production capacity. Likewise, some peptides and hydroxylated fatty acids have been shown to have a minor effect. However, none of these compare to the effects seen from organic acids. Though the mechanisms are not as well understood, wild yeast often affects the productivity of industrial ethanol fermentations, especially in unsterilized molasses and sugar cane fermentations. Wild yeasts are present on the cane at the time of the harvest and thus are introduced to the production facility from the very beginning of the fermentation. The most prominent yeast contaminants of biofuel fermentations include the following: Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis and C. intermedia. Of these, D. bruxellensis is the most widely studied and while acetic acid formation is associated with aerobic growth of D. bruxellensis, as with lactobacilli, there is not enough produced to completely explain the drop in ethanol production. Dekkera bruxellensis is robust, but slow-growing and typically does not present itself as a contaminant until the end of the fermentative process. Dekkera bruxellensis is able to utilize ethanol as a carbon source, which may contribute to the drop in ethanol that is observed in its presence. More research is needed to gain a better understanding of how D. bruxellensis is able to inhibit S. cerevisiae growth and cause a loss in ethanol production. Currently, the best defence against contamination is early detection. This is especially true for yeast contaminants, whose effects can be significantly minimized if caught early. Several methods have been proposed, all with varying degrees of detection success. DNA sequencing, PCR-rDNA restriction analysis and PCR-fingerprinting with single-primed amplification reactions using the (GTG) 5 primer are all currently being used to help detect contaminant yeasts. These procedures, however, are still somewhat expensive and must be performed in a laboratory setting; something that is not feasible for all production facilities. There is a need for simple and rapid onsite detection, and further research should be conducted to help solve this problem. 392 Letters in Applied Microbiology 53, ª 2011 The Society for Applied Microbiology

7 M. Beckner et al. Bioethanol contamination Conclusion The contamination of bioethanol fermentations with LAB and wild yeasts is a significant industrial problem causing production loss of anywhere from 2 to 22%. These micro-organisms impact the fermentations in a number of ways, and much work still needs to be carried out to understand their how they affect the fermentation and the best methods to control these chronic infections. Acknowledgments This project was supported by a grant from the Cooperative State Research, Education and Extension Service, US Department of Agriculture, under agreement no References Abbott, D.A. and Ingledew, W.M. (2005) The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera Brettanomyces yeasts. Appl Microbiol Biotechnol 69, Abbott, D.A., Hynes, S.H. and Ingledew, W.M. (2004) Growth rates of Dekkera Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentations. 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