Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine

The most important factors affecting microbial growth in an alcoholic beverage are the ethanol content and the low pH. The effectiveness of different organic acids in conjunction with ethanol concentration in controlling growth of yeasts was determined for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Brettanomyces lambicus, Pichia anomala, Zygosaccharomyces bailii, Saccharomycodes ludwigii and Kluyveromyces thermotolerans in malt extract broth (MEB). The results are summarized as undissociated concentrations of different acids required to inhibit growth of yeasts in MEB containing 10% (v/v) ethanol. About half the amount of undissociated malic or tartaric acid is necessary for inhibition of the yeasts, compared with acetic and lactic acid but the concentrations of acid necessary to inhibit growth were generally very high and unrealistic in wines for controlling growth of most of the yeasts tested. All the yeasts tested were able to grow in acidified non- or low alcoholic beverages but, at higher ethanol concentrations, Sacch. cerevisiae and Zygosacch. bailii have the greatest spoilage potential.     

Germanium toxicity in selected bacterial and yeast strains

Summary The toxicity of germanium dioxide (GeO₂) to 21 bacterial and 13 yeast strains was investigated in liquid broth medium to obtain information on strains tolerant to high (1 to 2 mg/ml) GeO₂ concentrations.Arthrobacter sp. NRC 32005,enterobacter aerogenes NRC 2926,Klebsiella aerogenes NCTC 418 andPseudomonas putida NRC 5019 were tolerant to 1 mg/ml GeO₂.Bacillus sp. RC607 was able to grow in the presence of 2 mg/ml GeO₂ at pH 10 in broth culture. The yeastsCandida guilliermondii, Candida shehatae andPachysolen tannophilus were the most sensitive to GeO₂ as evidenced by their diminished growth rates at a GeO₂ concentration as low as 0.1 mg/ml. None of the yeast strains tested exhibited growth in the presence of 1 mg/ml GeO₂. The high pH of the medium containing germanium may be partially responsible for the growth inhibition of the yeast cultures. Select bacterial cultures previously exposed to 1 mg/ml GeO₂ could tolerate and grow better at 2 mg/ml GeO₂, suggesting the existence of very efficient adaptive mechanisms. The pH of the medium could modulate GeO₂ tolerance and this effect was found to be strain-dependent.     

Potential of ochratoxin A production by Aspergillus carbonarius strains isolated from grapes at different ecological factors

Aspergillus carbonarius is known to colonize and produce ochratoxin A (OTA) on grapes and its derived products which is harmful to humans. We screened and tested A. carbonarius strains which isolated from grapes for production of OTA and selected three high OTA producing strains (ACSP1, ACSP2, ACSP3) for this study. These strains were further tested for their ability to produce OTA at different ecological factors [temperature 15, 25, 30, 35°C; water activity (a_w) 0.98, 0.95, 0.90, 0.88; and pH 4.0, 7.0, 9.0, 10.0]. Out of the three strains tested, A. carbonarius ACSP3 produced high levels of OTA than ACSP2 and ACSP1 in all the ecological factors. At 30°C A. carbonarius strains produced the highest OTA compared with other temperature regimes. With reference to water activity, a_w 0.98 favoured mycelial growth and accumulation of more OTA with all the three A. carbonarius strains. Further, pH 4.0 was encouraged the greatest production of OTA in all the strains. No growth was observed at a_w 0.88 and pH 10.0 in all the three strains except the strain ACSP3 at high pH. Our work demonstrated that temperature 30°C, a_w 0.98 and pH 4.0 is optimum for growth and production of OTA by A. carbonarius strains. Maximum amounts of OTA were found at earlier growth stages (7–9 days of incubation) in all the strains of A. carbonarius. The present study revealed that different ecological factors had great impact on OTA production by A. carbonarius which is useful for understanding OTA contamination and to develop proper management practices in future research programmes.     

