Academic literature on the topic 'Fungal flocculation'

We studied the flocculation behaviours of microbial communities in 21 soil, wastewater and activated sludge samples to clarify the effects of culture medium types on flocculation ability and screening efficiency, and to analyze diverse functions and microbial compositions. The bioflocculants produced by 33% of the microbial communities had flocculating efficiencies higher than 90%. Six out of the eight microbial communities with efficiencies over 94% were screened from the culture medium using dibutyl phthalate (DBP) as the carbon source. BF-BCT, which was derived from the Chinese cabbage soil sample, had the highest flocculating efficiency (99.6%), species diversity and uniformity. Nine highly efficient strains were separated and purified from seven different microbial communities, indicating that flocculating microorganisms are widely distributed in ecosystems. The 16S rRNA gene testing shows that the eight bacterial and the one fungal strains are common soil microorganisms. The flocculating abilities of BB11 (Sphingobacterium multivorum) and SE3 (Galactomyces geotrichum) have never been reported hitherto. Six strains, including the most flocculating-active TB13 and JB17, were screened from the culture medium using DBP as the sole carbon source. In particular, we compared the performance of culture media and analyzed analogous microbial communities with a Biolog automatic micro-analysis system for the first time.

Several river water fungal strains (RWF-1 to RWF-6) were isolated to investigate the potential of having coagulant properties from the metabolites produced by the fungus. The myco-coagulant produced from the liquid-state process was characterized and tested for flocculation of kaolin water. Molecular identification of the fungal strain isolated from river water and characterization of the myco-coagulant produced by the strain are presented in this paper. The genomic DNA of the fungal 18S ribosomal ribonucleic-acid (rRNA) and 28S rRNA genes were used and the species was identified as Lentinus squarrosulus strain 7-4-2 RWF-5. The characterization of myco-coagulant by Fourier-transform infrared spectroscopy (FTIR) showed that hydroxyl, carbonyl, amide and amine groups as principal functional groups were present in the new myco-coagulant. The mean zeta potential value of the myco-coagulant was −7.0 mV while the kaolin solution was −25.2 mV. Chemical analyses of the extracellular myco-coagulant revealed that it contained total sugar (5.17 g/L), total carbohydrate (237 mg/L), protein (295.4 mg/L), glucosamine (1.152 mg/L); and exhibited cellulase activity (20 units/L) and laccase activity (6.22 units/L). Elemental analyses of C, H, O, N and S showed that the weight fractions of each element in the myco-coagulant was 40.9, 6.0, 49.8, 1.7 and 1.4%, respectively. The myco-coagulant showed 97% flocculation activity at a dose of 1.8 mg/L, indicating good flocculation performance compared to that of polyaluminum chloride (PAC). The present work revealed that the fungal strain, L. squarrosulus 7-4-2 RWF-5 is able to produce cationic bio-coagulant. The flocculation mechanism of the novel myco-coagulant was a combination of polymer bridging and charge neutralization.

ABSTRACTEchinocandin drugs such as caspofungin (CASP), micafungin, and anidulafungin inhibit fungal cell wall biogenesis by blocking Fks1-mediated β-glucan deposition into the cell surface. Candins have become suitable drugs to treat life-threatening diseases caused by several fungal species, includingCandida albicans, that are pathogenic for humans.Here, we present the discovery of a novel CASP-induced flocculation phenotype ofC. albicans, which formed large cell aggregates in the presence of CASP. High concentrations of sugars such as mannose or glucose inhibit CASP-induced flocculation and improve survival ofC. albicanscells exposed to CASP. Notably, exposure ofC. albicanscells to CASP triggers Efg1-dependent expression of the adhesinALS1and induces invasive growth on agar plates. Indeed, cells lacking either Efg1 or Als1 show strongly diminished CASP-induced flocculation, and the absence of Efg1 leads to marked CASP hypersensitivity. On the other hand, CASP-induced invasive growth is enhanced in cells lacking Efg1. Hence, CASP stress drives an Efg1-dependent response, indicating that this multifunctional transcriptional regulator, which is otherwise involved in filamentation, white-to-opaque switching, and virulence, also modulates cell wall remodeling upon CASP challenge. Taken together, our data suggest that CASP-induced cell wall damage activates Efg1 in parallel with the known cell integrity stress signaling pathway to coordinate cell wall remodeling.

Chemical oxygen demand (COD) in domestic wastewater can be treated using flocculation-coagulation process with addition of Oyster mushroom (Pleurotus ostreatus) in powder form as biocoagulant. The fungal cell wall of Oyster mushroom comprises of chitin that is high polyelectrolyte and can be function as an absorbent of heavy metals in wastewater. The effectiveness of flocculation-coagulation process in treating wastewater depends on dose of coagulant and mixing rate. Therefore, this study aims to determine the best combination of three variation of dose of biocoagulant which are 600 mg/l, 1000 mg/l, and 2000 mg/l and mixing rate which are 100 rpm, 125 rpm, and 150 rpm that give the most reduction of COD in the wastewater. The result indicates that the combination of 1000 mg/l of biocoagulant and 100 rpm of mixing rate were found to be the most optimum combination to treat COD in the wastewater with COD reduction of 47.7%.

