Avibacterium paragallinarum is an avian pathogen that causes the upper respiratory disease Infectious coryza (IC) in chickens. This disease has the ability to cause vast economic losses due to a decrease in egg production. To date the factors contributing to pathogenicity, immunogenicity and serotyping are still not clearly understood. Vaccine failures are a major problem that occurs due to no or poor cross-protection occurring especially between the C-serovars of A. paragallinarum. This problem will be overcome by having a more accurate serotyping technique available for the diagnosis of IC. Therefore one of the aims of this study was the development of a molecular serotyping technique, where a serotyping PCR was developed which distinguished between the Modesto (C-2) and SA-3 (C-3) A. paragallinarum isolates which is the major cause of IC in South Africa. Reported in a recently published article was the presence of a HP2-like and Mu-like phage within the genome of the Modesto (C-2) strain of A. paragallinarum. Therefore another major question addressed during this study was whether there are prophage genes present in all of the reference isolates as well as in field isolates of A. paragallinarum and what the effect of these phages might be on the virulence and pathogenicity of these isolates. Phage genes that are important during lysogeny were selected and screened for by means of PCR. From the results it was able to determine that some of these genes are present in some of these isolates but no discernible patterns were detected in terms of the effect on pathogenicity. Therefore future studies will be conducted to mainly focus on the effect these phages might have on the virulence and pathogenicity as well as whether these phages are responsible for the occurrence of different A. paragallinarum serovars.

Fermentation has been used for over three thousand years as an effective and inexpensive means to preserve the quality and safety of foods. In Africa most of the traditional foods are fermented at household level before consumption. The cereals are fermented, and then cooked prior to consumption and this offers added safety advantages because pathogenic microorganisms are inactivated thereby increasing shelf life of product. However, some cereals are cooked first and then other sources of fermentable carbohydrates and amylase such as other cereals (malts) or roots from Rhynchosia spp. (generally called munkoyo roots) are added and then fermented prior to consumption. Chibwantu and munkoyo beverages, Zambiaâs important cereal based fermented foods are prepared in such a way. The substrates added after cooking of cereals are a major concern in terms of product quality and safety. In literature there is limited information on munkoyo roots. Knowledge on quality of these roots, their effect on quality and microbiological safety of the fermented food products prepared by their use is essential for the development of improved products for increased consumption, commercial production and marketing. The present research work aimed at contributing to closing up this knowledge gap by evaluating the microbiological quality and safety of chibwantu. This was done by first gathering information on the production and utilisation of indigenous cereal based fermented food products in Lusaka and Chongwe districts of Lusaka province, Zambia with special emphasis on chibwantu and munkoyo beverages prepared using munkoyo roots. Second to isolate and characterize the microorganisms associated with the munkoyo roots and maize grit used during the preparation of chibwantu and to establish whether the same microorganisms are involved during the fermentation process of the beverage. Third to study the survival of selected food borne enteropathogenic microorganisms during fermentation of chibwantu beverage and finally to investigate the effect of munkoyo roots on growth of selected microorganisms. From the information gathered during this study, it was found that different types of indigenous cereal based fermented foods especially the beverages are prepared in Zambian households. The foods contribute to the nutritional, economical and medical needs of the consumers, thereby making them relevant to the consumers and the country at large. The raw materials; maize grit and munkoyo roots, used in the preparation of the beverage chibwantu are associated with coliforms, lactic acid bacteria (LAB), yeasts and moulds, particularly the munkoyo roots. The groups of microorganisms; coliforms, LAB, Yeasts and moulds, associated with the raw materials used in the preparation of the beverage chibwantu, particularly from the roots, were also responsible for the fermentation of the beverage. Some microorganisms that could not be identified from the raw materials were also encountered during the fermentation process of chibwantu. Staphylococcus aureus, E. coli, Salmonella and Shigella species were not encountered during natural fermentation of chibwantu and growth of selected enteropathogenic microorganisms was significantly suppressed during the fermentation process and storage. Both the yellow and white munkoyo roots possess some antimicrobial activity against some enteropathogenic microorganisms as well as microbial growth stimulatory activity. This study forms basis for further studies on the active components of the roots for possible use in preservation for improved food quality and safety. The quality of the beverage is varied due to different methods of preparation and the microorganisms involved during fermentation which are dependent on the quality of the munkoyo roots. There is still a lot of work needed on the munkoyo roots and the fermented products prepared from them to be able to establish their safety convincingly. However, fermented foods generally have a very good safety record and chibwantu should be relatively safe to consume.