Environmental factors affectingin vitro nitrogenase activity of cyanobacteria isolated from rice-fields

The nitrogen-fixing capacity of four cyanobacterial strains was tested in relation to heterotrophic ability, tolerance to combined nitrogen and adaptive capacity to changes in light intensity and pH. Strains (Anabaena variabilis UAM 202;Calothrix marchica UAM 214;Nodularia spumigena UAM 204,Nostoc punctiforme UAM 205) were isolated from the rice-fields of Valencia (Spain).C. marchica, was the only strain able to grow and to fix dinitrogen under heterotrophic conditions, with fructose and glucose. Fructose was the best substrate supporting growth and dinitrogen fixation in mixotrophy (growth in the light under conditions where CO₂ and organic carbon are assimilated simultaneously), photoheterotrophy (growth in the light on an organic compound in the absence of net CO₂ fixation) and heterotrophy (growth on an organic compound in the dark). Ammonium repressed nitrogenase more than nitrate. Full repression was observed only at concentrations of combined nitrogen higher than those usually found in rice-fields. Carbohydrates had a protective effect on nitrogenase against ammonium inhibition inC. marchica. All four strains showed increased nitrogenase activity when the light intensity was increased during assays. Variations of pH normally occurring in rice fields led to no important changes in nitrogenase activity inC. marchica. This fact, together with its potential for heterotrophic growth and tolerance to combined nitrogen, make this the most suitable of the four strains for inoculation experiments in rice fields.     

Microbiological Characterization of Wet Wheat Distillers' Grain, with Focus on Isolation of Lactobacilli with Potential as Probiotics

Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species Lactobacillus amylolyticus, Lactobacillus panis, and Lactobacillus pontis, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50°C, with L. amylolyticus being able to grow at temperatures as high as 54°C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of L. panis and L. pontis showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.     

Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products

The aim of this study was to investigate some probiotic properties of 42 wild Lactobacillus plantarum strains isolated from different Italian foods of animal origin. The strains were first screened for their antibiotic resistance profile (chloramphenicol, erythromycin, gentamicin, and tetracycline), subsequently they were tested for their in vitro resistance to lysozyme (100 mg L^?1), low pH (3.0, 2.5 and 2.0) and bile salts (0.3, 0.5 and 1.0 %). Moreover, agglutination property was studied (adhesion to Saccharomyces cerevisiae cells), as well as the presence of bsh and msa genes. The strains with the best characteristics were subjected to a further trial in order to evaluate their ability to survive to multiple stresses over time (lysozyme, low pH and bile salts) and the effect of these treatments on adhesion to yeast cells. All the strains were susceptible to chloramphenicol, erythromycin and gentamicin, while 6 strains were excluded from further evaluation because of their resistant phenotype against tetracycline. All the strains were able to grow in presence of lysozyme, as well as in MRS broth at pH 3.0. Only 4 strains showed a growth rate lower than 80 % when grown in MRS broth at pH 2.5, while a relevant growth rate decrease was observed after exposure to pH 2.0. Bile salts didn’t affect the viability of the L. plantarum cells. Twenty-one strains out of 33 tested strains were able to adhere to S. cerevisiae cells. Presence of both bsh and msa genes was detected in 6 strains. The strains resistant to all the stresses, positive to agglutination with S. cerevisiae and showing bsh and msa genes were selected for further evaluation and subjected to different stress treatments over time. The assessment of growth rates showed that exposure to lysozyme significantly increased low pH resistance in L. plantarum. This increase ranged from 2.35 to 15.57 %. The consequential lysozyme and low pH exposures didn’t affect the growth rate values after bile salts treatment, as well as the ability of the strains to adhere to yeast cells wasn’t modified by previous treatments (lysozyme, low pH and bile salts). The present work allows to increase knowledge about non starter lactic acid bacteria from Italian food products. The studied L. plantarum strains showed a good potential for their use as probiotic cultures. However, more in vivo tests are necessary to confirm this potentiality.     