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

Many industrially important fungi can grow in the form of small spherical pellets.Such pellets reduce the viscosity and enhances mass transfer rates in culture broths.The pelleted morphology also influences the fungus’s metabolism, directing it tospecific metabolites. The pellets are easily harvested from the broth and recycled.These properties makes pelleted morphology very attractive.The pelleted morphology of four Zygomycetes strains was studies. Several differentnutrient media used by other researchers for achieving pelleted growth was tested.The influence of eight factors on pelletization of Rhizopus sp. in a completely definedmedium was determined using a fractional central composite design and logistic regression.Pelleted growth of all four Zygomycetes was achieved, with very good results for twoRhizomucor sp. strains. A simple medium containing calcium carbonate was foundto induce pelletization with very high reproducibility.Immobilization of yeast cells was attempted in pellets of Rhizomucor. It was foundthat a flocculating yeast can be immobilized inside pellets of fungal mycelium, forming”biocapsules”. This is accomplished by first using a medium that induces pelletizationof the filamentous fungus and does not allow for growth of the yeast. Thepellets are then inoculated into a second medium that induces growth and flocculationof the yeast and inhibits further growth of the filamentous fungus.Non-flocculating yeasts could not be immobilized, suggesting that the flocculin proteinsin the cell wall of flocculating strains are important for proper immobilization.The flocculation and immobilization arises due to expression of several differentFLO-genes and the importance of these genes for successful immobilization isdiscussed.The results demonstrate that the morphology of Zygomycetes can be controlled andthat this may be useful in industrial fermentation. The new immobilization techniquereveals the great importance of flocculation and cell surface hydrophobicity. Usingyeast strains that express certain FLO-genes may be beneficial in fermentation oflignocellulosic hydrolysates.Microscopy techniques were developed that allows for high quality microphotographyof pellets and thin cross-sections of pellets and biocapsules.
Program: MSc in Resource Recovery - Industrial Biotechnology

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: Flocculation can be defined as the asexual aggregation of yeast cells in a liquid environment. This aggregation of cells, also referred to as "floc formation", will in most cases lead to rapid settling or sedimentation. However, in so-called top-fermenting yeast strains, the floes can move to the surface of the liquid growth substrate to form a thin layer, called a "velum", that has been compared to other microbial biofilms. The factors that trigger flocculation can be divided into two groups, physical/chemical (e.g. sugar content, the presence of inorganic salts, organic solvents, ethanol concentration, pH, agitation etc.) and genetic factors (genes that encode for proteins that are either directly or indirectly involved in flocculation). In top-fermenting yeast strains, several physical and chemical factors that trigger the process have been described, including ethanol concentration, the presence of organic solvents, the absence of molecular oxygen and the presence of inorganic salts (Ca2+ and Mg2+). These factors appear to affect the cell hydrophobicity and the cell surface charge. As for genetic factors, no specific genes have thus far been associated with flocculation in top fermenting yeast strains. In bottom-fermenting yeast strains, the physical and chemical factors that affect the process are similar to the ones described for top-fermenting yeast strains, but include, more specifically, the concentration of hexoses in the media (mannose or glucose), which may inhibit the process. Indeed, flocculation in bottom-fermenting yeast strains has been divided into the NewFlo type (inhibited by both mannose and glucose) and the Fl01 type (inhibited by mannose) on the basis of the inhibitory effect of specific sugars. Various genes have been associated with the flocculation of bottom-fermenting yeast strains. Through genetic analysis, the genes have been categorised into dominant genes, semidominant genes and recessive genes. In order to better understand the role of some of the proteins responsible for flocculation in S. cerevisiae, and to create strains whose flocculation properties would correspond to those wanted in the wine and beer industries, three of the dominant flocculation genes, FL01, FL05 and FL011, were placed under the control of the promoters of the stationary phase-induced genes, ADH2 and HSP30. This was achieved by replacing the native promoters of the flocculation genes with the heterologous promoters through homologous recombination. The laboratory strain FY23, which is nonflocculent due to the absence of the transcription factor that is required for flocculation, F108p,was used as a model system. Some of the transformed strains showed high flocculation, especially when the genes were placed under control of the ADH2 promoter. In addition to this, the strains carrying a modified FL011 gene showed increased adhesion to solid agar media and were able to invade the growth substrate. These strains also showed an increased velum-forming ability when grown in media containing only non-fermentable carbon sources.
AFRIKAANSE OPSOMMING: Flokkulasie kan gedefinieër word as die ongeslagtelike aggregasie van gisselle in 'n vloeibare medium. Hierdie aggregasie van selle, kan ook na verwys word as flok formasie, en in meeste gevalle lei dit tot In vinnige sedimentering. In oppervlak-fermenterende giste, beweeg die flokke na die oppervlakte van die vloeibare medium om sodoende 'n flor -lagie te vorm. Hierdie verskynsel was ook al gevind in ander organismes. Verskeie faktore is verantwoordelik vir die effektiwiteit van flokkuklasie. Hierdie faktore kan in twee groepe verdeel word, nl. fisiese en chemiese faktore (byv. suikerkonsentrasie, die teenwoordigheid van anorganiese soute, organiese oplossings, etanol konsentrasie, pH, ens.) en genetiese faktore (gene wat kodeer vir die proteïene wat of direk of indirek betrokke is by flokkulasie). In oppervlak-fermenterende giste is daar al heelwat informasie beskikbaar omtrent fisies en chemiese faktore se effekte op flokkulasie. Van die faktore waarvan heelwat informasie beskikbaar is sluit in, etanol konsentrasie, die teenwoordigheid van organiese oplossings, die afwesigheid van molekulêre suurstof en die teenwoordigheid van anorganiese soute (Ca2+ en Mg2+). Hierdie faktore toon 'n effek of hidrofobisiteit en elektriese lading op die seloppervlakte. Geen genetiese faktore kon tot dusver gekoppel word aan flokkulasie in oppervlak-fermenterende giste nie. Benede-oppervlak fermenterende giste se fisies en chemiese faktore wat effektiwiteit van flokkulasie beïnvloed is dieselfde as die van oppervlak-fermenterende giste, maar sluit in meer spesifiek, die konsentrasie van heksoses in die media (nl. mannose en glukose), wat 'n inhiberende effek het op flokkulasie. Die benede-oppervlak fermenterende giste se flokkulasie kan in twee segmente verdeel word nl. die NewFlo tipe (word geïnhibeer deur die teenwoordigheid van mannose en glukose) en die Flo1-tipe (word geïnhibeer deur slegs die teenwoordigheid van mannose). Verskeie gene was ook al geidentifiseer wat die effektiwiteit van flokkulasie beïnvloed in benede-oppervlak fermenterende giste. Hierdie gene kan in drie kategorieë opverdeel word, nl dominante-, semi-dominante- en ressessiewe flokkulerende gene. Ten orde 'n beter begrip te kry rondom die proteïene verantwoordelik vir die meeste effektiwiteit ten opsigte van flokkulasie in S. cerevisiae, asook om giste te manipuleer om spesifieke flokkulasie eienskappe te toon volgens die belange van die wyn en bierindustrieë, was drie dominante flokkulerende gene, nl. FL01, FL05, en FL011, onder regulering van stationêre fase-geïnduseerde promotors, PADH2 en PHSP30, geplaas. Dit was verkry deur die vervanging van die wilde tipe promotors van die drie gene met die stationêre fase-geïnduseerde promotors deur middel van homoloë rekombinasie. Die laboratorium gisras, FY23, wat 'n nie-flokkulerende gisras is vanweë die afwesigheid van 'n transkripsionele faktor, Flo8p, wat verantwoordelik is vir die aktivering van belangrike gene in flokkulasie, was gebruik as 'n wilde tipe ras. Sommige van die transformante het In hoë mate van flokkulasie getoon, veral wanneer onder die regulering van die PADH2. Tesame met laasgenoemde verskynsel, was daar gevind dat FL011-transformante 'n verhoging in hul vermoeë het om te bind aan die agar en ook om die agar te penetreer. Laasgenoemde gisrasse het ook die vermoë getoon om 'n flor-lagie te vorm bo-op die oppervlakte van die medium, maar slegs wanneer dit in niefermenteerbare koolstofbronbevattende media opgegroei word.

Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009.
In many industrial fermentation processes, Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation a high suspended yeast count is of paramount importance to maintain a rapid fermentation rate, whilst efficient flocculation should ideally be initiated only on completion of the primary alcoholic fermentation, so as to enhance product clarification and recovery. Most commercial wine yeast strains are non-flocculent, probably because this trait was counter-selected to avoid fermentation problems. In this study, we assessed molecular strategies to optimise the flocculation behaviour of non-flocculent laboratory and wine yeast strains. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5 and FLO11, of a non-flocculent S. cerevisiae laboratory strain (FY23) and two commercial wine yeast strains (BM45 and VIN13) were placed under the transcriptional control of the stationary phase-inducible promoters of the S. cerevisiae ADH2 or HSP30 genes. Under standard laboratory media and culture conditions, all six promoter-gene combinations resulted in specific flocculation behaviours in terms of timing and intensity. The data show that the strategy resulted in the expected and stable expression patterns of these genes in both laboratory and industrial wine yeast strains. Most importantly, the data confirm that inducible expression of the native FLO1 and FLO5 open reading frames, albeit to varying degrees, are responsible for a quantifiable cell-cell adhesion phenotype that can be characterized as a Flo1 flocculation phenotype. On the other hand, we found that inducible expression of the native FLO11 ORF under these conditions resulted in flor/biofilm formation and invasive growth phenotypes. However, the specific impact of the expression of individual dominant FLO genes with regard to characteristics such as flocculation efficiency, cell wall hydrophobicity, biofilm formation and substrate adhesion properties showed significant differences between the commercial strains as well as between commercial and laboratory strains. These adhesion phenotype differences may at least in part be attributed to wine yeast FLO gene open reading frames containing significantly smaller intragenic repeat regions than laboratory strains. The data show that the ADH2 regulatory sequences employed in this study were unsuitable for the purpose of driving FLO gene expression under wine-making conditions. However, HSP30p-based FLO1 and FLO5 wine yeast transformants displayed similar flocculent phenotypes under both synthetic and authentic red wine-making conditions, and the intensities of these phenotypes were closely aligned to those observed under nutrient-rich YEPD conditions. The fermentation activities of HSP30p-based transgenic yeast strains were indistinguishable from that of their parental host wine yeast strains. The chemical composition of wines obtained using transgenic yeast strains were similar to those produced by parental strains. The BM45-derived HSP30p-FLO5 transformant in particular was capable of generating compacted or ‘caked’ lees fractions, thereby providing a distinct separation of the fermented wine product and lees fractions. Furthermore, in this study we report a novel FLO11 induced flocculation phenotype that seems to exclusively develop under authentic red wine-making conditions. This strong FLO11 flocculation phenotype was not wine yeast strain dependant, possessed both Ca2+-dependant and Ca2+-independent flocculation characteristics and was insensitive to inhibition by both glucose and mannose. A distinct advantage of this unique FLO11 phenotype was highlighted in its ability to dramatically promote faster lees settling rates. Moreover, wines produced by HSP30p-FLO11 wine yeast transformants were significantly less turbid than those produced by their wild type parental strains. The benefit of this attractive property is it facilitates simpler and faster recovery of wines and also promotes greater volume recovery of the wine product.