Aflatoxins are carcinogenic mycotoxins produced by certain species of the Aspergilli, with the most prominent producers being Aspergillus flavus and A. parasiticus. Exposure to aflatoxins causes liver cancer, immune suppression, retardation in growth and in extreme cases, even death. A. parasiticus and A. flavus infect a wide range of crops, including corn, cotton, peanuts and tree nuts. Consequently, aflatoxin contamination has a severe impact on public health, as well as the agricultural sector. Several control strategies are currently in place to combat aflatoxin contamination. Although great progress has been made in developing innovative methods for aflatoxin control, no strategy is completely efficient in eliminating aflatoxin contamination. Also, the application of the strategies is often limited by practical and economic factors. This necessitates the development of further aflatoxin control strategies. A yet under-exploited resource is the aflatoxin biosynthesis pathway. Aflatoxins are synthesised in a complex polyketide-initiated pathway that requires multiple enzymatic steps. The genes encoding the aflatoxin biosynthetic enzymes are located in a cluster which contains both the regulatory and structural genes. A novel approach to aflatoxin control is the direct inhibition of a target enzyme in the aflatoxin biosynthesis pathway. An ideal candidate is the Baeyer-Villiger monooxygenase (BVMO), MoxY, that catalyses the conversion of hydroxyversicolorone to versiconal hemiacetal acetate. BVMOs are flavin-containing proteins that catalyse the oxidation of ketones or cyclic ketones to esters or lactones, respectively. BVMOs occur only in the genomes of bacteria and fungi, rendering the MoxY enzyme as a suitable candidate for targeted enzyme inhibition. In silico analysis of the moxY gene indicated that MoxY may exist with an elongated N-terminus or an alternative C-terminus, due to alternative splicing of the mRNA. In addition to cloning the moxY gene, variations of the moxY gene were created that encode a protein with both the alternative termini or either an alternative N-terminus or an alternative C-terminus. The MoxY variants were heterologously expressed in E. coli and MoxY with an elongated N-terminus (MoxYAltN) was found to be the only active recombinant form of the MoxY enzyme. MoxYAltN is promiscuous with regard to the substrate accepted, converting linear, aromatic, substituted aromatic and bicyclic ketones. MoxYAltN was purified and the kinetic parameters determined with respect to the reaction with a surrogate substrate, phenylacetone. Purified MoxYAltN was demonstrated to convert synthetic [1â-2H]hydroxyversicolorone to versiconal hemiacetal acetate, confirming the role of MoxYAltN in the aflatoxin biosynthesis pathway. Intragenomic complementation of an impaired aflatoxin biosynthetic step is a common theme that has emerged from the experimental study of the aflatoxin biosynthetic pathway. Therefore, the complementation of the activity of MoxY(AltN) by another BVMO in the A. flavus genome was investigated. The genome contains 26 putative BVMOs and phylogenetic analysis indicted that six of these are closely-related to MoxY, which were selected for heterologous expression in E. coli. Three of the homologues expressed as soluble proteins and activity was observed for two. The substrate profiles of the homologues differ significantly from that of MoxYAltN, with the only common substrate converted being (±)-cis-bicyclo[3.2.0]hept-2-en-6-one. The enantio- and regioselective conversion of (±)-cis-bicyclo[3.2.0]hept-2-en-6-one by BVMOs is a well-characterised reaction. Chiral analysis indicated divergent biocatalytic profiles of MoxYAltN and the two homologues with respect to the conversion of (±)-cis-bicyclo[3.2.0]hept-2-en-6-one, which suggests that the substrate binding pockets of the enzymes are likely to differ. The ability of the homologues to complement the activity of MoxY(AltN) by converting [1â-2H]hydroxyversicolorone remains uncertain. However, the differential substrate profiles demonstrate that phylogenetic clustering is not an absolute indication of overlapping biocatalytic abilities. Therefore, more distantly related BVMOs in A. flavus have to be considered as candidates for intragenomic complementation as well.

