Search

This work aims to develop a new enzymatic method for analysing fumaric acid in wines. Fumaric acid is a very effective lactic acid bacteria inhibitor that is widely used in the food industry as an additive (E297). Using it to inhibit malolactic fermentation has recently been authorized by the International Organisation of Vine and Wine (OIV). However, the official analytical method for fumaric acid involves the classical HPLC method for organic acids, which is overly complicated for use in wineries. This research proposes a very simple and efficient enzymatic method that allows the simultaneous determination of L-malic acid and fumaric acid. The method uses a commercial enzymatic kit for L-malic acid and adds a supplementary step in which the fumarase enzyme is added to transform fumaric acid into L-malic acid. The results obtained show that this method could be used in different media (synthetic solution, white wine, red wine and white grape juice). This article also proposes a modification of the enzymatic method to be applied when the L-malic acid concentration of the sample is very high.

This review examines the different types of interactions between the microorganisms involved in the fermentation processes of alcoholic beverages produced all over the world from cereals or fruit juices. The alcoholic fermentation converting sugars into ethanol is usually carried out by yeasts, mainly Saccharomyces cerevisiae, which can grow directly using fruit sugars, such as those in grapes for wine or apples for cider, or on previously hydrolyzed starch of cereals, such as for beers. Some of these beverages, or the worts obtained from cereals, can be distilled to obtain spirits. Besides S. cerevisiae, all alcoholic beverages can contain other microorganisms and especially in spontaneous fermentation when starter cultures are not used. These other microbes are mostly lactic acid bacteria and other yeasts-the non-Saccharomyces yeasts. The interactions between all these microorganisms are very diverse and complex, as in any natural occurring ecosystem, including food fermentations. To describe them, we have followed a simplified ecological classification of the interactions. The negative ones are amensalism, by which a metabolic product of one species has a negative effect on others, and antagonism, by which one microbe competes directly with others. The positive interactions are commensalism, by which one species has benefits but no apparent effect on others, and synergism, by which there are benefits for all the microbes and also for the final product. The main interactions in alcoholic beverages are between S. cerevisiae and non-Saccharomyces and between yeasts and lactic acid bacteria. These interactions can be related to metabolites produced by fermentation such as ethanol, or to secondary metabolites such as proteinaceous toxins, or are feed-related, either by competition for nutrients or by benefit from released compounds during yeast autolysis. The positive or negative effects of these interactions on the organoleptic qualities of the final product are also revised. Focusing mainly on the alcoholic beverages produced by spontaneous fermentations, this paper reviews the interactions between the different yeasts and lactic acid bacteria in wine, cider, beer, and in spirits such as tequila, mezcal and cachaça.

Oenococcus oeni is the main agent of malolactic fermentation in wine. This fermentation takes place after alcoholic fermentation, in a low nutrient medium where ethanol and other inhibitor compounds are present. In addition, some yeast-derived compounds such as mannoproteins can be stimulatory for O. oeni. The mannoprotein concentration in wine depends on the fermenting yeasts, and non-Saccharomyces in particular can increase it. As a result of the hydrolytic activity of O. oeni, these macromolecules can be degraded, and the released mannose can be taken up and used as an energy source by the bacterium. Here we look at mannoprotein consumption and the expression of four O. oeni genes related to mannose uptake (manA, manB, ptsI, and ptsH) in a wine-like medium supplemented with mannoproteins and in natural wines fermented with different yeasts. We observe a general gene upregulation in response to wine-like conditions and different consumption patterns in the studied media. O. oeni was able to consume mannoproteins in all the wines. This consumption was notably higher in natural wines, especially in T. delbrueckii and S. cerevisiae 3D wines, which presented the highest mannoprotein levels. Regardless of the general upregulation, it seems that mannoprotein degradation is more closely related to the fermenting medium.

