Biofilm formation by bacterial contaminants of fuel ethanol production

Commercial fuel ethanol production facilities were previously shown to have characteristic populations of bacterial contaminants which reduce product yield and are difficult to eradicate. Bacterial contaminants were found, for the first time, to form biofilms under laboratory conditions. Fermentor samples from a commercial fuel ethanol production facility were used to inoculate a biofilm reactor and purified bacterial isolates were identified. Biofilms were composed of many of the same species present in production samples, with lactic acid bacteria predominating. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Antimicrobial susceptibility of <Emphasis Type="Italic">Lactobacillus</Emphasis> species isolated from commercial ethanol plants

Bacterial contamination of commercial fermentation cultures is a common and costly problem to the fuel ethanol industry. Antimicrobials such as virginiamycin (VIR) and penicillin (PEN) are frequently used to control contamination but there are little data available on the susceptibility of bacterial contaminants to these agents. A survey of bacterial contaminants from a wet-mill ethanol plant with no history of using antibiotics and a dry-grind facility that periodically doses with VIR found that the majority of contaminants were species of Lactobacillus. Thirty-seven isolates of Lactobacillus species from the wet-mill and 42 isolates from the dry-grind facility were tested for antimicrobial susceptibility using broth dilution and agar dilution methods. In general, the Lactobacillus isolates from the dry-grind plant had higher minimum inhibitory concentrations (MICs) for the tested agents than the isolates from the wet-mill facility. The MIC₉₀ for VIR was 4 μg/ml for the dry-grind isolates versus 0.25 μg/ml for the wet-mill isolates; and for PEN, the MIC₉₀’s were >8 and 2 μg/ml for the dry-grind and wet-mill isolates, respectively. Sixteen Lactobacillus isolates from the dry-grind plant but none from the wet-mill possessed vatE, a gene that encodes a streptogramin acetyltransferase associated with resistance to virginiamycin. Despite decreased susceptibility to virginiamycin, most dry-grind isolates had MICs lower than the maximal recommended application rate of 6 ppm. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Rapid evaluation of the antibiotic susceptibility of fuel ethanol contaminant biofilms

Bacterial contaminants from commercial fuel ethanol production facilities were previously shown to form biofilms as mixed cultures under laboratory conditions. In this study, a rapid assay was developed to simultaneously compare isolates for their ability to form biofilms as pure cultures. A total of 10 strains were isolated from a dry-grind fuel ethanol plant that routinely doses with virginiamycin. These were identified by sequence analysis as six strains of Lactobacillus fermentum, two strains of L. johnsonii, and one strain each of L. mucosae and L. amylovorus. Isolates exhibited a range of susceptibility to virginiamycin in a planktonic assay, with MIC’s (minimum inhibitory concentration) of ?0.5-16 μg/ml. Even though all strains were isolated from a mixed culture biofilm, they varied greatly in their ability to form biofilms as pure cultures. Surprisingly, growth as biofilms did not appear to provide resistance to virginiamycin, even if biofilms were grown for 144 h prior to antibiotic challenge.     

Construction of a flocculating yeast for fuel ethanol production

The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Saccharomyces. cerevisiae ZWA46. The transformant, ZWA46-F2, showed strong and stable flocculation ability during 20 serial batch cultivations. The flocculation onset of the strain is in the early stationary growth phase, not coincident with the glucose depletion in the culture medium. The flocculation ability of the transformant showed no difference with the initial pH ranging from 3.5 to 6.0. Furthermore, the ethanol concentration and other properties of the transformant strain ZWA46-F2 were similar to those of the wild-type strain ZWA46.     

