Academic literature on the topic 'Mixed culture fermentations'

ABSTRACT Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process.

In recent years, as a consequence of the re-evaluation of the role of non-Saccharomyces yeasts, several studies have been conducted on the use of controlled mixed fermentations with Saccharomyces and different non-Saccharomyces yeast species from the winemaking environment. To benefit from the metabolic particularities of some non-Saccharomyces yeasts, the management of a non-Saccharomyces strain in mixed fermentation is a crucial step, in particular the use of procedures addressed to increase the persistence of non-Saccharomyces strains during the fermentative process. The use of microencapsulation for cell immobilization might represent a strategy for enhancing the competitiveness of non-Saccharomyces yeasts during mixed fermentation. This study was aimed to assess the fermentative performance of a mixed starter culture, composed by a wild Hanseniaspora osmophila strain (ND1) and a commercial Saccharomyces cerevisiae strain (EC1118). For this purpose, free and microencapsulated cells of ND1 strain were tested in co-culture with EC1118 during mixed fermentations in order to evaluate the effect of the microencapsulation on fermentative behavior of mixed starter and final wine composition. The data have shown that H. osmophila cell formulation affects the persistence of both ND1 and EC1118 strains during fermentations and microencapsulation resulted in a suitable system to increase the fermentative efficiency of ND1 strain during mixed starter fermentation.

ABSTRACTWith the advent of the -omics era, classical technology platforms, such as hyphenated mass spectrometry, are currently undergoing a transformation toward high-throughput application. These novel platforms yield highly detailed metabolite profiles in large numbers of samples. Such profiles can be used as fingerprints for the accurate identification and classification of samples as well as for the study of effects of experimental conditions on the concentrations of specific metabolites. Challenges for the application of these methods lie in the acquisition of high-quality data, data normalization, and data mining. Here, a high-throughput fingerprinting approach based on analysis of headspace volatiles using ultrafast gas chromatography coupled to time of flight mass spectrometry (ultrafast GC/TOF-MS) was developed and evaluated for classification and screening purposes in food fermentation. GC-MS mass spectra of headspace samples of milk fermented by different mixed cultures of lactic acid bacteria (LAB) were collected and preprocessed in MetAlign, a dedicated software package for the preprocessing and comparison of liquid chromatography (LC)-MS and GC-MS data. The Random Forest algorithm was used to detect mass peaks that discriminated combinations of species or strains used in fermentations. Many of these mass peaks originated from key flavor compounds, indicating that the presence or absence of individual strains or combinations of strains significantly influenced the concentrations of these components. We demonstrate that the approach can be used for purposes like the selection of strains from collections based on flavor characteristics and the screening of (mixed) cultures for the presence or absence of strains. In addition, we show that strain-specific flavor characteristics can be traced back to genetic markers when comparative genome hybridization (CGH) data are available.

Consumers require high-quality beers with specific enhanced flavor profiles and non-conventional yeasts could represent a large source of bioflavoring diversity to obtain new beer styles. In this work, we investigated the use of three different non-conventional yeasts belonging to Lachancea thermotolerans, Wickerhamomyces anomalus, and Zygotorulaspora florentina species in pure and mixed fermentation with the Saccharomyces cerevisiae commercial starter US-05. All three non-conventional yeasts were competitive in co-cultures with the S. cerevisiae, and they dominated fermentations with 1:20 ratio (S. cerevisiae/non-conventional yeasts ratios). Pure non-conventional yeasts and co-cultures affected significantly the beer aroma. A general reduction in acetaldehyde content in all mixed fermentations was found. L. thermotolerans and Z. florentina in mixed and W. anomalus in pure cultures increased higher alcohols. L. thermotolerans led to a large reduction in pH value, producing, in pure culture, a large amount of lactic acid (1.83 g/L) while showing an enhancement of ethyl butyrate and ethyl acetate in all pure and mixed fermentations. W. anomalus decreased the main aroma compounds in comparison with the S. cerevisiae but showed a significant increase in ethyl butyrate and ethyl acetate. Beers produced with Z. florentina were characterized by an increase in the isoamyl acetate and α-terpineol content.

Fermented foods are of importance worldwide. Most are prepared under nonsterile conditions using mixed cultures, either deliberately or unavoidably. Fungal mixed cultures show interactive relations at various levels. In this paper, inhibitory effects among fungi owing to competition, formation of organic acids, toxic proteins, and mycotoxins are discussed. In addition, fungi show inhibitory effects towards bacteria and vice versa, through pH changes, and excretion of organic acids, antibiotics, peptides, etc. Stimulatory interactions among fungi and between fungi and bacteria relate mainly to carbon and nitrogen metabolism, and they play an important role in the inherent stability of mixed-culture systems maintained by enrichment techniques. Better understanding of natural mixed-culture fermentations has evolved into the development of the concept of cocultivation employing compatible microbial strains of complementary metabolic ability. Especially in the area of direct conversion of complex carbohydrates (e.g., starch, inulin, or lignocellulosic matter into ethanol), cocultivation has much to offer. Genetic modification of starter organisms offers opportunities to improve, for example, their ability to degrade substrate with a minimum of catabolite repression, and produce final products of superior quality. This is illustrated by recent recombinant DNA constructs for alcoholic fermentations. Key words: food, fungi, interaction, inhibition, stimulation, cocultivation.