The use of non-Saccharomyces yeast together with S. cerevisiae in winemaking is a current trend. Apart from the organoleptic modulation of the wine, the composition of the resulting yeast lees is different and may thus impact malolactic fermentation (MLF). Yeasts of Saccharomyces cerevisiae, Torulaspora delbrueckii and Metschnikowia pulcherrima were inactivated and added to a synthetic wine. Three different strains of Oenococcus oeni were inoculated and MLF was monitored. Non-Saccharomyces lees, especially from some strains of T. delbrueckii, showed higher compatibility with some O. oeni strains, with a shorter MLF and a maintained bacterial cell viability. The supplementation of lees increased nitrogen compounds available by O. oeni. A lower mannoprotein consumption was related with longer MLF. Amino acid assimilation by O. oeni was strain specific. There may be many other compounds regulating these yeast lees-O. oeni interactions apart from the well-known mannoproteins and amino acids. This is the first study of MLF with different O. oeni strains in the presence of S. cerevisiae and non-Saccharomyces yeast lees to report a strain-specific interaction between them.

Non-Saccharomyces yeasts have increasingly been used in vinification recently. This is particularly true of Torulaspora delbrueckii and Metschnikowia pulcherrima, which are inoculated before S. cerevisiae, to complete a sequential alcoholic fermentation. This paper aims to study the effects of these two non-Saccharomyces yeasts on malolactic fermentation (MLF) carried out by two strains of Oenococcus oeni, under cellar conditions. Oenological parameters, and volatile and phenolic compounds were analysed in wines. The wines were tasted, and the microorganisms identified. In general, non-Saccharomyces created more MLF friendly conditions, largely because of lower concentrations of SO2 and medium chain fatty acids. The most favourable results were observed in wines inoculated with T. delbrueckii, that seemed to promote the development of O. oeni and improve MLF performance.

Oenococcus oeni, the lactic acid bacterium (LAB) mainly responsible for malolactic fermentation, has been repeatedly isolated from wines, but hardly ever from grapes. In this study, a large survey of autochthonous LAB from the Catalan wine region of Priorat was made. A total of 761 LAB isolates, 254 from Grenache and Carignan grape berries and 507 from wines, were identified and typed. Around 70% of the isolates were O. oeni, mostly from wines, but remarkably, 53 of them were isolated from grapes. A minimum spanning tree of O. oeni strains constructed from the multilocus variable number tandem repeat analysis showed considerable phylogenetic diversity. Other non-Oenococcus species were also identified and typed, Lactobacillus plantarum being predominant in grapes. Other LAB isolates were Pediococcus pentosaceus, Fructobacillus tropaeoli, L. mali, L. lindneri and L. sanfranciscensis. High-throughput sequencing (HTS) analysis was also carried out with grape samples, and Oenococcus and Lactobacillus were detected in significant quantities, which corroborates the culturing results from the same samples.

Interest in some non-Saccharomyces yeasts has increased recently, because they have been associated with an improvement in wine quality. Nevertheless, little attention has been paid to the effect that the use of these yeasts may have on malolactic fermentation (MLF). In this study, the strains Torulaspora delbrueckii Biodiva and Metschnikowia pulcherrima Flavia were evaluated by co-inoculation and sequential fermentation with S. cerevisiae QA23. A fermentation with S. cerevisiae as a single starter was also performed as a control, then MLF was performed inoculating Oenococcus oeni PSU-1 in all wines. Finally, the wines obtained after alcoholic fermentation and MLF were characterised. The results of the coinoculated fermentations were similar to those of the S. cerevisiae control fermentations. Nevertheless, significant differences were observed in sequential fermentations in terms of lower content of acetic, L-malic and succinic acids. These differences were particularly noticeable in fermentations carried out with T. delbrueckii.

Using Torulaspora delbrueckii as starter culture for alcoholic fermentation (AF) is a current trend for enhancing the quality of red wines. As red winemaking usually requires subsequent malolactic fermentation (MLF), the compatibility of this yeast and Oenococcus oeni is a key factor for a successful fermentative process. In this work we study the interactions of T. delbrueckii and O. oeni in wines from grapes with different degrees of maturity. The results showed higher total polyphenolic index (TPI) values in T. delbrueckii wines. Moreover, the aromatic characteristics were improved in these wines, compared to the wines inoculated only with Saccharomyces cerevisiae. There was also a reduction in some inhibitor compounds for O. oeni, for instance medium chain fatty acids, as a result of the fermentation with this non-Saccharomyces. Overall, the use of T. delbrueckii resulted in better MLF performances.