Survey of US fuel ethanol plants

The ethanol industry is growing in response to increased consumer demands for fuel as well as the renewable fuel standard. Corn ethanol processing creates the following products: 1/3 ethanol, 1/3 distillers grains, and 1/3 carbon dioxide. As the production of ethanol increases so does the generation of its coproducts, and viable uses continually need to be developed. A survey was mailed to operational US ethanol plants to determine current practices. It inquired about processes, equipment used, end products, and desired future directions for coproducts. Results indicated that approximately one-third of plant managers surveyed expressed a willingness to alter current drying time and temperature if it could result in a higher quality coproduct. Other managers indicated hesitation, based on lack of economic incentives, potential cost and return, and capital required. Respondents also reported the desire to use their coproducts in some of the following products: fuels, extrusion, pellets, plastics, and human food applications. These results provide a snapshot of the industry, and indicate that operational changes to the current production of DDGS must be based upon the potential for positive economic returns.     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

The role of nisin in fuel ethanol production with Saccharomyces cerevisiae

Aims: To investigate the effects of nisin on lactobacilli contamination of yeast during ethanol fermentation and to determine the appropriate concentration required to control the growth of selected lactobacilli in a YP/glucose media fermentation model. Methods and Results: The lowest concentration of nisin tested (5 IU ml^?1) effectively controlled the contamination of YP/glucose media with 10⁶ CFU ml^?1 lactobacilli. Lactic acid yield decreased from 5·0 to 2·0 g l^?1 and potential ethanol yield losses owing to the growth and metabolism of Lactobacillus plantarum and Lactobacillus brevis were reduced by 11 and 7·8%, respectively. Approximately, equal concentrations of lactic acid were produced by Lact. plantarum and Lact. brevis in the presence of 5 and 2 IU ml^?1 nisin, respectively, thus demonstrating the relatively higher nisin sensitivity of Lact. brevis for the strains in this study. No differences were observed in the final ethanol concentrations produced by yeast in the absence of bacteria at any of the nisin concentrations tested. Conclusions: Metabolism of contaminating bacteria was reduced in the presence of 5 IU ml^?1 nisin, resulting in reduced lactic acid production and increased ethanol production by the yeast. Significance and Impact of the Study: Bacteriocins represent an alternative to the use of antibiotics for the control of bacterial contamination in fuel ethanol plants and may be important in preventing the emergence of antibiotic‐resistant contaminating strains.     

Continuous ethanol production byZymomonas mobilis andSaccharomyces cerevisiae in biofilm reactors

Continuous ethanol fermentations were performed in duplicate for 60 days withZymomonas mobilis ATCC 331821 orSaccharomyces cerevisiae ATCC 24859 in packed-bed reactors with polypropylene or plastic composite-supports. The plastic composite-supports used contained polypropylene (75%) with ground soybean-hulls (20%) and zein (5%) forZ. mobilis, or with ground soybean-hulls (20%) and soybean flour (5%) forS. cerevisiae. Maximum ethanol productivities of 536 gL^–1 h^–1 (39% yield) and 499 gL^–1 h^–1 (37% yield) were obtained withZ. mobilis on polypropylene and plastic composite-supports of soybean hull-zein, respectively. ForZ. mobilis, and optimal yield of 50% was observed at a 1.92h^–1 dilution rate for soybean hull-zein plastic composite-supports with a productivity of 96gL^–1h^–1, whereas with polypropylene-supports the yield was 32% and the productivity was 60gL^–1h^–1. With aS. cerevisiae fermentation, the ethanol production was less, with a maximum productivity of 76gL^–1h^–1 on the plastic composite-support at a 2.88h^–1 dilution rate with a 45% yield. Polypropylene-support bioreactors were discontinued due to reactor plugging by the cell mass accumulation. Support shape (3-mm chips) was responsible for bioreactor plugging due to extensive biofilm development on the plastic composite-supports. With suspensionculture continuous fermentations in continuously-stirred benchtop fermentors, maximum productivities of 5gL^–1h^–1 were obtained with a yield of 24 and 26% withS. cerevisiae andZ. mobilis, respectively. Cell washout in suspensionculture continuous fermentations was observed at a 1.0h^–1 dilution rate. Therefore, for continuous ethanol fermentations, biofilm reactors out-performed suspension-culture reactors, with 15 to 100-fold higher productivities (gL^–1h^–1) and with higher percentage yields forS. cerevisiae andZ. mobilis, respectively. Further research is needed with these novel supports to evaluate different support shapes and medium compositions that will permit medium flow, stimulate biofilm formation, reduce fermentation costs, and produce maximum yields and productivities.This is Journal Paper No. J-16357 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253     