Bacterial infections, a major problem in the poultry industry, are controlled through the use of antibiotics. Due to the increase in antibiotic resistance and the restrictions placed on the use of antibiotics in animals, the poultry industry is slowly heading for a post-antibiotic era. The use of disinfectants, like quaternary ammonium compounds (QACs), could possibly be the last resort in the fight against bacterial infections. Resistance against QACs has been observed but needs to be investigated in order to prevent similar resistance problems. The overall aim of this study was to understand bacterial resistance to QACs, using the following objectives: ï· To examine the presence of qac resistance genes in field isolates and determining if the number of genes present confer higher resistance. ï· To study the expression of one of these qac resistance genes in the presence of increasing QAC concentration. ï· To study the efflux system, to determine the uptake and efflux of disinfectant, and to determine if the bacteria causes structural changes of the disinfectant. Bacterial resistance is conferred through acquisition of resistance genes; therefore qac resistance genes were studied to understand resistance to QACs. Screening of field isolates using conventional PCR for qac resistance genes (smr, qacJ, qacH and qacG) showed that one strain could contain more than one resistance gene. This could not be correlated with the minimum inhibitory concentration (MIC) of the three QACs tested and possession of more genes did not necessarily make the strain more resistant. Staphylococcus aureus strains known to contain at least one of the resistance genes were also screened for these qac genes. Conventional PCR showed that all the genes could only be detected in the strain when it was exposed to QAC. Conversely, real-time PCR showed that the genes could be detected even in the absence of QAC, and was detected in susceptible strains as well. Therefore containing the genes does not necessarily confer resistance. Relative quantitative real-time PCR was used to determine the expression of the qacJ gene in the S. aureus strains VB3_qacJ and ATCC 25923. It was hypothesised that the expression of the qacJ gene would increase with increasing didecyldimethylammonium chloride (DDAC) concentration. However, in the VB3_qacJ strain there was no significant difference in expression when induced with different DDAC concentrations. Expression of known qac resistance genes needs to be investigated simultaneously to determine whether a relationship exists between the qac genes conferring resistance. The mechanism of resistance is mainly efflux of the disinfectants; thereby reducing the disinfectant concentration inside the cell. The efflux mechanism of S. aureus was also studied using liquid chromatography-mass spectrometry (LC-MS). The hypothesis was that the bacteria are able to alter the structure of QAC before extruding it from the cell. Samples were prepared for LC-MS using solid phase extraction (SPE). The SPE protocol was optimised for extraction DDAC from growth media. It was shown that the DDAC concentration in the growth media decreased after 18 hrs of growth. It was not determined if structural changes of DDAC occurred. Resistance to disinfectants is much more complex than originally thought, and therefore resistance is still not fully understood. Further research is required to understand the full mechanism of resistance against QACs, so that similar problems associated with antibiotic resistance can be prevented.