The thioredoxin system protects against oxidative stress through the reversible oxidation of the thioredoxin active center dithiol to a disulphide. The genome of Oenococcus oeni PSU-1 contains three thioredoxin genes (trxA1, trxA2, trxA3), one thioredoxin reductase (trxB) and one ferredoxin reductase (fdr) which, until recently, was annotated as a second thioredoxin reductase. For the first time, the entire thioredoxin system in several O. oeni strains isolated from wine has been analysed. Comparisons at the DNA and protein levels have been undertaken between sequences from O. oeni and other genera and species, and the genera Leuconostoc and Lactobacillus were found to present the highest similarities. The gene most frequently absent from a collection of 34 strains and the sequences annotated in the NCBI database was trxA1. Moreover, phylogenetic analysis suggested that this gene was horizontally transferred from Lactobacillus to O. oeni. Strain-dependent expression profiles were determined in rich and in wine-like media. General over-expression was detected after inoculation into wine-like medium, with trxA3 being the most highly expressed gene. The increased transcriptional levels of the thioredoxin genes are indicative of the crucial role of this system in the O. oeni response to wine harsh conditions.

FIliació URV: SIInclòs a la memòria: SI Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were able to import GSH, substantial variability among them was detected. To assess the physiological function of GSH, three strains with different GSH-import capacities were selected. Significant changes in membrane fatty acids composition were observed due to GSH addition. The most relevant was the increase of cyclopropane fatty acids in cell membrane, in both the exponential and the stationary phases. Cells grown with GSH showed an improved survival against ethanol shock (14% v/v). GSH addition also increased biomass production during the adaptation to wine stress conditions (pH 4, pH 3.4 and 6% ethanol). The results suggest that GSH enrichment could improve the resistance to stress to O. oeni, which could be useful for the adaptation of MLF starter cultures.

Interest in using non-Saccharomyces yeasts in winemaking has increased in recent years due to their ability to improve wine quality. However, little information has been published regarding the possible effect on malolactic fermentation (MLF), carried out mostly by Oenococcus oeni. The aim of this paper is therefore to evaluate the effect of the most representative non-Saccharomyces species on O. oeni and wine MLF. Different strains of Torulaspora delbrueckii, Metschnikowia pulcherrima, Hanseniaspora uvarum, Hanseniaspora vineae and Starmerella bacillaris (syn. Candida zemplinina) were used in sequential alcoholic fermentation with Saccharomyces cerevisiae. The resulting wines were inoculated with four O. oeni strains. The action of non-Saccharomyces affected the final wine composition and the later role of O. oeni. Some of its strains could not perform MLF in H. uvarum wine due to high SO2 concentrations. In some cases, MLF was inhibited in wines inoculated with S. bacillaris. All the H. uvarum and H. vineae strains notably increased acetic acid concentrations, thus threatening wine quality. The best conditions for MLF were provided by some T. delbruecckii and M. pulcherrima strains, which showed increased concentrations of mannoproteins - compounds described as MLF activators -, no production of SO2, and low consumption of L-malic acid. In conclusion, non-Saccharomyces yeasts have diverse effects on O. oeni and MLF depending on the species, with T. delbrueckii and M. pulcherrima being those that showed the best compatibility with MLF development.

Trace amounts of the carcinogenic ethyl carbamate can appear in wine as a result of a reaction between ethanol and citrulline, which is produced from arginine degradation by some bacteria used in winemaking. In this study, arginine deiminase (ADI) pathway genes were evaluated in 44 Oenococcus oeni strains from wines originating from several locations in order to establish the relationship between the ability of a strain to degrade arginine and the presence of related genes. To detect the presence of arc genes of the ADI pathway in O. oeni, pairs of primers were designed to amplify arcA, arcB, arcC and arcD1 sequences. All strains contained these four genes. The same primers were used to confirm the organization of these genes in an arcABCD1 operon. Nevertheless, considerable variability in the ability to degrade arginine among these O. oeni strains was observed. Therefore, despite the presence of the arc genes in all strains, the expression patterns of individual genes must be strain dependent and influenced by the different wine conditions. Additionally, the presence of arc genes was also determined in the 57 sequenced strains of O. oeni available in GenBank, and the complete operon was found in 83 % of strains derived from wine. The other strains were found to lack the arcB, arcC and arcD genes, but all contained sequences homologous to arcA, and some of them had also ADI activity.