ABSTRACTSelection of a starter culture with excellent viability and metabolic activity is important for inoculated fermentation of traditional food. To obtain a suitable starter culture for making Chinese sesame-flavored liquor, the yeast and bacterium community structures were investigated during spontaneous and solid-state fermentations of this type of liquor. Five dominant species in spontaneous fermentation were identified:Saccharomyces cerevisiae,Pichia membranaefaciens,Issatchenkia orientalis,Bacillus licheniformis, andBacillus amyloliquefaciens. The metabolic activity of each species in mixed and inoculated fermentations of liquor was investigated in 14 different cocultures that used different combinations of these species. The relationships between the microbial species and volatile metabolites were analyzed by partial least-squares (PLS) regression analysis. We found thatS. cerevisiaewas positively correlated to nonanal, andB. licheniformiswas positively associated with 2,3-butanediol, isobutyric acid, guaiacol, and 4-vinyl guaiacol, whileI. orientaliswas positively correlated to butyric acid, isovaleric acid, hexanoic acid, and 2,3-butanediol. These three species are excellent flavor producers for Chinese liquor. AlthoughP. membranaefaciensandB. amyloliquefacienswere not efficient flavor producers, the addition of them alleviated competition among the other three species and altered their growth rates and flavor production. As a result, the coculture of all five dominant species produced the largest amount of flavor compounds. The result indicates that flavor producers and microbial interaction regulators are important for inoculated fermentation of Chinese sesame-flavored liquor.

One-step fermentation involving two microorganisms inoculated at the same time and two-step fermentations involving two inoculations of different microbes at different times were used to enrich corn meal. Starch in corn meal was first hydrolyzed by amylases of B. stearothermophilus, E. fibuligera or A. oryzae followed by the growth of C. utilis. The combination of E. fibuligera and C. utilis produced a significant (P<0.05) increase in lysine, methionine, tryptophan. The relative nutritive value (%), which reflected the amino acid balance, increased significantly (P<0.05) with this sequence of microorganisms. Niacin, riboflavin, and thiamin increased significantly (P<0.05) when mixed cultures of A. oryzae and E. fibuligera in combination with C. utilis were employed. When E. fibuligera alone was grown, no significant change was observed in thiamin content but significant increases occurred in niacin and riboflavin. A. oryzae by itself produced significant (P<0.05) changes in niacin, riboflavin and thiamin.

Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with the yeast S. cerevisiae in multistarter wine fermentations has emerged as a useful tool to modulate wine aroma and/or to decrease the concentration of undesirable compounds. However, upon inoculation, these yeast species do not co-exist passively, but interact in various ways. While competition for nutrients and the excretion of killer toxins in an antagonistic relationship are obvious and well established types of interactions, some studies have suggested the existence of other forms of cellular or molecular interactions. One of these includes physical cell-cell contact and to our knowledge, only one previous study has confirmed its existence in wine yeasts. Yeast interactions are also influenced by other factors, such as ethanol concentration, however some studies have highlighted the role that dissolved oxygen plays on the survival of non-Saccharomyces yeasts and their ability to compete for space with S. cerevisiae and little research has focused on this. This study aimed to investigate the occurrence of a physical cell-cell and/or metabolic interaction between S. cerevisiae and L. thermotolerans in mixed culture fermentations of synthetic grape must. For this purpose, fermentations in a Double Compartment Bioreactor (DCB) which separates yeast population through the use of a membrane were compared to mixed fermentations in the absence of the membrane, using the same reactor. Furthermore, the impact of oxygen supply on yeast behaviour was also assessed. Following mixed culture fermentations in a DCB, it was observed that the presence of S. cerevisiae led to a significant decline in viability in L. thermotolerans. This decline was significantly less prominent in mixed cultures where the cells were in indirect contact. Together, the data provided evidence for both cell-cell and metabolic interactions whereby S. cerevisiae had a strong negative influence on the growth of L. thermotolerans. However, it was also observed that L. thermotolerans had some negative impact on the growth of S. cerevisiae, leading to a reduction in biomass (when in indirect contact) and a reduced maximum CFU/mL compared to pure cultures. The data also suggest that direct physical contact may increase the production of glycerol and propanol, but this needs further investigation. By decreasing the frequency at which oxygen pulses were provided, a reduction in biomass and increase in fermentation duration was observed for all fermentations. However, this effect was somewhat reduced in mixed cultures. Here, no impact on fermentation duration was observed and the decrease in biomass was less compared to pure cultures. The impact of these oxygen pulses was also greater on L. thermotolerans. In the latter yeast’s pure culture a slight increase in glycerol was observed when less oxygen was provided and in general there appeared to be no impact on acetic acid production. Furthermore, there was little or no impact on volatile production, however, more repeats might reveal different results and therefore more research is needed to confirm these results. To our knowledge, this is the first study of its kind to confirm a physical cell-cell interaction between the yeast pair S. cerevisiae and L. thermotolerans.
AFRIKAANSE OPSOMMING: Die gebruik van nie-Saccharomyces gis saam met die gis S. cerevisiae in multi-inokuleringskulture het die afgelope paar jaar as n goeie hulpmiddel na vore gekom om wyn aroma te moduleer en/of om die konsentrasie van ongewensde verbindings te verminder. Sodra inokulasie plaasgevind het, het hierdie gis die potensiaal om op verskeie maniere teenoor mekaar te reageer. Kompetisie vir nutriente en die afskeiding van toksiese verbindings in n antagonistiese verhouding is alreeds goed beskryf in die literatuur. Somige studies het, alhoewel, die bestaan van ander vorme van sellulêre of molekulêre interaksies voorgestel. Een van hierdie sluit in n fisiese sell-sell interaksie en so ver as wat ons kennis strek, het nog net een studie van tevore so ‘n interaksie bevestig tussen wyn giste. Gis interaksies word ook beïnvloed deur ander faktore, soos byvoorbeeld etanol konsentrasie. Terwyl sommige studies die rol wat opgelosde suurstof speel in die oorlewing van nie-Saccharomyces gis en hulle vermoë om te kompeteer vir spasie met S. cerevisiae alreeds beklemtoon, het min navorsing al hierop gefokus. Hierdie studie het gestreef om die voorkoms van n fisiese sell-sell en/of metaboliese interaksie tussen S. cerevisie en L. thermotolerans in gemengde kultuur fermentasies van sintetiese druiwe sap te ondersoek. Vir hierdie doeleinde was fermentasies uitgevoer met behulp van ‘n Dubbel Kompartement Bioreaktor (DKB) wat gis populasies skei deur middel van ‘n membraan en hierdie was vergelyk met gemengde kultuur fermentasies sonder die membraan in dieselfde reaktor sisteem. Verder was die impak van suurstof toevoer op gis gedrag ook geassesseer. Na afloop van gemengde kultuur fermentasies in ‘n DKB, was daar waargeneem dat die teenwoordigheid van S. cerevisiae gelei het tot ‘n betekenisvolle afname in lewensvatbaarheid in L. thermotolerans. Hierdie afname was aansienlik minder in gemengde kulture waar die gis in indirekte kontak was. Saam verskaf hierdie data bewyse vir n sell-sell asook metaboliese interaksie waardeur S. cerevisiae ‘n sterk, negatiewe invloed op die groei van L. thermotolerans gehad het. Daar was egter ook waargeneem dat L. thermotolerans tot ‘n mindere mate ‘n negatiewe impak op die groei van S. cerevisiae gehad het en dat dit gelei het tot ‘n verlaging in biomassa (toe die gis in indirekte kontak was) en ‘n verlaagde maksimum CFU/mL in vergelyking met suiwer kulture. Die data dui ook aan dat fisiese kontak kon gelei het tot ‘n verhoging in gliserol en propanol produksie, maar hierdie kort verdere ondersoek. Deur die frekwensie te verminder waardeur suurstof pulse aan die fermentasies verskaf was, was ‘n verlaging in biomassa produksie en ‘n verlenging in fermentasie tydperk waargeneem. Hierdie tendense was waargeneem in almal, behalwe die gemengde kultuur fermentasies. Die effek van suurstof puls verlaging was minder op hierdie fermentasies aangesien daar geen impak op fermentasie tydperk was nie en die verlaging in biomassa minder was. Die impak van hierdie suurstof pulse was ook groter op L. thermotolerans. ‘n Klein toename in gliserol produksie was waargeneem in laasgenoemde gis se suiwer kultuur toe minder suurstof beskikbaar was en oor die algemeen was asynsuur onveranderd. Verder was daar ‘n klein of geen impak op vlugtige verbindings nie, alhoewel, meer herhalings mag verskillende resultate lewer en daarom is meer navorsing nodig om hierde resultate te bevestig. So ver as wat ons kennis strek is hierdie die eerste studie van sy soort om ‘n fisiese sell-sell interaksie tussen die gispaar S. cerevisiae en L. thermotolerans te bevestig.