Philosophiae Doctor - PhD
Biofuel production from microalgae is currently not economically competitive with fossil fuels due to high operational costs. A sustainable system needs to be developed which considers cultivation, harvesting and conversion to fuels as a single loop. The harvesting step has been identified as a major bottleneck within the biofuel production process, contributing to a significant proportion of the operational cost (20-30%). Chemical flocculation is a more affordable alternative to centrifugation and filtration. Chemical flocculants however negatively impact the quality of biomass and conversion efficiency to biofuel by increasing biomass ash content. Bioflocculation with biopolymers or microbes have a minimal impact on the quality of biomass. In this study, the interaction between the filamentous fungus Isaria fumosorosea and the microalgae C. sorokiniana is investigated. Under strict autotrophic conditions at pH 7-8, co-culture of microalgae (2-20 μm) with fungal blastospores resulted in theidevelopment of large pellets (1-2 mm) which may be easily harvested by sedimentation or filtration at 95% harvesting efficiency. Fungal assisted bioflocculation was compared to other harvesting methods with respect to cost and impact on the hydrothermal conversion process. Low cost carbon sources, including waste hydrothermal nutrients, minimal sugar concentrations and algal exudate may reduce fungal cultivation costs. Waste products, such as organic carbon, N, P, CO₂ and trace metals can be recycled and used for algae and fungal cultivation, closing the loop to make the system sustainable.
National Research Foundation; Swiss Government

A three part study was carried out to examine rheological, colloidal and floe microstructural aspects of industrial brewing yeast strains. Following a review of the literature, the rheological properties of four yeast strains (two flocculent ale and lager types and their non-flocculent variants) were examined. In related colloidal studies, orthokinetic flocculation rates of these strains as well as their cell surface charge were determined. Floc microstructure was characterized using both light and scanning electron microscopy. In a summary chapter, the cell floc model (a modification of Hunter's elastic floc model) was used to the explain the rheological and colloidal behaviour of brewing yeast suspensions. Flow behaviour studies of the commercial yeast suspensions suspended in a calcium-containing sodium acetate buffer revealed that yeast flocculent characteristics had an important influence on their suspension flow behaviour. As cell concentrations increased, suspension flow properties become increasingly non-Newtonian and could be described by the Casson model at low rates of shear and the Bingham model at shear rates above 100 s⁻¹. The cell floc model was proposed to explain the Bingham flow behaviour of these csuspensions. The Bingham yield stress in these suspensions was believed to be a function of the orthokinetic capture coefficient, cell volume and the energy to break up doublet cells. Increasing temperature tended to lower the Bingham yield stress in lager strains and increase the yield stress in ale strains. A semi-empirical explanation for the viscosity increase of deflocculated cell suspensions and the estimation of pseudo-capture coefficients was presented. Furthermore, studies of the flow behaviour of yeast strains suspended in decarbonated ale and lager beer revealed that: 1) suspensions of flocculent strains show higher yield stress values than their non-flocculent variants, 2) ale strain suspensions tended to have higher yield values than the lager strains and 3) yeast dispersed in beer had higher yield stress values than when suspended in buffered calcium suspensions. This last observation was believed to reflect the influence of ethanol on the cell binding process which has important implications for future measurements of yeast flocculation. Colloidal studies revealed for the first time, that the orthokinetic rate of flocculation of brewing yeast cells could be modelled by a first order equation, as predicted by fundamental colloid theory. While subject to considerable variation, measured rate constants led to the calculation of orthokinetic capture coefficients. Yeast cell zeta potential values generally agreed with literature data but could not be employed in the DLVO model of colloid flocculation to explain measured orthokinetic capture coefficient values. Examination of the cell zeta potential data indicated that the data had non-normal distributions. SEM examination of the four industrial yeast strains suggested that a number of distinct structures mediated cell-to-cell interaction and that intra-strain differences occurred. These findings, along with the observation of non-normal surface charge distributions, indicated that these industrially pure strains had undergone substantial variation. Treatment of the flocculent cells with pronase tended to reduce cell-to-cell contacts. In the summary chapter the cell floe model was employed to describe the rheological behaviour of the yeast suspensions. Estimation of the force needed to separate doublet yeast cells were made using critical shear rate data (i.e., the point at which Bingham flow begins). This estimate was similar to that reported for single antibody bonds and may be due to the presence of lectin-like structures on the yeast cell wall.
Land and Food Systems, Faculty of
Graduate