This work is a short review of the interactions between oenological yeasts and lactic acid bacteria (LAB), especially Oenococcus oeni, the main species carrying out the malolactic fermentation (MLF). The emphasis has been placed on non-Saccharomyces effects due to their recent increased interest in winemaking. Those interactions are variable, ranging from inhibitory, to neutral and stimulatory and are mediated by some known compounds, which will be discussed. One phenomena responsible of inhibitory interactions is the media exhaustion by yeasts, and particularly a decrease in L-malic acid by some non-Saccharomyces. Clearly ethanol is the main inhibitory compound of LAB produced by S. cerevisiae, but non-Saccharomyces can be used to decrease it. Sulfur dioxide and medium chain fatty acids (MCFAs) produced by yeasts can exhibit inhibitory effect upon LAB or even result lethal. Interestingly mixed fermentations with non-Saccharomyces present less MCFA concentration. Among organic acids derived as result of yeast metabolism, succinic acid seems to be the most related with MLF inhibition. Several protein factors produced by S. cerevisiae inhibiting O. oeni have been described, but they have not been studied in non-Saccharomyces. According to the stimulatory effects, the use of non-Saccharomyces can increase the concentration of favorable mediators such as citric acid, pyruvic acid, or other compounds derived of yeast autolysis such as peptides, glucans, or mannoproteins. The emergence of non-Saccharomyces in winemaking present a new scenario in which MLF has to take place. For this reason, new tools and approaches should be explored to better understand this new winemaking context., This work was supported by grant AGL2015-70378-R awarded by the Spanish Ministry of Economy and Competitiveness. The research leading to these results has received funding from “la Caixa” Foundation and Triptolemos Foundation. AiB was grateful to the predoctoral fellowship from the Universitat Rovira i Virgili.

Filiació URV: SIInclòs a la memòria: SI Although Oenococcus oeni is the main species that is responsible for malolactic fermentation (MLF), harsh wine conditions can limit its performance. Although several mechanisms underlying the response to stress have been studied in this species, little is known regarding the cellular systems that protect against oxidative stress in other bacteria, such as glutathione (GSH). O. oeni cannot synthesize GSH but contains several genes related to its utilization. In this study, the relative expression (RE) of the seven genes involved in the GSH redox system found in O. oeni PSU-1 (gshR, gpo, three glutaredoxin-like genes and two subunits of an hypothetical transporter) has been measured. The study was performed using three strains, with each exhibiting a different GSH uptake capacity. The strains were grown in a stress-adaptation medium supplemented with 5 mM GSH and under different adaptation stress conditions (pH 4 and 6% ethanol). The RE showed that only some of these genes, including one for a possible glutaredoxin (OEOE_RS04215) and cydC for a subunit of a putative GSH transporter (OEOE_RS1995), responded to the addition of GSH. The presence of ethanol had a relevant effect on gene expression. Among the studied genes, the one for a NrdH-redoxin (OEOE_RS00645) showed a common response to ethanol in the strains, being over-expressed when grown with GSH. In most cases, the transcriptional changes were more evident for the strain with a higher capacity of GSH uptake. Malolactic performance of the three strains after pre-adaptation was evaluated in wine-like media (12% ethanol and pH 3.4). It was observed that the addition of GSH during pre-adaptation growth had a protective role in the cells exposed to low pH and ethanol, resulting in a