In the last decades increasing attention has been paid on syngas, produced through the thermal gasification of biomass, for energy recovery. Syngas components, mainly CO and H2, can be used as substrate for the conversion into important chemicals and fuels, in this case specifically methane, by a wide range of microorganisms. This study focused on the exploitation of an anaerobic mixed culture, collected from an anaerobic sludge of the Denmark Technical University (DTU) Bioengineering laboratory, in the fermentation of syngas for the production of CH4. A series of enrichment experiments with different initial partial pressure of CO and H2 were performed, in mesophilic and thermophilic conditions. Both yields and production rates resulted to be higher in thermophilic conditions; experiments with H2 tended to be faster than those without and the mmols of produced methane, over fed carbon (mmol/mmol), were higher than when only CO was present as substrate. A second experimental test has also been executed in thermophilic conditions (since they better performed in the previous experiment) to better evaluate which reactions are those which mainly took place during the syngas fermentation process. Experiments were performed both in the absence and in the occurrence of 2-bromoethanesulfonic acid as an effective inhibitor of the methanogens. Results showed that acetate mainly came from Acetogenesis via CO, being however negligible, while methane was produced either by hydrogenotrophic methanogens either via H2/CO. Production of bio hydrogen was also present, and it is usually further consumed to produce CH4. To conclude, findings of this projects may be considered as a further step towards the knowledge of biogas production through the use of microbial catalysts, giving important information both about the optimal operating conditions and about microbial metabolism.

The removal of multiple substrates in a defined mixed culture process was investigated in the treatment of brewery wastewater. The study was conducted using both batch and a semi-continuous reactor system called self-cycling fermentation. Batch experiments were conducted using a synthetic brewery wastewater containing glucose, ethanol and maltose. Activated sludge from a municipal wastewater treatment plant was acclimatized in the synthetic brewery wastewater. The microbes capable of degrading this wastewater were analyzed by a combination of microscopy, spread plating, and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and identified as Acinetobacter sp., Enterobacter sp. and Candida sp. From the pure culture batch experiments, it was determined that Enterobacter could degrade glucose and maltose but no ethanol, while Acinetobacter and Candida could degrade all three carbon sources. In mixed culture batch experiments, Enterobacter was dominant in degrading the sugar concentrations to levels permissible for Acinetobacter to degrade ethanol. PCR-DGGE was found to be effective in identifying the dominant species but selective carbon source plating was required to determine viability and track the population dynamics. Kinetic experiments were carried out in a semi-continuous, self-cycling fermentation process using the defined mixed culture in media containing glucose and various initial concentrations of ethanol and maltose. The overall rate of substrate removal was attributable to both the suspended culture and the biofilm formed during the process. A rate expression was developed for this system for the range of substrate concentrations tested. The data indicated that substrate removal by the suspended culture was a function of only the biomass concentration. However, substrate removal by the biofilm was found to be limited to the surface cells and determined to be a function of substrate concentration only.

Thesis submitted in fulfilment of the requirements for the degree of Doctor Technologiae: Chemical Engineering, Faculty of Engineering at the Cape Peninsula University of Technology - Cape Town, South Africa
Polycyclic aromatic hydrocarbons (PAHs) are one of the most common and recalcitrant environmental contaminants – known for their potential toxicity, mutagenicity, and carcinogenicity to humans. Biosurfactant application can enhance the biodegradation of PAHs. The main object of this work was to explore the novelty of biosurfactant produced by the isolated strains of Bacillus sp and Pseudomonas aeruginosa grown exclusively on Beta vulgaris, and the modification of the zeolites nanoparticles by the biosurfactant, for enhanced biodegradation of PAHs in soil. Novel biosurfactant-producing strains were isolated from hydrocarbon-contaminated environments, while several agrowaste were screened as primary carbon sources for the expression of biosurfactants, which were quantified using various standardized methods......