Technological properties of indigenous wine yeast strains isolated from wine production regions of Turkey

The purpose of this study was to evaluate the important technological and fermentative properties of wine yeast strains previously isolated from different wine producing regions of Turkey. The determination of the following important properties was made: growth at high temperatures; fermentative capability in the presence of high sugar concentration; fermentation rate; hydrogen sulfide production; killer activity; resistance to high ethanol and sulfur dioxide; foam production; and enzymatic profiles. Ten local wine yeast strains belonging to Saccharomyces, and one commercial active dry yeast as a reference strain were evaluated. Fermentation characteristics were evaluated in terms of kinetic parameters, including ethanol yield (Y_P/S), biomass yield (Y_X/S), theoretical ethanol yield (%), specific ethanol production rate (q_p; g/gh), specific glucose uptake rate (q_s; g/gh), and the substrate conversion (%). All tested strains were able to grow at 37 °C and to start fermentation at 30° Brix, and were resistant to high concentrations of sulfur dioxide. 60 % of the strains were weak H₂S producers, while the others produced high levels. Foam production was high, and no strains had killer activity. Six of the tested strains had the ability to grow and ferment at concentrations of 14 % ethanol. Except for one strain, all fermented most of the media sugars at a high rate, producing 11.0–12.4 % (v/v) ethanol. Although all but one strain had suitable characteristics for wine production, they possessed poor activities of glycosidase, esterase and proteinase enzymes of oenological interest. Nine of the ten local yeast strains were selected for their good oenological properties and their suitability as a wine starter culture.     

Production of Kluyveromyces spp. and environmental tolerance induction against Aspergillus flavus

The viability and biomass production of three isolates of Kluyveromyces spp. in six different growth media were studied. All yeast isolates showed good growth in all of the media tested, but nutrient yeast dextrose broth (NYDB 75 %) and molasses soy meal (MSB) media were selected for further analyses. The adaptive response of the yeasts to heat shock and water stress was studied, revealing that 60 min of incubation at 45 °C and a water activity value of 0.95 a_w were the appropriate conditions to adapt these yeasts for subsequent analyses. The physiological adaptation did not affect the ecological similarity between biocontrol agents and pathogen. The adapted yeasts also had a negative influence on the growth of Aspergillus flavus RCM89 mycelia and the accumulation of aflatoxin B₁ levels in vitro. These results have important implications for optimizing the formulation process of proven biocontrol agents against A. flavus. In addition, the applications of physiological methods are necessary for increasing the performance of biocontrol agents. Moreover, the physiological methods could enhance survival under environmental stress conditions of biological control agents.     

Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka, India

A total of 14 Azotobacter strains were isolated from different paddy cultivating soils with pH ranging from 6.5 to 9.5 by using serial dilution agar plate method. The strains were Gram negative, rod shaped, cyst forming and developed brown to black colored colonies, which were glistening, smooth, slimy on Ashby’s agar plates. Biochemically they were positive for biochemical tests namely, indole production, citrate, catalase, carbohydrate fermentation and Voges–Proskauer test. Further, sequence analysis of PCR amplicons obtained from these cultures revealed the presence of five different Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris, Azotobacter sp., Azotobacter nigricans subsp. nigricans and Azotobacter tropicalis. Phylogenetically these strains were grouped into two distinct clusters. These strains were tested for their ability to grow on a media containing four different pesticides such as pendimethalin, glyphosate, chloropyrifos and phorate, which are commonly used for the paddy. Out of 14 strains tested, 13 strains were able to grow on a media containing herbicides such as pendimethalin, glyphosate and insecticides like chloropyrifos and phorate. However, five Azotobacter strains were able to grow at higher concentration of 5 % pesticides, without affecting their growth rate. Further, the effect of pesticides on the indole acetic acid (IAA) production by Azotobacter strains was also estimated. Azotobacter-16 strain was found to produce 34.4 μg ml^?l of IAA in a media supplemented with 1,000 mg of tryptophan and 5 % of pendimethalin. Present study reveals that species of Azotobacter are able to grow and survive in the presence of pesticides and no significant effects were observed on the metabolic activities of Azotobacter species.     