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: In all cellular systems the ability to alter eellular programs in response to extracellular cues is essential for survival. This involves the integration of signals triggered by membrane bound receptors in order to adjust the expression of target genes and enzyme activities and consequently phenotypic outcome. The yeast Saccharomyces cerevisiae has evolved several adaptations, such as, sporulation and pseudohyphal differentiation, in order to survive changes in the surrounding environment. Pseudohyphal differentiation and the related phenotype, invasive growth, are proposed to be adaptations that enable the yeast to forage for scarce nutrients or escape from a detrimental environment. This dimorphic transition is associated with a change from the normal "yeast" form to a pseudohyphal form, which involves changes in budding pattern, cell-cycle progression, cellular elongation, and cell-eell and cell-substrate adherence. The outcome of these changes is elongated eells, which bud in a unipolar fashion and do not separate after budding to form chains of cells referred to as pseudohyphae. These pseudohyphae are able to penetrate the surface of agar containing growth medium, a process referred to as invasive growth. Nutrient-induced adaptations, such as pseudohyphal growth, have been extensively studied in S. cere visiae , and several factors have been implicated in the regulation thereof, many of which are part of specific signalling pathways. The most clearly defined are the filamentous growth specific MAP kinase cascade and the Gpa2p-cAMP-PKA pathway. MUC1/FL011, encoding a member of a family of cell wall associated proteins involved in cellcell/ cell-substrate adhesion, is regulated by these pathways and considered to be critical in the establishment of pseudohyphal differentiation and invasive growth. The promoter region of MUC1/FL011 represents one of the largest yeast promoters identified to date, with cis-acting elements present up to 2.4 kb upstream from the first coding triplet. The upstream regulatory region of MUC1/FL011 is almost identical to that of the STA2 gene, which encodes an extracellular glucoamylase required for the utilisation of extracellular starch. As suggested by the extent of homology between these two promoters, MUC1/FL011 and STA2 are co-regulated to a large degree and both require the same transcription factors. Mss11p plays a central role in the regulation of MUC1/FL011 and STA2 and consequently starch metabolism and pseudohyphaI differentiation. The regulation conferred by MSS11 on the transcriptional levels of MUC1/FL011 and STA2 also appears to be dependent on signals generated specifically in the presence of low nitrogen and glucose. Mss11p does not have significant homology to any other yeast protein, with the exception of limited homology to the transcriptional activator F108p. However, several distinctive domains have been identified in the MSS11 gene product. Firstly, Mss11p contains polyglutamine and poly-asparagine domains. It also contains a putative ATP- or GTP-binding domain (P-Ioop), commonly found in proteins such as kinases, ATPases or GTPases. Two short stretches close to the N-terminal, labelled H1 and H2, share significant homology to the transcriptional activator, F108p. Both the H2 domain and the extreme C-terminal of Mss11p are able to stimulate RNA polymerase II dependent transcription. Furthermore, the H1 domain together with the P-Ioop negatively regulates the activation potential of the H2 domain. This study presents further insight into the functioning of Mss11p and the involvement of the separate activation and regulatory domains in mediating transcriptional activation and pseudohyphal differentiation in response to nutrient limitation. Genetic interactions between Mss11p and other factors involved in the regulation of pseudohyphal growth and starch degradation were revealed, and specific regions of Mss11p were shown to be required by these factors in order to achieve their required function. In addition, results obtained in this study implicates Mss11p in the regulation of Ca2+-dependent flocculation and suggest that the FL01 gene is also regulated by Mss11p in this capacity.
AFRIKAANSE OPSOMMING: Die vermoë om sellulêre programme in reaksie op ekstrasellulêre seine te verander, is 'n essensiële vereiste vir alle sellulêre sisteme. Dit behels die integrasie van seine gegenereer deur membraan-gebonde reseptore om ekspressie van teikengene en ensiemaktiwiteite sodanig aan te pas dat gewenste fenotipise uitkomste bewerkstellig kan word. Die gis Saccharomyces cerevisiae het verskeie aanpassingsmeganismes ontwikkel, soos byvoorbeeld sporulasie en pseudohifeforming, om veranderinge in die omgewing te kan oorleef. Pseudohifevorming en die verwante fenotipe, penetrasiegroei, word beskou as aanpassings te wees wat die gis in staat stel om van 'n skadelike omgewing weg te kom, of dit in staat te stelom by skaars voedingstowwe uit te kom. Hierdie dimorfiese transisie word geassosieer met 'n verandering van die normale "gisvorm" tot pseudohifevorming wat veranderinge in die botpatroon, selsiklusprogressie, selverlenging, sel-sel en sel-substraat aanhegting behels. Die uitkoms van hierdie verandering is verlengde selle, wat unipolêr bot en nie van mekaar skei nie om sodoende kettings van selle te vorm en waarna verwys word as pseudohifes. Hierdie pseudohifes is ook in staat om die oppervlak van 'n agar bevattende groeimedium te penetreer, 'n proses waarna verwys word as penetrasiegroei. Aanpassings soos pseudohitevorminq is die afgelope dekade intensief nagevors, en verskeie faktore en seintransduksienetwerke is in die regulering daarvan geïmpliseer. Onder hierdie seintransduksienetwerke is die bes gedefiniëerde paaie die filamentasie-spesifieke MAP-kinasekaskade en die Gpa2p-cAMP-PKA pad. MUC1/FL011 kodeer vir 'n lid van 'n geenfamilie wat met sel-sel/sel-substraat aanhegting geasosieer word en dit word deur hierdie seintransduksie netwerke gereguleer. MUC1/FL011 word as essensieel vir pseudohife vorming beskou. MUC1/FL011 word gereguleer deur die grootste gispromoter wat tot op hede geïdentifiseer is, met cis-werkende elemente so ver as 2.4 kb stroom-op van ATG. Die MUC1/FL011 promoter is feitlik identies tot die van die STA2-geen, wat kodeer vir 'n ekstrasellulêre glukoamilase wat die gis in staat stelom ekstrasellulêre stysel te benut. Weens die homologie tussen die twee promoters, word MUC1/FL011 en STA2 tot In groot mate ge-koreguleer en beide benodig dieselfde transkripsiefaktore. Mss11p speel In sentrale rol in die regulering van MUC1/FL011 en STA2 en dus ook in die regulering van pseudohifevorming en styselmetabolisme. Die regulering wat deur Mss11p of MUC1/FL011 en STA2 uitgeofen word, blyk verder onderhewig te wees aan seine wat gegenereer word spesifiek in die teenwoordigheid van lae konsentrasies glukose en stikstof. Mss11p het nie betekenisvolle homologie met enige ander gisproteïen nie, behalwe vir beperkte homologie met die tranksripsionele aktiveerder F108p. Verskeie onderskeidbare domeine is egter in die MSS11 geenproduk teenwoordig. Eerstens, Mss11p bevat kenmerkende poliglutamien en poli-asparagien domeine. Verder bevat Mss11p ook In voorspelde ATP- of GTP-bindings domein (P-Ius), wat algemeen in proteïene soos kinases, ATPasaes en GTPases voorkom. Twee kort areas naby die N-terminaal, aangedui as H1 en H2, het betekenisvolle homologie met die transkripsiefaktor F108p. Beide die H2 domein en die ektreme C-terminaal van Mss11p is in staat om RNA polimerase " afhanklike transkripsie te stimuleer. Verder het die H1-domein in samewerking met die P-Ius In negatiewe uitwerking op die aktiveringspotensiaal van die H2-domein. Hierdie studie bied verdere insig tot die werking van Mss11p en die betrokkenheid van die verskeie aktiverings- en reguleringsdomeine by die oemiddetlinq van transkripsionele aktivering en pseudohifevorming in reaksie op beperking van voedingstowwe. Genetiese interaksies tussen Mss11p en ander faktore betrokke met die regulering van pseudohifevorming en styselafbraak is in hierdie studie aangetoon. Voorts is daar ook gewys dat spesifieke areas van Mss11p benodig word deur hierdie faktore om hulle biologiese funksie uit te oefen. Daar is ook In rol vir Mss11p in die regulering van Ca2+-afhanklike flokkulasie aangetoon en daar is bewys dat die FL01 geen deur Mss11p benodig word om hierdie effek uit te oefen.