Table olives are one of the main fermented vegetables in the world. Olives can be processed as treated or natural. Both have to be fermented but treated green olives have to undergo an alkaline treatment before they are placed in brine to start their fermentation. It has been generally established that lactic acid bacteria (LAB) are responsible for the fermentation of treated olives. However, LAB and yeasts compete for the fermentation of natural olives. Yeasts play a minor role in some cases, contributing to the flavour and aroma of table olives and in LAB development. The main microbial genus isolated in table olives is Lactobacillus. Other genera of LAB have also been isolated but to a lesser extent. Lactobacillus plantarum and Lactobacillus pentosus are the predominant species in most fermentations. Factors influencing the correct development of fermentation and LAB, such as pH, temperature, the amount of NaCl, the polyphenol content or the availability of nutrients are also reviewed. Finally, current research topics on LAB from table olives are reviewed, such as using starters, methods of detection and identification of LAB, their production of bacteriocins, and the possibility of using table olives as probiotics.

Strains of Oenococcus oeni performing malolactic fermentation might prove an interesting source of β-glucosidase enzymes, which can release aromatic volatile compounds from their flavorless glycoconjugate precursors present in wine. Many studies have been conducted using strains cultured in winelike media, but little is known about the activity of these strains in real wine. In this work, real Cabernet Sauvignon red wine was used as the culture medium to test β-glucosidase activity and bgl gene expression of four O. oeni strains. Assays in wine were compared with winelike medium and mixtures of wine and MRS broth medium. β-Glucosidase activity was detected in both whole-cell and supernatant fractions for all four strains tested, although results varied depending on the conditions. The highest activity was found in wine (4 U/mg) and the activity was inhibited in the presence of fructose. To measure bgl expression, the RNA of O. oeni was extracted directly from red wine for the first time. Using real-time qPCR, the relative expression of the bgl gene was evaluated. The expression of this gene varied depending on the strain and it was overexpressed in samples of wine diluted with MRS. These results could be of importance to the wine industry and in studies of strain adaptation to wine for the selection of starters.

Summary The production of ethyl carbamate (EC) and its precursor citrulline by two strains of Oenococcus oeni and one of Lactobacillus plantarum during malolactic fermentation (MLF) was studied in different conditions of pH, temperature, and ethanol and l-malic acid concentrations. The potential EC, defined as the concentration of EC after wine is heated at 80 °C for 48 h, was also investigated. The O. oeni strains were able to degrade arginine in the conditions studied and to excrete some citrulline. In these strains, the conditions that led to a slight increase in EC were a high ethanol concentration, low pH and a high l-malic acid concentration. However, the potential EC increased with higher pH. In the case of L. plantarum, arginine was not degraded and citrulline was not produced, although the potential EC was comparable with that of the O. oeni strains studied.

Trace amounts of the carcinogen ethyl carbamate can appear in wine by the reaction of ethanol with compounds such as citrulline and carbamyl phosphate, which are produced from arginine degradation by some wine lactic acid bacteria (LAB). In this work, the presence of arc genes for the arginine-deiminase pathway was studied in several strains of different species of LAB. Their ability to degrade arginine was also studied. To detect the presence of arc genes, degenerate primers were designed from the alignment of protein sequences in already sequenced LAB. The usefulness of these degenerate primers has been proven by sequencing some of the amplified PCR fragments and searching for homologies with published sequences of the same species and related ones. Correlation was found between the presence of genes and the ability to degrade arginine. Degrading strains included all heterofermentative lactobacilli, Oenococcus oeni , Pediococcus pentosaceus , and some strains of Leuconostoc mesenteroides and Lactobacillus plantarum .

Summary The effects of aeration and temperature during alcoholic fermentation (AF) on spontaneous and inoculated malolactic fermentation (MLF) of wine have been analysed by following the population dynamics of Oenococcus oeni strains with multiplex random amplified polymorphic DNA-polymerase chain reaction. It has enabled us to follow precisely the proportion of different autochthonous strains and the starter strain through fermentations. Lack of aeration led to delays in AF, which meant that autochthonous lactic acid bacteria could develop earlier and prevented the starter strain from developing correctly. Temperature was not found to lead to any differences. Two strains were isolated in the same spontaneous MLF, suggesting that, in some cases, multiple strains might be responsible for the degradation of malic acid in wine. It can be concluded that delays in the AF can negatively affect the control of MLF and that this can be studied by following the development of the different strains of O. oeni.