Preliminary characterization of some Streptomyces species isolated from a composting process and their antimicrobial potential

The aim of this study was to screen Streptomycetes isolates with antimicrobial and antiviral activity, in a search for new metabolites. The isolates were obtained from a composting process, and identified based on morphological characteristics and molecular biological methods. The antimicrobial activity was determined using the double-layer agar method against 53 test organisms (bacteria, yeasts, and filamentous fungi). All isolates were grown in submerged culture, in mineral salts-starch-casein (SC) broth and ISP2 media, and the filtrate cultures were used in the assays for antibacterial and antiviral activity. Bovine Herpes virus (BoHV-I) was used for the antiviral activity. The morphological and molecular characteristics confirmed that all 25 isolates belonged to the genus Streptomyces. In the assay for antimicrobial activity, 80% of the Streptomyces isolates were able to inhibit at least one of the test organisms. Of these, 80% were active against bacteria and 45% against fungi. Eight of the isolates showed a broad spectrum of inhibitory activity; of these, the isolate Streptomyces spp. 1S was able to inhibit 46 of the test organisms, and, most importantly, the 16 Gram-negative strains were inhibited. Of the 25 isolates, 44.4% of the isolates were able to grow and produce bioactive metabolites when grown in submerged culture. Four extracts showed a cytopathic effect in 10 CCID₅₀ MDBK cell, even though no viricidal effect was observed. The results obtained with these isolates indicated good biotechnological potential of these Streptomyces strains.     

Utilisation of steviol glycosides from Stevia rebaudiana (Bertoni) by lactobacilli and bifidobacteria in in vitro conditions

In the current study, eight strains of bifidobacteria and seven strains of lactobacilli were tested for their ability to grow in the presence of rebaudioside A and steviol glycosides from the sweetener Natusweet M001 originating from herb Stevia rebaudiana (Bertoni). Stevia is gaining popularity as a natural, non-caloric sugar substitute, and recently, it was allowed as a food additive by European Union too. Utilisation of steviol glycosides by intestinal microbiota suggests that they might have potential prebiotic effect. Based on the evaluation of bacterial density and pH values in our in vitro study, it was found that lactobacilli and bifidobacteria tested were able to utilise steviol glycosides as a carbon source only to a very limited extent. All strains tested showed significantly lower change in the absorbance A₅₄₀ (P?<?0.05) and pH decrease of the growth media as compared with the positive controls (medium containing glucose as a carbon source and de Man Rogosa Sharpe broth). We concluded that a suggested prebiotic effect was not confirmed either in the case of rebaudioside A or in the case of the sweetener Natusweet M001 containing a mixture of steviol glycosides.     

Growth and sporulation of entomopathogenic Beauveria bassiana,Metarhizium anisopliae,Isaria farinosa and Isaria fumosorosea strains in relation to water activity and temperature interactions

This study examined six strains of Beauveria bassiana s.l. and Isaria farinosa, one strain of Isaria fumosorosea and five strains of Metarhizium anisopliae s.l. to identify the ability for (1) growth and (2) sporulation under interacting environmental factors of water activity (a_w) and temperature stress. Growth on Sabouraud Dextrose Agar (SDA; water activity, a_w = 0.995) or SDA modified with glycerol to 0.98, 0.96 and 0.94 a_w was measured at four different temperatures (25, 30, 35 and 37°C). All M. anisopliae strains grew at 25–35°C and 0.995 a_w while only two strains tolerated extreme water stress at 0.94 a_w.Three strains of B. bassiana were able to grow at 25–37°C and 0.995 a_w. Only one strain of I. farinosa was able to grow at 25–37°C and 0.995 a_w. A_w and temperature interactions resulted in different strain-dependent responses, in terms of growth and sporulation. Only one strain of I. farinosa and three of M. anisopliae grew at 0.94 a_w and none of the B. bassiana strains tolerated such water stress. At 0.96 and 0.94 a_w and 35–37°C, sporulation by all the strains of the three species were significantly affected. Under elevated temperatures and drought stress, very few of these strains of entomopathogenic fungi are able to grow and sporulate. Indeed, the B. bassiana strains were unable to tolerate the extreme conditions examined. Resilience to such abiotic interactions is critical for selecting strains for formulations. Tolerance to water and temperature stress could be good criteria for selection of strains with secondary spread potential for use as part of an integrated pest management system where secondary cycling may be important, especially in sub-tropical and tropical environments.     