Orientador: Silvio Roberto Andrietta
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
Made available in DSpace on 2018-08-10T10:19:18Z (GMT). No. of bitstreams: 1 Galassi_GabrielRamos_M.pdf: 1009774 bytes, checksum: 86a841b010532edff0ac4939c19472f4 (MD5) Previous issue date: 2007
Resumo: Este trabalho tem por objetivo a realização de um estudo aprofundado do processo de fermentação alcoólica contínua em que são utilizados reatores tubulares tipo torre e células auto-imobilizadas. Buscou-se nesse projeto a seleção entre quatro cepas com características floculantes e perfis metabólicos adequados ao trabalho em tais reatores. O anseio por melhorias no sistema de fermentação contínua com células auto-imobilizadas se dá em função da busca por métodos com reduzido custo de produção do etanol, o que ocorre nesse sistema, onde inexiste a necessidade da utilização de unidades de separação de células (centrifugas), reduzindo assim em torno de 15% o custo final do preço do produto acabado. As linhagens utilizadas foram quatro cepas do gênero Saccharomyces cerevisiae com características floculantes e formadoras de partículas esféricas (pellets) quando cultivadas em mesa agitadora circulatória. Duas dessas cepas selecionadas foram obtidas da unidade de fermentação industrial da Usina Junqueira (Igarapava/SP), isoladas durante a safra de janeiro de 2000, são elas as cepas JU C2 e JU C4, outras duas cepas foram obtidas através de recentes isolamentos no Departamento de Biotecnologia e Processos do CPQBA/Unicamp, cepas G1 e G2. No processo de otimização foram utilizados dois reatores tubulares em série, um primeiro reator (R1) recebia um substrato concentrado de sacarose, diluído em linha visando atingir concentrações pré-determinadas para o uso nos experimentos e um segundo reator (R2) que recebia o efluente (vinho fermentado) proveniente de R1. Foram realizados quatro experimentos, cada qual com uma linhagem diferente. Os experimentos tiveram entre 20 a 25 dias de duração cada, porém, para fins de padronização foram selecionados 12 dias de ensaios. Durante os experimentos, diferentes vazões de alimentação aos reatores foram testadas, estas alternaram de forma crescente entre 1,2; 1,5; 1,8 e 2,1 L/h de substrato. Amostras foram retiradas do sistema e analisadas quanto à quantidade de sólidos solúveis, quantidade de ART, etanol produzido e vazão real do sistema. Ao término de cada experimento foram realizadas análises de cariotipagem, potencial de floculação por absorbância e determinação da floculação pela expressão dos genes FLO5 e FLO10. De forma geral, todas as linhagens estudadas mostraram-se hábeis ao trabalho em biorreatores fluidizados, desde que sejam respeitados seus limites metabólicos atuando nesse tipo de reatores. As linhagens JU C4 e G1 sobressaíram-se entre as demais pelo comportamento flexível e adaptável diante das situações impostas ao longo do projeto. Foram capazes de formar leitos celulares estáveis e com bom desempenho na conversão de açúcares á etanol. As linhagens JU C2 e G2 apresentaram também bons resultados na produção de etanol, no entanto, seus perfis como linhagens floculantes exigem uma maior cautela na utilização das mesmas em processos fermentativos. JU C2 com baixo poder floculante, formando por vezes leitos pouco estáveis e G2 com grande poder floculante, formando por vezes leitos compactos com canais preferenciais de passagem de substrato
Abstract: using flocculation yeasts of Saccharomyces cerevisiae in fixed bed tower reactors for ethanol production. We sought, through this job, the selection between four flocculent yeasts strains with appropriate metabolic profile to work in this kind of reactor. The selected yeasts strains present strong flocculent characteristics and there is no need to use cell separation unity (centrifuge) after the fermentation process reducing around 15% the costs of ethanol production. Two of these strains, JU C2 and JU C4, were collected from an industrial fermentation unity at Usina Junqueira (Igarapava/SP) during the crop of 2000. The other two strains, G1 and G2, were collected from CPQBA/Unicamp. In the operational optimization were used two fixed bed type tower reactors connected in series, a first one (R1) receive a concentrated sucrose medium diluted in line for reach predetermined concentrations and a second one (R2), that receive fermented wine from the first (R1). The fermentations experiments had between 20 or 25 days, but for standardization of the work 12 days of assays were selected. During the experiments, different feed outflow were tested in the reactors, these had been alternated in a increasing way between 1,2; 1,5; 1,8 and 2,1 L/h of medium. Samples were collected from the system and analyzed the amount of soluble solids, amount of reducing sugars, ethanol and real outflow of the system. In the ending of each experiment were analyzed the karyotyping profile and flocculating potential by absorbance method and expression of the FLO1 and FLO10 genes. The results confirm that all the studied strains are able to work in fluidized bioreactors since their metabolic limits where respected. The best results were obtained with JU C4 and G1 strains. They show a flexible and adaptable behavior ahead the tested situations in this job. They were capable to form steady beds and demonstrate good performance in sugar conversion to ethanol. The other strains, JU C2 and G2, showed nice results in the production of ethanol, however their flocculent profiles demand caution when used in fermentation processes. JU C2 has a low flocculent power forming sometimes unstable cell beds and G2 has a high flocculent power forming sometimes compact cell beds with preferential channels of medium passage
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química