Beak and feather disease (BFD), caused by Beak and feather disease virus (BFDV) is a dermatological condition afflicting parrot species. It is becoming increasingly difficult to ignore not only the significant negative economic impact that the virus has on the parrot breeding industry but also the detrimental effect it has on the survival of the endemic Cape parrot (Poicephalus robustus). The virus, a member of the Circoviridae, is known to possess a non-enveloped, circular, single-stranded DNA genome. Two major open reading frames (ORFs) encode the replication associated protein (Rep) and the coat protein (CP). The study was set out to evaluate and improve the current diagnostic strategy for BFDV, with both molecular and serological techniques. The following objectives were attempted: 1. To evaluate polymerase chain reaction (PCR) and quantitative real-time polymerase chain reaction (qPCR) as diagnostic tools for BFDV. Detection of BFDV with conventional PCR is not always sensitive, especially in birds without clinical symptoms. Furthermore, genetic variance was suggested to have a detrimental effect on primer hybridisation. A real-time assay was designed to address these problems. It amplified a 115 bp fragment of ORF V1 and was able to quantify viral load. 2. To recombinantly express BFDV coat protein. A sustainable source of the main immunogen, coat protein, was needed for use in serological test development. Bacterial expression of BFDV CP was unsuccessful; however, BFDV CP from an alternative expression study was used as a serological diagnostic antigen. 3. To develop serological diagnostic tests for BFDV. A novel slide agglutination test was developed and will serve as an initial screening tool in serological diagnosis. Steps were made in the development of a competitive Enzyme Linked Immunosorbent Assay (ELISA) for a quantitative indication of immune response to BFDV. A significant proportion of asymptomatic BFDV infections exist. Using a combination approach of both molecular and serological tests increases the capacity to detect infections or exposure to virus. New techniques described should be used in conjunction with existing tests and should not completely replace conventional techniques for diagnosis of BFDV infection or detection of exposure to the virus.

In order to study the decrease of yogurt fermentation time, the effects of a wide range of supplements on yogurt fermentation time were evaluated. YE was identified as the only supplement which showed potential. Unfortunately it resulted in a product with an unacceptable flavour. It was therefore important to identify and isolate the specific component responsible for the decrease in yogurt fermentation time. YE was fractionated with size exclusion chromatography and it was subsequently determined that the accelerating fraction had a low molecular weight. It was important to establish whether the accelerating component was of mineral, vitamin or amino acid origin. Three cocktails containing the most common minerals, vitamins and amino acids were prepared and their respective effects on yogurt fermentation were determined. Results indicated that when compared to the respective controls, no decrease in fermentation time was observed with the mineral and vitamin cocktails. A decrease in fermentation time was observed with the amino acid cocktail, indicating that the accelerating component present in YE was of amino acid origin. It was however not clear whether it was a single amino acid or a peptide. The accelerating fraction was further analysed by SDS-PAGE and due to no visible bands in the respective region (<1kDa), it could not be analysed with mass spectrometry. The fraction obtained directly after size exclusion chromatography was however analysed by using mass spectrometry in order to determine the total amino acid content of the accelerating fraction after which it was evident that the fraction containing the accelerant contained an abundance of peptides. The individual effects of the 17 identified amino acids were determined in respective yogurt fermentations. Results indicated that no single amino acid was responsible for the decrease in yogurt fermentation time. Although a combination of the 17 amino acids in one fermentation run resulted in a decrease in fermentation time in comparison to unsupplemented yogurt, the decrease was not as considerable as that of the accelerating fraction obtained after size exclusion chromatography. Due to the fact that it was not practical to evaluate the effect of all possible combinations of the 17 amino acids, the combinations that were evaluated were based on literature reports on stimulation of Streptococcus thermophilus growth. Focus was placed on the growth of Streptococcus thermophilus due to this organism being the growth limiting organism between the two starter organisms used for yogurt fermentation. None of the evaluated amino acid combinations decreased yogurt fermentation time, and it was therefore concluded that the accelerating component was a peptide and not a free amino acid. In order to establish the mechanism of acceleration of the isolated YE fraction, it was important to determine whether the addition of YE to milk increased the rate of starter bacteria growth or whether it completed the growth requirements of starter bacteria. The latter could either result in earlier initiation of lactic acid production or in increased levels of lactic acid production. Results indicated that YE had no effect on the total growth rate of the starter bacteria. However, when examining the effect on the respective bacteria individually, it became clear that YE increased Streptococcus thermophilus cell numbers in comparison to the unsupplemented control. Using resazurin, it seems YE increased the metabolic rate of the starter bacteria. Although supplementation of yogurt with YE did not influence combined starter bacteria growth, it influenced lactic acid production. The addition of YE to yogurt resulted in increased lactic acid levels as well as an increase in lactic acid production rate. Lactic acid was also initiated earlier in the fermentation process, resulting in higher lactic acid levels in comparison to unsupplemented yogurt. It was therefore evident from this study that YE did not provide nutrients that are not already present in milk, but rather provided nutrients in a readily available form at the beginning of the yogurt fermentation process resulting in the reduction of the lag phase of lactic acid production.