Oxidation of Gaseous and Volatile Hydrocarbons by Selected Alkene-Utilizing Bacteria

Eleven strains of alkene-utilizing bacteria belonging to the genera Mycobacterium, Nocardia, and Xanthobacter were tested for their ability to grow with C₁ to C₆ alkanes, C₂ to C₆ alkenes, alkadienes, and monoterpenes furnished individually as sole sources of carbon and energy in a mineral salts medium. A limited number of alkenes and alkanes supported growth of the bacteria; some bacteria were unable to grow on any of the saturated hydrocarbons tested. Monoterpenes were frequently used as carbon and energy sources by alkene-utilizing bacteria belonging to the genera Mycobacterium and Nocardia. Washed cell suspensions of alkene-grown bacteria attacked the whole range of alkenes tested, whereas only three strains were able to oxidize alkanes as well. The alkenes tested were oxidized either to water and carbon dioxide or to epoxyalkanes. Few epoxides accumulated in stoichiometric amounts from the corresponding alkenes, because most epoxides formed were further converted to other compounds like alkanediols.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

Relation of the Heat Resistance of Salmonellae to the Water Activity of the Environment

The effect of water activity (a_w) on the heat resistance of eight strains of Salmonella was studied. Heat resistance of the organisms increased as the a_w of the heating menstruum was reduced. Sucrose afforded the cells a greater degree of protection than did fructose, glycerol, and sorbitol. A direct correlation between a_w and heat resistance could not be established over the range of a_w levels tested in this study. There was variation among the strains of salmonellae in the magnitude of the increase in heat resistance as the a_w level was reduced. All strains of Salmonella tested showed a greater increase in heat resistance than S. senftenberg 775W as the environment became drier. Washed cells had D values 25 to 75% lower than unwashed cells. Prior growth of the organisms in media with a reduced a_w increased the heat resistance of the organisms when glycerol, but not when sucrose, was the controlling substance.     

Killer activity of Saccharomyces cerevisiae strains: partial characterization and strategies to improve the biocontrol efficacy in winemaking

Killer yeasts are considered potential biocontrol agents to avoid or reduce wine spoilage by undesirable species. In this study two Saccharomyces cerevisiae strains (Cf8 and M12) producing killer toxin were partially characterized and new strategies to improve their activity in winemaking were evaluated. Killer toxins were characterized by biochemical tests and growth inhibition of sensitive yeasts. Also genes encoding killer toxin were detected in the chromosomes of both strains by PCR. Both toxins showed optimal activity and production at conditions used during the wine-making process (pH 3.5 and temperatures of 15–25 °C). In addition, production of both toxins was higher when a nitrogen source was added. To improve killer activity different strategies of inoculation were studied, with the sequential inoculation of killer strains the best combination to control the growth of undesired yeasts. Sequential inoculation of Cf8–M12 showed a 45 % increase of killer activity on sensitive S. cerevisiae and spoilage yeasts. In the presence of ethanol (5–12 %) and SO₂ (50 mg/L) the killer activity of both toxins was increased, especially for toxin Cf8. Characteristics of both killer strains support their future application as starter cultures and biocontrol agents to produce wines of controlled quality.     

Effects of temperature,water activity and gas atmosphere on mycelial growth of tempe fungi Rhizopus microsporus var. microsporus and R. microsporus var. oligosporus