"Le phénomène de floculation des souches kluyveromyces lactis haploides est sous l'influence des facteurs nutritionnels et physicochimiques liés à l'environnement. Ce phénomène observe au milieu de la phase de croissance se produit entre le PH 4,0 et 4,5. La présence de glucose est indispensable pour l'apparition de la floculation, alors que le galactose et ses dérivés sont des inhibiteurs potentiels. Les ions CA**(2+) à 4 MM induisent une floculation optimale. Les ions MG**(2+) peuvent partiellement substituer les ions CA**(2+), mais les ions NA**(+) et K**(+) sont des antagonistes. Le mécanisme d'action des ions CA**(2+) sur la floculation est en relation avec leur capacité à se lier sur les phosphopeptidomannannes. Cette fixation se réalise très probablement sur les groupements carboxyles comme le montre l'incapacité des molécules de phosphopeptidomannannes modifiés chimiquement à fixer les ions CA**(2+). Une composante lectinique intervient aussi dans ce mécanisme de floculation. Cette composante ayant un poids moléculaire apparent de 70. 000 est localisée sur la surface cellulaire au niveau d'un récepteur ayant une configuration stéréochimique reconnaissant l'alpha -D-galactopyranose. Dans le mécanisme de floculation, cette "composante lectine" jouerait le rôle de ligand, par contre la partie polyosidique des phosphopeptidomannannes jouerait le rôle du récepteur et les ions CA**(2+) joueraient un rôle de cofacteur ou d'activateur. Les ions CA**(2+) sont reconnus aussi comme stabilisateurs des liaisons mannannes-lectine en se fixant sur les groupements carboxyles des parties peptidiques des phosphopeptidomannannes. "