Evaluation of plastic composite-supports for enhanced ethanol production in biofilm reactors

Biofilms are a natural form of cell immobilization that result from microbial attachment to solid supports. Biofilm reactors with polypropylene composite-supports containing up to 25% (w/w) of various agricultural materials (corn hulls, cellulose, oat hulls, soybean hulls or starch) and nutrients (soybean flour or zein) were used for ethanol production. Pure cultures ofZymomonas mobilis, ATCC 31821 orSaccharomyces cerevisiae ATCC 24859 and mixed cultures with either of these ethanol-producing microorganisms and the biofilm-formingStreptomyces viridosporus T7A ATCC 39115 were evaluated. An ethanol productivity of 374g L^–1 h^–1 (44% yield) was obtained on polypropylene composite-supports of soybean hull-zein-polypropylene by usingZ. mobilis, whereas mixed-culture fermentations withS. viridosporus resulted in ethanol productivity of 147.5 g L^–1 h^–1 when polypropylene composite-supports of corn starch-soybean flour were used. WithS. cerevisiae, maximum productivity of 40 g L^–1 h^–1 (47% yield) was obtained on polypropylene composite-supports of soybean hull-soybean flour, whereas mixed-culture fermentation withS. viridosporus resulted in ethanol productivity of 190g L^–1 h^–1 (35% yield) when polypropylene composite-supports of oat hull-polypropylene were used. The maximum productivities obtained without supports (suspension culture) were 124 g L^–1 h^–1 and 5 g L^–1 h^–1 withZ. mobilis andS. cerevisiae, respectively. Therefore, forZ. mobilis andS. cerevisiae, ethanol productivities in biofilm fermentations were three- and eight-fold higher than suspension culture fermentations, respectively. Biofilm formation on the chips was detected by weight change and Gram staining of the support material at the end of the fermentation. The ethanol production rate and concentrations were consistently greater in biofilm reactors than in suspension cultures.This is Journal Paper No. J-16356 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 3253     

Paddy straw as substrate for ethanol production

Pretreatment of paddy straw with 2% sodium hydroxide at 15 psi for 1 h resulted in 83% delignification. The hydrolysis of alkali treated paddy straw with a commercial preparation of cellulase for 2 h at 50°C resulted in release of 65% total reducing sugars. Maximum sugars were released at enzyme loading of 1.5% (v/v). The fermentation of hydrolysate supplemented with nutrients by S. cerevisiae resulted in the production of 20–30 g L⁻¹ ethanol after 48 h incubation which was further improved with addition of yeast nitrogen base and inoculated with 1% (w/v) yeast cells.     

Modeling bacterial contamination of fuel ethanol fermentation

The emergence of antibiotic‐resistant bacteria may limit the effectiveness of antibiotics to treat bacterial contamination in fuel ethanol plants, and therefore, new antibacterial intervention methods and tools to test their application are needed. Using shake‐flask cultures of Saccharomyces cerevisiae grown on saccharified corn mash and strains of lactic acid bacteria isolated from a dry‐grind ethanol facility, a simple model to simulate bacterial contamination and infection was developed. Challenging the model with 10⁸ CFU/mL Lactobacillus fermentum decreased ethanol yield by 27% and increased residual glucose from 6.2 to 45.5 g/L. The magnitude of the effect was proportional to the initial bacterial load, with 10⁵ CFU/mL L. fermentum still producing an 8% decrease in ethanol and a 3.2‐fold increase in residual glucose. Infection was also dependent on the bacterial species used to challenge the fermentation, as neither L. delbrueckii ATCC 4797 nor L. amylovorus 0315‐7B produced a significant decrease in ethanol when inoculated at a density of 10⁸ CFU/mL. In the shake‐flask model, treatment with 2 µg/mL virginiamycin mitigated the infection when challenged with a susceptible strain of L. fermentum (MIC for virginiamycin ≤2 ppm), but treatment was ineffective at treating infection by a resistant strain of L. fermentum (MIC = 16 ppm). The model may find application in developing new antibacterial agents and management practices for use in controlling contamination in the fuel ethanol industry. Biotechnol. Bioeng. 2009;103: 117–122. Published 2008 Wiley Periodicals, Inc.     