Rhizopus microsporus var. microsporus and var. oligosporus are used in the manufacture of various Asian fermented foods (tempe, black oncom, sufu). In view of solid-substrate fermentation (SSF) control, mycelial growth of strains of both varieties was tested for sensitivity to fluctuations of temperature, water activity and interstitial gas composition. This was achieved by measuring radial growth as well as biomass dry weight of pre-germinated microcolonies on defined media. The optimum conditions were temperature 40 °C, a _w 0.995 and a gas composition of air for the growth of both strains on a model medium. Whereas radial growth rates of var. microsporus and var. oligosporus were similar, biomass growth rates of var. oligosporus were higher than those of var. microsporus under optimum conditions. The temperature-dependent growth of Rhizopus spp. at a _w > 0.98 could be described by the Ratkowsky Equation. Carbon dioxide (5–10% v/v) inhibited the growth of Rhizopus spp. at non-limiting levels of oxygen. The two strains were able to grow at low (0.5% v/v) oxygen levels, but the mycelial density was rather low. No interrelation of water activity and gas composition was observed, but at high water activity the fungi were more sensitive to changes of temperature. The implications for process control are discussed. This revised version was published online in November 2006 with corrections to the Cover Date.     

Biochemical characterization and growth patterns of new yeast isolates

African sorghum opaque beers play a vital role in the diet of millions of consumers. In the current study we investigated the growth profiles of yeast strains isolated from kpete-kpete, a traditional starter used to produce tchoukoutou, an opaque sorghum beer in Benin. 10 yeast strains were isolated from sorghum beer starters and cultivated under both liquid and solid media for phenotypic growth characterization. All yeast isolates were able to grow both on solid and liquid media. Based on their growth profiles, the isolates were clustered into three groups: (i) the aggressive growth pattern (30 %), (ii) the moderate growth pattern (50 %), and (iii) the slow growth pattern (20 %). Based on gene expression pattern, absorbance (A_600nm) and diameter of growth in both liquid and solid media respectively, yeast strains YK34, YK15 and YK48 were clustered in the first group, and referred to as the most aggressive growth strains, followed by group 2 (YK24, YK5, YK12, YK20, YK2) and group 3 (YK37, YK41). This growth pattern was confirmed by Invertase gene expression profiling of the yeasts showing group 1 with high level of Invertase gene expression followed by group 2 and group 3 respectively. Our results suggest that YK34, YK15 and YK48 and YK2 yeast strains constitute the best candidates in fermentation of sorghum beer production based on growth rate and assimilation of carbon and nitrogen sources.     

Survey of nonconventional yeasts for lipid and hydrocarbon biotechnology

Nonconventional yeasts have an untapped potential to expand biotechnology and enable process development necessary for a circular economy. They are especially convenient for the field of lipid and hydrocarbon biotechnology because they offer faster growth than plants and easier scalability than microalgae and exhibit increased tolerance relative to some bacteria. The ability of industrial organisms to import and metabolically transform lipids and hydrocarbons is crucial in such applications. Here, we assessed the ability of 14 yeasts to utilize 18 model lipids and hydrocarbons from six functional groups and three carbon chain lengths. The studied strains covered 12 genera from nine families. Nine nonconventional yeasts performed better than Saccharomyces cerevisiae, the most common industrial yeast. Rhodotorula toruloides, Candida maltosa, Scheffersomyces stipitis, and Yarrowia lipolytica were observed to grow significantly better and on more types of lipids and lipid molecules than other strains. They were all able to utilize mid- to long-chain fatty acids, fatty alcohols, alkanes, alkenes, and dicarboxylic acids, including 28 previously unreported substrates across the four yeasts. Interestingly, a phylogenetic analysis showed a short evolutionary distance between the R. toruloides, C. maltosa, and S. stipitis, even though R. toruloides is classified under a different phylum. This work provides valuable insight into the lipid substrate range of nonconventional yeasts that can inform species selection decisions and viability of lipid feedstocks.     

Sinorhizobium meliloti Cells Require Biotin and either Cobalt or Methionine for Growth

Sinorhizobium meliloti is usually cultured in rich media containing yeast extract. It has been suggested that some components of yeast extract are also required for growth in minimal medium. We tested 27 strains of this bacterium and found that none were able to grow in minimal medium when methods to limit carryover of yeast extract were used during inoculation. By fractionation of yeast extract, two required growth factors were identified. Biotin was found to be absolutely required for growth, whereas previously the need for this vitamin was considered to be strain specific. All strains also required supplementation with cobalt or methionine, consistent with the requirement for a vitamin B₁₂-dependent homocysteine methyltransferase for methionine biosynthesis.