The goal of this study was to analyze how the profiles of membrane proteins of Oenococcus oeni change under particular stress conditions of wine. Sodium dodecyl sulfate polyacrylamide gel electrophoresis protein profiles of membrane fraction showed that a 40-kDa protein was overexpressed in the presence of SO2. The sequence of its N-terminal fragment showed a significant identity with glyceraldehyde-3-phosphate dehydrogenases (GAPDHs), but the protein showed no GAPDH activity. This sequence was compared with those of other GAPDHs with ClustalW alignment, and it was found to be somewhat similar to that of the cell-wall and membrane proteins of other lactic acid bacteria.

Agrobacterium MC501 and the mixed culture composed of Agrobacterium MC501, Xanthomonas MC701, Corynebacterium sp. MC401, and Corynebacterium sp. MC402, all previously isolated from a coal mine area by an enrichment culture with dibenzothiophene (DBT), were used to study DBT sulfone desulfurization. Both cultures were able to use DBT sulfone as a sole source of sulfur for growth. This compound was metabolized to 2-hydroxybiphenyl (2PP) and sulfate. Agrobacterium MC501 and the mixed culture could also utilize a wide range of organic and inorganic sulfur compounds as aources of sulfur such as DBT, thianthrene, diphenylsulfide, thiophene-2-carboxylate, dibutilsulfide, methionine, cysteine, sulfate, and sulfite. Based on these results, the above-mentioned strains can be used to characterize and study coal desulfurization.

The accumulation of citrulline and ornithine in wine or beer as a result of the arginine catabolism of some lactic acid bacteria (LAB) species increases the risk of ethyl carbamate and putrescine formation, respectively. Several LAB species, which are found as spoilage bacteria in alcoholic beverages, have been reported to be arginine degrading. This study evaluates the effect of ethanol content and low pH on the excretion of citrulline and ornithine by two strains belonging to the potential contaminant species Lactobacillus brevis and Pediococcus pentosaceus. In the conditions that most affected cell viability, arginine consumption per cell increased noticeably, indicating that arginine utilization may be a stress responsive mechanism. L. brevis showed a higher accumulation of ornithine in the media than P. pentosaceus. In the presence of ethanol, a higher expression of the arcC gene was found in P. pentosaceus, which resulted in a lower excretion of citrulline and ornithine than in L. brevis. This suggests that L. brevis is more likely to produce these amino acids, which are precursors of ethyl carbamate and putrescine.

Four out of 187 strains, from enrichment cultures of dibenzothiophene (DBT), grew on DBT or thiophene 2-carboxylate as S sources. The four isolates, presumptively identified as Agrobacterium sp., Xanthomonas sp. and Corynebacterium spp., individually and together desulphurized DBT, producing 2-hydroxybiphenyl and sulphate.

The microbial degradation of dibenzothiophene (DBT) and other organosulphur compounds such as thiophene-2-carboxylate (T2C) is of interest for the potential desulphurization of coal. The feasibility of degradation of DBT and T2C by Pseudomonas putida and other bacteria was analysed. Pseudomonas putida oxidized sulphur from DBT in the presence of yeast extract, but it did not when DBT was the sole source of carbon.

This research studied the influence of sodium chloride on bacteriocin activity of table olives’ strain Lactobacillus pentosus B96. The strain was cultured in MRS under different NaCl concentrations (0, 4, 6 and 8%, in w/v). In MRS, maximum bacteriocin activity was achieved 9 h later. A medium containing 4 or 6% NaCl (w/v) increased the total bioactivity of the strain and an 8% NaCl reduced it. Real-time PCR was used to monitor the genetic expression of the bacteriocin genes plnA, plnB, plnC, plnE/F, plnJ, plnK, plnN and plantaricin S. Cultured in MRS, plantaricin S reached its maximum expression during the lag phase while plnE/F expresses during the exponential phase. The presence of sodium chloride in the medium moved the maximum expression of plantaricin S to the stationary phase, independently of the concentration. 4% (w/v) of NaCl didn’t affect the expression pattern of plnE/F while promotes the expression of plnN during both the lag and the exponential phases. More sodium chloride, 6% (w/v) maintained the expression of plnN in the pag phase but not in the exponential and moved plnE/F expression to the stationary phase. Plantaricin S, plnE/F and plnN over-expressed during the stationary phase in the higher sodium chloride concentration assayed, 8% (w/v). The relative expression level of plsA was 1000-fold higher than that of the plnE/F and plnN genes and even the ldhD constitutive gene used. Under our conditions, expression of plnA, plnB, plnC, plnJ and plnK genes was not observed.