This thesis aimed at investigating a process based on anaerobic mixed culture fermentation (AMCF) of organic waste to produce carboxylates, molecular hydrogen and ethanol. The novelty of this study was to estimate the potential production of these bulk chemicals from selected renewable feedstocks and to experimentally investigate their production from the AMCF of three distinct types of feedstock without physicochemical pre-treatment: grass, a lignocellulosic substrate; cheese whey, a lactose-rich residue; and fish processing wastewater, a complex effluent containing proteins and fats. The estimation showed that AMCF of a small fraction (less than 1.6%) of the selected feedstock would be required to suffice the current production of carboxylates from oil-based feedstock, whilst up to 51% of the feedstock would be needed to satisfy the current production of ethanol and molecular hydrogen. In the experiments with AMCF of grass, low organic loading rates and long solids retention times were required to increase the substrate conversion because of the hard-biodegradability found in the substrate without any physicochemical pre-treatment. For the AMCF of cheese whey, the production of carboxylates decreased the pH to low levels and consequently inhibited the fermentation. For the AMCF of fish processing wastewater, the feedstock showed high biodegradability with production of biogas under batch condition and stable production of carboxylates under semi-continuous condition. The release of ammonium during the degradation of proteins was considered one of the main factors to avoid the pH drop when carboxylates were produced throughout the fermentation. In conclusion, the study showed that the feedstock nature and the operating condition play a key role in how to drive the AMCF to the production of these chemicals and avoid methanogenesis. Whilst hydrolysis limits the fermentation for hardly biodegradable feedstock, volatile fatty acids formation and pH inhibit the AMCF of more easily biodegradable feedstock.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Grape must gives rise to various stress conditions for the yeast inoculated for alcoholic fermentation. These include hyperosmotic stress due to the high initial sugar concentration and redox imbalances due to the fast depletion of oxygen. Under these stress conditions, Saccharomyces cerevisiae tends to produce glycerol as an osmoprotectant and to regenerate reducing equivalents. However, the production of glycerol often leads to increased acetic acid production. According to literature, it seems that many non-Saccharomyces yeasts have a different metabolic response to the above-mentioned stress conditions, especially since it has been found that they produce low levels of acetic acid. Only recently non-Saccharomyces yeasts were researched to be used as starter cultures in wine fermentations. It is found that they can confer beneficial characteristics to the resulting wine. However, most of the non-Saccharomyces yeasts lead to stuck fermentations as confirmed by this study. Therefore, if the positive characteristics of these yeasts were to be exploited in wine making they need to be inoculated together with S. cerevisiae. When two yeasts are inoculated together, they affect each other and consequently the wine. In this context, the aim of this study was to investigate the metabolic response to hyperosmotic stress during wine fermentation of the following wine-related non-Saccharomyces yeasts: Lachancea thermotolerans, Torulaspora delbrueckii and Starmerella bacillaris. Fermentations were performed in a synthetic grape must medium with pure cultures of the mentioned strains as well as mixed cultures of each non-Saccharomyces yeast with S. cerevisiae. The fermentation behaviour was monitored and concentrations of various wine-related metabolites were determined. Concerning polyol concentrations, S. cerevisiae produced only glycerol while the non-Saccharomyces yeasts also produced other polyols. The low production of acetic acid in the non-Saccharomyces fermentations was confirmed especially in the case of L. thermotolerans. Moreover, this yeast produced high levels of the higher alcohols butanol and propanol. St. bacillaris produced significant levels of acetoin and isobutyric acid and T. delbrueckii produced an increased concentration of succinic acid. All these metabolites might play a role in maintaining intracellular redox balance. However, a more extensive systematic study is needed to investigate the extent of their involvement. The mixed cultures completed fermentation and had higher final glycerol levels than the control and lower acetic acid concentrations and therefore can contribute positively to the wine aroma. Furthermore, the mixed culture fermentations showed the potential of lowering the ethanol concentrations of wine. Furthermore it has been shown in literature that the yeasts present in the mixed culture can affect each other on gene expression level as well. However, there is little genetic information available on non-Saccharomyces yeasts. In this study, we sequenced the genes involved in glycerol and acetic acid biosynthesis of L. thermotolerans and T. delbrueckii. The gene sequences are fairly homologous with only a few differences. These gene sequences can be used to study gene expression of GPD1 and ALD6 from fermentation samples in order to determine to what extent the yeasts in a mixed culture influence the gene expression of one another.
AFRIKAANSE OPSOMMING: Druiwemos gee oorsprong aan verskeie strestoestande vir die gis wat vir alkoholiese fermentasie geïnokuleer word. Hierdie strestoestande sluit hiper-osmotiese stres, as gevolg van die hoë suiker konsentrasie, in asook redoks wanbalanse toegeskryf aan die vinnige afname in beskikbare suurstof. Tydens hierdie toestande is Saccharomyces cerevisiae geneig om gliserol as beskerming teen die osmotiese stres te produseer, sowel as vir die regenereering van reduserings ekwivalente. Die produksie van gliserol lei egter dikwels tot toenemende asynsuur produksie. Volgens literatuur kom dit voor asof menige nie-Saccharomyces giste 'n ander metabolise reaksie tot die bogenoemde stresse het, omdat daar gevind is dat hulle laer vlakke van asynsuur produseer. Eers onlangs is navorsing gedoen op die potensiële gebruik van nie-Saccharomyces giste in gemengde kulture tydens wynfermentasies. Daar is bevind dat hulle voordelige eienskappe aan die wyn kan verleen. Meeste van die nie-Saccharomyces giste lei egter tot onvolledige fermentasies soos bevesting deur hierdie studie. Dus, indien die positiewe eienskappe van hierdie giste sou benut word in wynmaak sal hulle saam met S. cerevisiae geïnokuleer moet word. Wanneer twee giste saam geïnokuleer word, beïnvloed hulle mekaar en gevolglik die wyn. In hierdie konteks was die doel van die betrokke studie om die metaboliese reaksie tot hiperosmotiese stress tydens wynfermentasies te ondersoek in die volgende wyn verwante nie-Saccharomyces giste: Lachancea thermotolerans, Torulaspora delbrueckii en Starmerella bacillaris. Fermentasies was in sintetiese druiwemos medium uitgevoer met rein kulture van die genoemde gisrasse, sowel as gemengde kulture van elke nie-Saccharomyces gis met S. cerevisiae. Die fermentasiegedarg is gemonitor en die konsentrasies van verskeie wyn verwante metaboliete is bepaal. Wat die poliol konsentrasies betref, het S. cerevisiae slegs gliserol geproduseer terwyl die nie-Saccharomyces giste additionele poliole ook geproduseer het. Die lae produksie van asynsuur in die nie-Saccharomyces fermentasies is bevestig, veral in die geval van L. thermotolerans. Verder produseer hierdie gis hoë vlakke van asetoïen en iso-bottersuur en T. delbrueckii produseer 'n hoër konsentrasie van suksiensuur. Al hierdie metaboliete mag 'n rol speel in die handhawing van intrasellulêre redoksbalans. 'n Meer uitgebreide, sistematiese studie is egter nodig om die mate van hul betrokkenheid te ondersoek. Die gemengde kulture het hul fermentasies voltooi en het hoër finale gliserol vlakke as die kontrole gehad, asook laer asynsuur konsentrasies en kan dus positief bydra tot die wyn aroma. Verder het die gemengde kultuur fermentasies die potensiaal om die etanol vlakke van wyn te verlaag, getoon. Daar is verder in die literatuur gevind dat die giste teenwoordig in die gemengde kultuur mekaar op geenuitdrukkings vlak ook kan beïnvloed. Daar is egter min genetiese inligting beskikbaar vir die nie-Saccharomyces giste. In hierdie studie het ons die gene betrokke by die produksie van gliserol en asynsuur van L. thermotolerans en T. delbrueckii se nukleotied volgordes bepaal. Die gevolglike nukleotied volgordes is redelik homoloog met net 'n paar verskille. Hierdie volgordes kan gebruik word om die geenuitdrukking van GPD1 en ALD6 vanaf fermentasie monsters te bestudeer om sodoende te bepaal tot watter mate die giste in 'n gemengde kultuur mekaar se geenuitdukking kan beïnvloed.