Availability of substratum enhances ethanol production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Novel additives that act as substratum for attachment of the yeast cells, increased ethanol production in Saccharomyces cerevisiae. The addition of 2 g rice husk, straw, wood shavings, plastic pieces or silica gel to 100 ml medium enhanced ethanol production by 30–40 (v/v). Six distillery strains showed an average enhancement of 34 from 4.1 (v/v) in control to 5.5 (v/v) on addition of rice husk. The cell wall bound glycogen increased by 40–50 mg g ^–1 dry yeast while intracellular glycogen decreased by 10–12 mg g^–1 dry yeast in cells grown in presence of substratum     

Mathematical modelling of ethanol production from glucose/xylose mixtures by recombinant Zymomonas mobilis

A model has been developed for the fermentation of mixtures of glucose and xylose by recombinant Zymomonas mobilis strain ZM4(pZB5), containing additional genes for xylose assimilation and metabolism. A two-substrate model based on substrate limitation, substrate inhibition, and product (ethanol) inhibition was evaluated, and experimental data was compared with model simulations using a Microsoft EXCEL based program and methods of statistical analysis for error minimization. From the results it was established that the model provides good predictions of experimental batch culture data for 25/25, 50/50, and 65/65 g l^–1 glucose/xylose media.     

Cellulose production by Gluconacetobacter hansenii in a medium containing ethanol

The addition of 1% (v/v) ethanol to the basal medium inhibited growth of Gluconacetobacter hansenii but decreased the numbers of non-cellulose producing cells. Cellulose production increased 1.7 times to approx. 2.5 g l(-1) and showed a pattern of mixed growth-associated production. Microbial cells produced rigid pellicle-type bacterial cellulose as the shell of a large lump of bacterial cellulose like a static culture. The inoculum cultivated for 3 d maintained cellulose production by the fifth batch.     

Economic analysis of fuel ethanol production from winter hulled barley by the EDGE (Enhanced Dry Grind Enzymatic) process

A process and cost model was developed for fuel ethanol production from winter barley based on the EDGE (Enhanced Dry Grind Enzymatic) process. In this process, in addition to β-glucanases, which are added to reduce the viscosity of the mash, β-glucosidase is also added to completely hydrolyze the oligomers obtained during the hydrolysis of β-glucans to glucose. The model allows determination of capital costs, operating costs, and ethanol production cost for a plant producing 40 million gallons of denatured fuel ethanol annually. A sensitivity study was also performed to examine the effects of β-glucosidase and barley costs on the final ethanol production cost. The results of this study clearly demonstrate the economic benefit of adding β-glucosidase. Lower ethanol production cost was obtained compared to that obtained without β-glucosidase addition in all cases except one where highest β-glucosidase cost allowance and lowest barley cost were used.     

Ethane production by Methanosarcina barkeri during growth in ethanol supplemented medium