High rate algal ponds (HRAPs) can be utilised as an efficient and economical wastewater treatment method while also producing algal biomass. This study focused on the use of HRAPs to assimilate nutrients from secondary lagoon effluent and investigated various methods in which to enhance the algal biomass productivity of the HRAPs. The natural operation, productive potential, biomass production, nutrient removal capacity and environmental conditions were observed. From these findings, three experiments were proposed to enhance biomass production and in turn, the nutrient removal of the HRAPs. The first experiment was the addition of three separate algal cultures to the HRAPs during winter. Two of the algal species enhanced biomass production, however, there was no significant difference in nutrient removal during any of these experiments. The second set of experiments controlled the pH of the HRAPs utilising an inorganic and organic acid to determine if it was solely the control of pH which enhanced biomass production or if the addition of carbon that played a significant role. It was found that under high algal productivity conditions utilising inorganic acid to control pH negatively impacted algal growth whereas utilising organic acid significantly enhanced algal growth. The third experiment compared secondary lagoon effluent and primary lagoon effluent as the media sources. Secondary lagoon effluent was found to have higher biomass productivity by 106mg/L. This was thought to be a result of the primary lagoon effluents high colloidal turbidity. The results from the biomass enhancement experiments alongside the natural operation of the HRAPs were utilised to develop a simple and accurate algal growth model which utilised readily available data. The model aims to determine the biomass production of HRAPs in the south-eastern Australian climate which operated under elevated pH levels. The model was validated against the use of both secondary and primary lagoon effluent in the HRAPs and returned an R-squared value 0.98, suggesting a high accuracy. Following this work, two algal harvesting methods were investigated; membrane filtration and fungal flocculation. Three different membrane filtration systems were trialled and compared; ceramic crossflow system, polytetrafluoroethylene (PTFE) submerged system and a metal crossflow system. The PTFE membrane was found to be the most effective of the membranes tested for harvesting algae due to its low fouling tendency, low cost and relatively constant flux. The flocculation capability of fungi to flocculate algae was examined. Aspergillus oryzae was found to be the most effective fungi species trialled for monoculture flocculation with over 95% removal for all algal species tested. The fungal flocculation of mixed algal communities in wastewater samples was also investigated and removal values of 70-100% were achieved. Overall, the work conducted provides valuable information on the operation and enhancement of HRAPs. Furthermore, the simple model developed can be utilised to help identify the potential of an area for algal biomass production and the feasibility of incorporating HRAP systems into an existing wastewater treatment facility. The two harvesting techniques trialled offer new and vital insight into the often-difficult process of algal harvesting.

If a product is synthesized intracellularly and not secreted by the producing cell, or if the product is to be extracted from plant, animal, or fungal tissue, it is necessary to remove the product from the cell or tissue by force. The choice of procedure is highly dependent on the nature of the product and the nature of the cell or tissue. It was seen in Chapter 1 that bioproducts represent a wide variety of chemical species. In this chapter, we also see that the sources of bioproducts—cells and tissues—are widely varied. For this reason, there exists a wide variety of methods for breaking, or lysing, cells and tissues, broadly classified as “chemical” and “physical” methods. Once cells have been suspended and/or broken open, the resulting suspension of solids must be separated from the liquid in which it is suspended. This separation process, filtration and/or sedimentation (the subjects of the next two chapters), is enhanced by having larger particles. Larger particles can be achieved by flocculation, a process whereby particles are aggregated into clusters, or flocs. In recent years, it has become desirable to isolate specific cell types from mixtures of suspended cells and to deliver the resulting cell subpopulation(s) to a process for which they, and only they, are required. Most examples come from in vivo sources such as blood and dispersed tissue cells. This aspect of cell processing, namely, cell purification, places special demands on separation processes that are capable of handling particulate matter under conditions that allow cells to remain alive. This chapter presents two major elements of cell processing: the science and engineering of cell rupture by physical and chemical methods and the flocculation of cells and subcellular particles in aqueous suspension. First, however, it is helpful to develop a broad appreciation for the variety and compositions of cells that are likely to be encountered in downstream bioprocessing.

Water from surface sources is often contaminated by microbes, whereas groundwater is normally safer, but even groundwater can be contaminated by harmful chemicals from human activities or from the natural environment. The purification process of water may reduce the concentration of particulate matter including suspended particles, parasites, bacteria, algae, viruses, fungi, and a range of dissolved and particulate material derived from the surfaces. Water purification is the process of removing undesirable chemicals, materials, and biological contaminants from contaminated water. Most water is purified for human consumption (drinking water), but water purification may also be designed for a variety of other purposes, such as medical, pharmacology, chemical, and industrial applications. In general, the methods used include physical processes such as filtration and sedimentation, biological processes such as slow sand filters or activated sludge, chemical processes such as flocculation and chlorination, and the use of electromagnetic radiation such as ultraviolet light.