Milk is a complex and complete food for the specific nutritional requirements of the neonate. For the dairy industry, milk is a suitable raw material for the production of other high value products. Although extensive research has been carried out on milk of economically exploited dairy animals such as cow, goat, sheep, buffalo and camel, there are properties which are not explicit in the milk of these, that are not completely understood. Research of the milk from non-dairy animals, in which these properties are explicit, may provide answers. One of the unique properties is the content of oligosaccharides, which is low in the dairy animals, but high in some species. This property points to a specialized saccharide synthesis in the latter, where the whey protein α-lactalbumin may play a role. Another unique property is the structure of casein micelles, which in the dairy animals is stabilized by the presence of a specific ratio of four casein types, as well as their specific structural properties. The most important is the κ-casein with its amphipathic nature. In some non-dairy species, stable casein micelles are formed in spite of the absence, or low content, of some of the casein types, specifically the κ- casein. The milk of the African elephant (Loxodonta africana) displays several unique properties. In this research the proteome of its milk was investigated, with a focus on α-lactalbumin and the caseins, in order to shed light on the mentioned unique properties. The proteomics approach was used, which includes gel electrophoresis and mass spectrometry. Computer modeling was also employed. The major proteins αs1-, αs2-, β-casein, α-lactalbumin, β-lactoglobulin and serum albumin of African elephant milk were identified with one-dimensional electrophoresis and mass spectrometry. Better results were obtained with two-dimensional electrophoresis and orbitrap mass spectrometry, with which α-lactalbumin, lactoferrin, β- and κ-casein were identified. The multiple sequence alignment of α-lactalbumins showed that there are six amino acid positions that are unique to that of African elephant. Most of the amino acid substitutions in this protein were found to be conserved, and the structure model of African elephant α-lactalbumin was found to be homologous to the X-ray crystallography structures of several species. Consequently the structure models of β- 1,4-galactosyltransferase 1 and the lactose synthase complex were built, again showing homology to crystallographic data from other species. It may therefore be concluded that structures of α-lactalbumin and β-1,4-galactosyltransferase 1 are highly conserved amongst species. The saccharide synthesis in the African elephant milk would probably not differ from that of other mammals, and may therefore not be the reason for high levels of oligosaccharides in its milk. The comparison of amino acid sequences and hydropathy plots of African elephant β- casein with that of other species showed that it would self-aggregate and interact via hydrophobic interactions with other caseins to form casein micelles, similar to the model proposed for cowâs milk micelles. However, the African elephant β-casein displays several more hydrophilic regions, compared to the cowâs protein. The amino acid sequence and hydropathy plots predicted that African elephant κ- casein would function in the same way as the equivalent of other species. The ratio of African elephant milk κ-casein to β-casein was calculated to be approximately 1:8.5, which is in the same order as camel and rat milk, compared to the 3:8 of cowâs milk. This means that there would be very little κ-casein on the surface to effect repulsion of the micelles. There should therefore be other protein regions protruding from the micelle surface to aid in this function. It is suggested that in African elephant milk the hydrophilic regions of β-casein carry out this role.