Methanosarcina barkeri strain 227 produced ethane during growth on H₂/CO₂ when ethanol was added to the medium in concentrations of 89–974 mM; ethane production varied from 14 to 38 nmoles per tube (20 ml gas phase, 5.7 ml liquid) with increasing ethanol concentrations. Cells grown to mid-logarithmic phase (A₆₀₀ 0.46, protein = 64 g/ml) on H₂/CO₂, thoroughly flushed with H₂/CO₂, then exposed to ethanol, produced maximal ethane levels (at 585 and 974 mM ethanol) of about 215 nmoles per tube, with an ethane/methane ratio of 1×10^-3. Mid-logarithmic-phase cultures of Methanosarcina barkeri strain Fusaro also produced ethane (up to 20 nmoles per tube) when exposed to ethanol. Cultures of strain 227 growing on methanol in the absence of H₂ produced 6 nmoles per tube of ethane when supplemented with ethanol whereas those lacking ethanol but containing H₂ and/or methanol produced 1.6 nmoles per tube. Cultures of Methanococcus deltae strains LH and RC, Methanospirillum hungatei or Methanobacterium thermoautotrophicum produced 5 nmoles ethane per tube when grown in medium containing ethanol. Ethanol concentrations of 177–886 mM were inhibitory to growth of all methanogens examined. Production of ethane by Methanosarcina was inhibited by >62 mM methanol, and both methanogenic inhibitors tested, CCl₄ and Br–CH₂–CH₂–SO _inf3 ^sup- , inhibited ethane and methane production concurrently. The data suggest that ethanol is converted to ethane by Methanosarcina species using the terminal portion of the methanol-to-methane pathway.     

Improved ethanol production from various carbohydrates through anaerobic thermophilic co-culture

Saccharification is one of the most critical steps in producing lignocellulose-based bio-ethanol through consolidated bioprocessing (CBP). However, extreme pH and ethanol concentration are commonly considered as potential inhibitors for the application of Clostridium sp. in CBP. The fermentations of several saccharides derived from lignocellulosics were investigated with a co-culture consisting of Clostridium themocellum and Clostridium thermolacticum. Alkali environments proved to be more favorable for ethanol production. Fermentation inhibition was observed at high ethanol concentrations and extreme pH. However, low levels of initial ethanol addition resulted in an unexpected stimulatory impact on the final ethanol productions for all cultures under selected conditions. The co-culture was able to actively ferment glucose, xylose, cellulose and micro-crystallized cellulose (MCC). The ethanol yield observed in the co-culture was higher (up to twofold) than in mono-cultures, especially in MCC fermentation. The highest ethanol yield (as a percentage of the theoretical maximum) observed was 75% (w/w) for MCC and 90% (w/w) for xylose.     

High production of prodigiosin by Serratia marcescens grown on ethanol

Serratia marcescens S389, isolated as an ethanol-utilizing bacterium, produced prodigiosin at up to 3 mg ml^–1 when grown on ethanol and with the omission of inorganic phosphate and NaCl from the medium. This yield was some 200-fold greater than that previously reported.     

Bacterial community changes in TCE biodegradation detected in microcosm experiments

Laboratory microcosm experiments were set up to model biodegradation of trichloroethylene (TCE). Groundwater samples from two contaminated sites were taken, one of them with low (70 mg L⁻¹), the other with high sulfate (685 mg L⁻¹) concentration. In order to assess the biodegradative potential of natural microbiota, supplementary substrates (whey or molasses) were added to the bottles. At day 54, 98, 155, and 318, chemical and bacteriological parameters (i.e., Dehalococcoides test) were investigated. Terminal restriction fragment length polymorphism (T-RFLP) based diversity assessments were carried out to observe the bacterial community changes. Whey and molasses enhanced degradation at different rates. In the case of samples with high sulfate content and amended with whey, no ethylene, ethane, or methane was generated. Both ethylene and methane production was detected in samples of low sulfate content with added whey. The results of Dehalococcoides tests were positive for all control and amended samples. Based on T-RFLP analysis, the bacterial communities of high sulfate concentration groundwater microcosms amended with molasses or whey were very similar, while the communities of groundwater samples with low sulfate concentration were different when supplemented with whey or molasses. The rRNA and rDNA based investigations suggest that the proportions of the active microbes and the microbes present in the microcosms differ.     

Preliminary evaluation of the ethanol production from enzymatically hydrolyzed sugarcane bagasse

Ethanol production from sugarcane bagasse using immobilizedSaccharomyces cerevisiae was increased when coupled to a saccharification process involving the cellulase fromTrichoderma viride. However, the fixed costs of production, estimated to be about $US 0.4/tonne 95% ethanol, were high. Some key areas that influence these costs were identified.