Mestrado em Biotecnologia
Polyhydroxyalkanoates (PHA) emerge as a promising alternative to the conventional plastics due to its biodegradability, biocompatibility and thermochemical properties. Several strategies have been studied in order to decrease PHA production costs and enlarge their commercialization, such as the use of pre-treated renewable substrates and the development of economically and environmentally sustainable extraction procedures. The first part of this work aimed to compare two different reactor operation modes for the production of short-chain organic acids (SCOA), preferred substrates for polyhydroxyalkanoates (PHA) production. Acidogenic fermentation was performed by mixed microbial cultures (MMC) in a continuously stirred tank reactor (CSTR) and in a sequential batch reactor (SBR) operated for 130 days and submitted to the same operational conditions. Both reactors achieved full fermentation (>90%) for an OLR of 10gCOD/L·d, with a different range and relative concentrations of fermentation products (FP). Ethanol (EtOH) was the dominant FP in the CSTR and acetic acid (HAc) the main SCOA produced, while the SBR presented butyric acid (HBu) as dominant FP, slightly higher than the HAc concentration. Although the CSTR showed a higher yield of SCOA than the SBR – 0.671 and 0.604, respectively -, the SBR presented a higher conversion of substrate into SCOA – 48% over 38%. The second part of the work focused on the PHA extraction from pure cultures of C. necator and MMC with surfactants and an enzymatic solution from the hydrolysate of the fungus P. variotii. An extraction protocol was developed for application of the extractive agents studied. Afterwards, a systematic extraction of PHA from the microbial cultures was performed and several concentrations and temperatures tested. Among the surfactants used, BRIJ 30 presented the most promising results with recoveries above 30% for the pure cultures and above 20% for the MMC. The enzymatic solution enabled the PHA extraction when used at a concentration of 10% but not in inferior percentages, indicating that application of higher concentrations should be studied.
Os polihidroxialcanoatos (PHA) apresentam-se como uma alternativa promissora aos plásticos convencionais, devido à sua biodegradabilidade, biocompatibilidade e propriedades termoquímicas. Várias estratégias têm sido estudadas para reduzir os custos de produção de PHA e ampliar a sua comercialização, como o uso substratos renováveis sujeitos a um pré-tratamento e o desenvolvimento de procedimentos de extração económica e ambientalmente sustentáveis. A primeira parte do trabalho consistiu na comparação de dois modos de operação de reator distintos para a produção de ácidos orgânicos de cadeia curta (AOCC), substratos preferidos para a produção de PHA. A fermentação acidogénica foi protagonizada por culturas microbianas mistas (CMM), num reator contínuo perfeitamente agitado (CSTR) e num reator descontínuo sequencial (SBR), operados durante 130 dias sob as mesmas condições operacionais. Ambos os reatores atingiram fermentação total (>90%) para a carga orgânica de 10gCOD/L·d, com gama de produtos de fermentação (PF) e respetivas concentrações relativas distintas. Etanol foi o PF dominante no CSTR e ácido acético (HAc) o principal AOCC produzido, enquanto o SBR apresentou dominância pelo ácido butírico (HBu), com concentração pouco acima do HAc. Apesar do CSTR ter tido maior rendimento de AOCC que o SBR - 0.671 e 0.604, respetivamente - o SBR teve uma conversão de substrato em AOCC superior – 48% comparativamente a 38%. A segunda parte do trabalho incidiu na extração de PHA de culturas puras de C. necator e CMM com aplicação de surfatantes e solução enzimática de hidrolisado do fungo P. variotii. Inicialmente, foi desenvolvido um protocolo para aplicação dos agentes extrativos em estudo. Após, foi feita a extração sistemática de PHA das culturas microbianas seguindo o protocolo desenvolvido e combinando valores de temperatura e concentração. Dos surfatantes utilizados, o BRIJ 30 apresentou resultados mais promissores com recuperações de polímero acima de 30% para as culturas puras e acima de 20% para as CMM. A solução enzimática permitiu extração de PHA em concentração de 10% mas não em concentrações inferiores, indicando que concentrações mais elevadas deverão ser estudadas.