The correct performance of wine malolactic fermentation (MLF) depends on the metabolic characteristics of the Oenococcus oeni strain/s responsible for this process. This study characterizes four O. oeni strains, which behave differently in terms of malolactic performance, the citric acid use related to acetic acid production, and stress adaptation. Metabolic evolution and its associated enzymatic activities were studied and the transcriptional response of the genes related to MLF, citrate metabolism and stress response was compared among strains. A higher initial expression of both the malolactic enzyme and the encoding gene mleA may be related to faster MLF. The initial transcriptional levels of citrate lyase (citE) proved indicative of early citrate consumption. Moreover, the strains that performed best in wine-like conditions presented a much higher relative expression of several stress responsive genes, particularly hsp18, clpP, ctsR and rmlB. Finally, an inter-strain comparison of the transcriptional levels of selected genes at different times during MLF proved a useful tool in characterizing strains based on their metabolic behaviour.

The growth and oxygen consumption of a variety of thermophilic, acidophilic bacteria in the presence of thiophene-2-carboxylate (T2C) and dibenzothiophene (DBT) have been determined. T2C was extremely toxic to the acidophiles in comparison with neutrophiles, but appeared to be degraded by a heterotrophicSulfolobus- like thermophile. DBT proved to be unstable at high temperatures, even in the absence of bacteria, and was not a substrate for the thermophiles.

The production of Arbequina naturally green olives is a traditional and spontaneous process in which lactic acid bacteria (LAB) and yeasts are present. To better control the fermentation of olives, strains of LAB and yeasts that had been isolated from brines were used in this study. A strain of Lactobacillus pentosus selected from an industrial olive fermentation was used as a starter culture for the traditional fermentation of Arbequina naturally green olives. Three more strains isolated from Arbequina olive brines were selected: one yeast, (Candida diddensiae), and two Lactobacillus (one L. plantarum and the other L. pentosus). The individual fermentation profile of all the strains and the co-inoculation profile of each one of the three with the first selected L. pentosus were studied in pilot-scale fermentations. The results showed that all the strains used as a starter, and particularly the yeast C. diddensiae, reduced the Enterobacteriaceae survival period in comparison with the spontaneous process. Only when a L. pentosus strain was inoculated were the LAB counts above 106 cfu ml−1 throughout the process. The C. diddensiae starter failed to colonize the brine until the end of the process and no LAB were detected. Results of rep-PCR using the primer GTG5 showed that both L. pentosus starters were able to colonize the brine by the end of the process but when they were co-inoculated only one strain was dominant. The L. plantarum starter failed to colonize the brine. In the control fermentation, various autochthonous strains of L. pentosus and L. plantarum were detected. The pH only reached desirable levels when a L. pentosus strain was inoculated. From the results of the sensory evaluation, panellists found significant differences between the different starters used for fermenting olives.

The consumption of citrate by the malolactic bacterium Oenococcus oeni changes the aromatic profile of wines due to the production of volatile compounds such as diacetyl and acetic acid. In this study, the expression of genes related to citrate utilization in the O. oeni strain PSU-1 was investigated to further understand the role of this metabolic pathway in the adaptation to wine environment and its impact on organoleptic qualities. Different conditions of ethanol content (0% and 10%) and pH (3.5 and 4.0) were assayed to evaluate the transcriptional response to both these stress factors. In the presence of ethanol, metabolic and transcriptional behavior was different than the observed when ethanol was absent. The expression of citrate pathway genes was mainly affected by ethanol, while pH showed a lower effect. Among the studied genes, citE, ackA and alsD were the genes revealing a distinctive transcriptional response. The differences observed in gene expression were in correlation with the different content of end products such as acetic acid and diacetyl. The increment of gene expression observed in the presence of ethanol at low pH suggests the participation of citrate metabolism in the response to stress conditions.

