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Pleurotus ostreatus (oyster mushrooms) are the second edible mushroom produced worldwide. They have great nutritional value, their production is sustainable and uses a variety of cheap lignocellulosic substrates, including agricultural and industrial wastes. This article aims to discuss the various methods of substrates disinfection for growing Pleurotus ostreatus, which is one of the most critical step in mushroom cultivation. The main characteristics of thermal pasteurization and other disinfection methods such as microwave and ultraviolet radiation have been compared and discussed. In terms of the production of Pleurotus, the most common substrate disinfection methods have been compared with particular emphasis on the yield and the biological efficiency. The ratio time/Temperature in thermal pasteurization has also been focused because of its major influence on the degradation of substrate nutrients and the equipment used for pasteurization has also been described. The main conclusion of this review is that thermal pasteurization is the best option for disinfection as it eliminates the major competitive microorganisms from the substrates, retain major nutrients, and allows some beneficial microorganisms to survive. Among several pasteurization methods, scalding at 80ºC for short times less than 30 min seems the best option, and substrate processing tunnels seems to be the most suitable equipment.

Fermentation processes have proceeded without microbiological control until starter cultures came up in 19th Century. However, in some processes, such as wine making a widespread use of starter cultures did not come up until the end of the 20th Century, when the cellar-friendly active dry wine yeast (ADWY) were available. However, this practice was challenged for the uniformity that brought a limited number of commercially available presentations and new tendencies in wine making have withdrawn the use of ADWY. The return to non-controlled wine fermentations may have considerable set backs especially in terms of economical losses, as these wines have much higher risks of presenting different levels of spoilage (presence of unwanted compounds that will be organoleptically detectable) that will not be acceptable for the consumer. The WILDWINE Project (EU contract 315065) proposed a system to overcome the criticism on the uniformity after use of ADWY by bringing “wild” microorganisms to the starter culture practice. The Project proposed mixed cultures as an alternative to single strain cultures that is the usual practice for starter cultures. This multi-strain and multi-species starter cultures aimed at reproducing the vineyard natural microbiota with the advantage of a selection of those strains and species that could add complexity to the final wines. The WILDWINE Project has focused the selection of this “wild” microorganism in 5 worldwide-recognised wine regions: Nemea and Crete (Greece), Piedmont (Italy), Bordeaux (France) and Priorat (Spain). The basic protocol was the same for all the regions: (i) to establish the natural biodiversity of the regions and determine the “microbial fingerprint” of the region, (ii) oenological screening of all the strains and species isolates to determine their quality for the wine making process and (iii) to use the microbial cocktails at pilot plants and in commercial cellars to determine the “microbial footprint” that the “wild” microorganisms leave on the final wines. In the Priorat region, a limited number of different strains from Saccharomyces cerevisiae were detected after sampling cellars and vineyards in two different harvests (2012 and 2013). These low number of S. cerevisiae strains is in sharp contrast with the high biodiversity observed in the same area 18 years before when a selection of strains of this species was preformed to provide a commercial “autochthonous” strain for the Priorat region. Furthermore, the main species found in the vineyards were the yeast-like fungus Aerobasidium pullulans and Hanseniaspora uvarum. A. pullulans disappeared when grapes were turned into must and then H. uvarum and Candida zemplinina were the main species. Minority species found in musts and later in wines were Metschnokowia pulcherrima and Torulaspora delbrueckii.

Selected starter cultures of Oenococcus oeni are widely used to initiate malolactic fermentation (MLF) in wine. Nevertheless, the inoculated culture does not always develop as expected and undesired strains can grow causing wine spoilage. Therefore, methods that can reliably differentiate O. oeni strains are essential to monitor the population dynamics of MLF. This work presents a new multiplex PCR method that allows the simultaneous species identification and strain typification of O. oeni, based on the combined use of species-specific PCR primers and a Random Polymorphic DNA (RAPD)-PCR primer. This method represents an useful tool for the control of wine MLF.

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.

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 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.

Aims: The goal of this study was to develop a reproducible method for molecular typing strains of Oenococcus oeni, and also to apply it in the study of population dynamics of these strains during malolactic fermentation of wine. Methods and Results: A new method of multiplex randomly amplified polymorphic DNA (RAPD)-PCR has been developed, based on the combination of one random 10-mer and one specific 23-mer oligonucleotide in a single PCR. This method generates unique and discriminant DNA profiles for strains of O. oeni. The strains of this species were also clearly distinguished from other species of lactic acid bacteria. The method was applied to study the dynamics of O. oeni strains during malolactic fermentation, in three vintages in the same cellar. Conclusions: A fast and reliable method for typing strains of O. oeni has been designed and optimized. It improves the reproducibility and rapidity of conventional RAPD-PCR, and it has been validated monitoring the population dynamics during malolactic fermentation. Significance and Impact of the Study: This method will be a good tool to study the population dynamics of bacteria during malolactic fermentation and to evaluate the performance of new malolactic starter cultures and their dominance over the native microbiota.

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.

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.