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyl fatty acids, which are accumulated in microbial cells as carbon/energy reserves. PHAs are bio-based and biodegradable and display a wide range of thermoplastic properties, being a promising alternative to conventional plastics. Presently, industrial PHA production was primarily based on pure microbial cultures. Although this process has high PHA production efficiency, it presents high costs associated with the use of chemically-defined feedstocks, and to the need for sterility. An attractive feature of mixed microbial cultures (MMCs) PHAs production is the ability to use waste/surplus feedstocks. Many industrial wastes are seasonally produced making it necessary find the best method of utilization of this feedstock on PHA production process. Two different approaches might be taken account: (1) stock of industrial wastes during their production for their use throughout the year. However, the high fermentability of these agro-industrial wastes makes them susceptible to degradation during storage period; (2) the use of different feedstocks over the year according its availability. It is thus important to study MMC’s response to different feedstocks. The aim of this work is study how MMC PHA production process is affected by a feedstock shift, using cheese whey (CW) and sugar cane molasses (SCM) as model feedstocks. The use of waste based feedstock by MMCs requires a previous conversion of sugars to organic acids (OAs), which is achieved through anaerobic fermentation. In this study, a three-stage MMC PHA process was used, comprising: (1) anaerobic fermentation of surplus feedstocks to produce OAs in a membrane bioreactor (AnMBR); (2) PHA accumulating culture selection in a sequencing batch reactor (SBR) under feast and famine conditions using fermented feedstocks; and (3) PHA production using the selected cultures and the OAs produced in the earlier stages. Initially the effect of both feedstocks (CW and, SCM) in the acidogenic fermentation (stage 1) was assessed. Firstly, the AnMBR was operated under steady state with CW. When the feedstock was changed to SCM an adaption period of about 10 to 15 days was observed. When SCM was replaced by CW a faster adaptation response, approximately 7 days, was observed. The AnMBR reached similar OAs profiles in both phases when CW was fed (% g-COD HAA/g-COD OAs): 65% acetate, 10% propionate, 22% butyrate, 2% valerate, and 1% lactate. These results demonstrate that the system’s performance is reproducible. On the other hand, the anaerobic fermentation of a different feedstock, SCM, resulted in a different OAs profile (%g-COD HAA/g-COD OAs): 24% acetate, 38% propionate, 19% butyrate, and 19% valerate. In a second phase, different fermented feedstocks were used in the selection of PHA-storing organisms under a feast and famine regime in a SBR (stage 2). Initially the SBR was fed with a synthetic OAs solution; then fermented SCM (fSCM) and fermented CW (fCW) were subsequently fed as they were produced in the AnMBR. The adaption of the MMC to fSCM was faster than the adaptation to fCW. Whenever steady state was reached, PHA accumulation tests were performed using the enriched MMC fed with the corresponding feedstock (stage 3), namely synthetic OAs solution, fSCM, and fCW. Storage yields of 0.74, 0.49, and 0.73 C-mol PHA/ C-mol OAs were obtained with synthetic OAs solution, fermented molasses, and fermented CW, respectively. The culture reached a maximum PHA content of 60%, 56% and 65%, when feedstock fed were synthetic OAs solution, fSCM and fCW, respectively. A direct relation between the used feedstock and the polymers composition was observed, which was related with the different OAs profile. Even though, the shift of complex feedstock in three-stage MMC PHA process is still at a very early stage of development, this work illustrates the advantage of favoring the selection of cultures with the capacity to adapt its metabolism to different feedstocks. This will offer the possibility of using numerous substrates and improving strategies to optimize acidogenic fermentation, culture selection and polymer production.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory profile of most red wines as well as some white wines like Chardonnay. This process is conducted by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine environment. MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A further reason for conducting MLF in wine includes the improvement of microbial stability due to the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or modification of certain aroma compounds. In order for wine LAB to conduct MLF, they need to be able to survive the harsh and challenging wine environment. Conditions in South African wines are particularly challenging due to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to struggle in conducting and completing successful MLF. Many of the commercial starter cultures that are currently available contain LAB that have not been isolated from South African wine and are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also the single LAB culture present in all commercially available starter cultures. The overriding goal of this study was to create a MLF starter culture containing a mixture of LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine aroma profile. Lactobacillus plantarum has previously been considered for possible use in a commercial starter culture. The LAB isolates used in this study were selected from the Institute for Wine Biotechnology culture collection as well as isolated from spontaneous MLF. The first objective was to characterise these LAB strains for important traits and for possible use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were screened for the presence of genes encoding for enzymes responsible for biogenic amine production and were found to contain none of the genes associated with the formation of histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD). The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD. The second objective was to perform small-scale fermentations with the individual LAB isolates. The individual isolates were evaluated in Pinotage and based on these results; three strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to investigate the changes in the aroma profile that could be attributed to the presence of the mixed LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification, three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in fermentation periods comparable to that of a commercial culture used as control. The different LAB cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF. The results generated by this novel study clearly indicate the potential of a mixed LAB starter culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive contribution to the wine aroma profile.
AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse, wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay. AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied. Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB), spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te beïnvloed deur die produksie/modifisering van sekere aroma komponente. Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing. Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied, maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die enkele MSB kultuur wat in alle kommersiële kulture gebruik word. Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed. Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie. Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies. Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19 Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14 isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is, naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van β-glukosidase en fenoliese suurdekarboksilase. Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG. Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.