Aims: The goal of this study was to examine the growth of Oenococcus oeni in the presence of phenolic compounds under wine conditions and to see how these compounds affect bacterial metabolism. Methods and Results: Phenolic compounds have been added to a basal medium that simulates the composition of wine. Fifty milligrams per litre or more of phenolic compounds stimulated bacterial growth. Oenococcus oeni seemed to use citric acid and trehalose, if they were present, before glucose and fructose. Citrate was completely exhausted in three days and the yield of acetate was higher when phenolic compounds were present. Conclusions: Phenolic compounds reduced the rate of sugar consumption and enhanced citric acid consumption, increasing the yield of acetic acid. Significance and Impact of the Study: This study allows a better knowledge of co-metabolism of citric acid and sugars by O. oeni in the presence of phenolic compounds of wine.

Malolactic fermentation (MLF) is carried out by Oenococcus oeni under very harsh conditions. This paper shows that stress compounds in wine such as SO(2), fatty acids and copper have an inhibitory effect on cell growth and MLF duration, and relates this effect to an inhibition of ATPase activity. Of the stress compounds, SO(2) and dodecanoic acid had the strongest effect, decreasing the ATPase specific activity to 37% and 58%, respectively. It can be concluded that ATPase is a good indicator of the physiological state of the cells and their ability to lead MLF.

This study shows that the growth of Oenococcus oeni CECT 4100 in a synthetic medium is affected by phenolic compounds in different ways, depending on their type and concentration. Generally they have no effects at low concentrations, but hydroxycinnamic acids are inhibitory at high concentrations. Malolactic fermentation was stimulated in the presence of catechin and quercetin, but increasingly delayed with increasing amounts of p-coumaric acid. Gallic acid appeared to delay or inhibit the formation of acetic acid from citric acid. This could lead to a better control of malolactic fermentation and suppress the increase in volatile acidity, which is undesirable in the wine-making process.

A survey has been made of the accumulation of nickel by resting cells of 32 strains selected as Ni‐resistant among a population of bacteria isolated from activated sludges and polluted sediments, with a view to obtaining a strain suitable for removing nickel from industrial wastes. The best strain was classified as Pseudomonas and removed about 5% of the metal from the solution. Other nine selected strains were different genera of Enterobacteriaceas.

Phenol-degrading strain QT 31 ofPseudomonas sp. was selected among other phenol-resistant bacteria from activated sludges of wastewater treatment plant of an oil refinery. Its capacity of degradation was studied at different periods of adaptation, reaching a phenol biodegradation rate of 28 mg/l phenol per hour, from minimal, medium with 1000 mg/l phenol, after adaptation for 20 days.

A survey has been made of the copper accumulation by resting cells of bacteria selected as copper-resistant, isolated from activated sludges. The best selected strain, classified asBacillus, retained copper at up to 3.8% of its cell dry weight. These values were lower in the presence of glucose, unlike a type culture ofBacillus cereus, in which the retention of copper was higher when glucose was present. Possible reasons for these changes in uptake of both strains are suggested.

A Technicon AutoAnalyzer has been adapted to analyse lysine of fermentation broths from lysine-producer mutants, with a photometric method with ninhydrin. Linearity and accuracy have been tested, with errors lower than 5.5%. The great advantadge is its speed, analysing one sample every 5 minutes, automatically.

A strain of Pseudomonas has been studied for its nickel accumulation capacity. Most assays were carried out exposing cells to the metal in a resting state. Results indicate an extracellular, metabolically independent adsorption that is decreased by the presence of acetate. A possible exchange of nickel for magnesium from the outer membrane is suggested. Nickel tolerance of this strain in minimal medium is limited.