This is a book on the state of the art of scientific research applied to the fields of viticulture, management of the foliage, manual and mechanical harvesting, vinification techniques, the use of mixed yeast cultures, malolactic fermentation and microoxygenation. It synthetically illustrates the lines of research that the Consorzio Tuscania has co-ordinated in the four-year period 2007-2010. Each of these lines of research has been conducted in liaison with some of the most important scientific institutions in the sector: CNR Ibimet, DIPROVE (University of Milan), IASMA-FEM, University of Florence (DEISTAF, DIPSA, DIBA Departments), SAIFET (University-Polytechnic of the Marches) and DISAABA (University of Sassari). The work offer useful cues for updating the production techniques for quality red wines.

The food industrial sector generates large amounts of waste, which are often used for animal feed, for agriculture or landfilled. However, these wastes have a very reach composition in carbon and other compounds, which make them very attractive for valorization through biotechnological processes. Added value compounds, such as volatile fatty acids (VFAs), can be produced by anaerobic fermentation using pure cultures or mixed microbial cultures and food waste as carbon source. Research on valuable applications for VFAs, such as polyhydroxyalkanoates, bioenergy or biological nutrient removal, towards a circular economy is emerging. This enhances the sustainability and the economic value of food waste. This chapter reviews the various types of food waste used for VFAs production using mixed microbial cultures, the anaerobic processes, involved and the main applications for the produced VFAs. The main parameters affecting VFAs production are also discussed.

Filamentous fungi play important roles in the production of a variety of cheeses. The most famous are the blue cheeses, such as Roquefort or Gorgonzola, in which Penicillium roqueforti is the principal mold, and the moldy soft cheeses, such as Camembert or Brie, in which production involves Penicillium camemberti. There are also other filamentous fungi associated with certain types of cheeses, such as Mucor spp., Trichothecium roseum, and Fusarium domesticum in Saint Nectaire, Sporendonema casei in Cantal, Salers and Rodez cheeses, Scopulariopsis species in various French and Austrian cheeses, and Mucor mucedo and Mucor racemosus in the traditional Norwegian cheese Gamalost. These fungi are either inoculated on the cheese as a starter culture or stand out in mixed cultures during spontaneous fermentation. This chapter reviews the filamentous fungi used to produce different kinds of cheeses in terms of taxonomy, physiology, ecology, and mycotoxins, and the microbiological or biochemical effects of these fungi on cheese production.

In this paper the research about applied bio-hydrogen production engineering is introduced. The advantages, disadvantages and characteristics of bio-hydrogen production systems and some technical issues on anaerobic fermentative bio-hydrogen producing systems will be discussed and focused on the schematic processing, designing strategies and engineering control of fermentation parameters and also the technical means to increase the evolved hydrogen and hydrogen evolution rate. The technology of bio-hydrogen production based on ethanol-type fermentative theory has been established. The mixed continuous culture and pure batch culture processes were proposed for hydrogen production.