Previous unpublished research by Kock and co-workers indicated that the antimalarial drug chloroquine (CQ) stimulates yeast sexual stages (biosensors). Consequently several indicator yeasts (Eremothecium ashbyi, Dipodascopsis uninucleata var. uninucelata, Lipomyces yamadae and Scheffersomyces stipitis) were exposed to concentration gradients of CQ in the Anti-mitochondrial Antifungal Assay (3A) system and their ascospore release mechanics were subjected to Auger architectomics. Auger architectomics is the study of the structure and atomic composition of cells by making use of Nano Scanning Auger Microscopy (NanoSAM) as well as other techniques (http://en.wikipedia.org/wiki/Auger_architectomics). Investigation of the ascospore release mechanics revealed that L. yamadae and S. stipitis were sequestrate (making use of passive ascospore release) while E. ashbyi and D. uninucleata made use of active ascospore release. The sensors of L. yamadae have smooth, spherical ascospores that are released by destruction of the sensor wall. The spherical sensors of S. stipitis each contain two brimmed (âhatâ-shaped) ascospores that are released when the sensor wall breaks apart. The sensors of E. ashbyi are mostly intercalary in long chains with up to sixteen sickle-shaped ascospores in each ellipsoidal sensor. The V-shaped fins at the base of each ascospore of E. ashbyi are coated with 3-hydroxy (3-OH) oxylipins, making them hydrophobic. This facilitates the movement of ascospores by water flow. The tapered tips of the ascospores pierce through the sensor wall to allow release (http://vimeo.com/61521401). In D. Uninucleata, the inside surface of the sensors are lined with flexible sheaths, surrounding the ascospores inside the central channel. Inflation of the sheaths due to water uptake, generates turgor pressure that forces the ascospores out of the sensor. This is in sharp contrast to the morphologically similar yeast Dipodascus geniculatus, where the inflation of sheaths surrounding each individual ascospore is responsible for ascospore release. In all cases the sensors with ascospores were observed to have increased mitochondrial activity compared to surrounding cells. It was found with the 3A system that CQ is indeed a potent yeast fertility drug, having pro-fertility effects on all the yeast sensors used, including yeasts with decreased ability to sporulate due to preservation by sub-cultivation. Chloroquine caused increased formation of mature sensors of L. yamadae and S. stipitis, and increased the rate of ascospore release from the sensors of E. ashbyi and D. uninucleata. This data becomes even more compelling when considered that the S. stipitis strain used had lost the ability to sporulate prior to this study. An investigation of the relevant literature showed that the pro-fertility effects of CQ are highly conserved in the Eukarya, having similar effects on mammals (including humans) as well as the malaria causing parasite Plasmodium falciparum. This highlights the need to re-evaluate future and current CQ based treatment regimes.

The nutritional composition of agave flowers was determined and the following nutrients were analysed: moisture (86.62%); energy (226 kJ/100 g); proteins (1.71 g/100 g); fat (0.46 g/100 g); dietary fibre (2.12 g/100 g); K (207.77 mg/100 g); Mg (53.06 mg/100 g); Ca (48.33 mg/100 g); P (32.12 mg/100 g); Na (1.27 mg/100 g); Fe (1.03 mg/100 g); Zn (0.66 mg/100 g); Cu (0.04 mg/100 g); and Mn (0.15 mg/100 g). In contrast to many vegetables, the flower samples contained sugars in the form of sucrose (0.52 g/100 g), glucose (0.77 g/100 g), fructose (1.06 g/100 g) and maltose (0.69 g/100 g). The vitamin C content was 1.03 mg /100 g, but no vitamin A was detected. When compared to other flower vegetables, the agave flower had the lowest contents for protein, P, K, Mn, Na, Cu and vitamin C, but the highest value for fat. The agave flower and artichoke had similar values for energy, moisture and Mg, while the cauliflower and agave compared well in regard to Ca and Zn contents. Broccoli had similar contents for protein, fibre, P and Cu. The agave had higher values for energy, and fat than cauliflower, and higher fat and Ca, Fe, Mg, P and Zn values than broccoli. Artichokes had lower contents for moisture, fat, Ca and Zn than the agave flowers. Broccoli and cauliflower were moister than the agaves. Descriptive sensory analysis was used to investigate how various treatments would influence the sensory properties of Agave americana flowers. Blanched and unblanched flowers, subjected to three treatment methods (steaming, stir frying and pickling), were analyzed by ten trained panelists, in three replications. The data was analyzed using principle component analysis. A lexicon of 20 attributes was generated, including 11 for the steamed treatment, an additional six for the stir fried treatment and another four for the pickled treatment. Of these, three descriptors were for the attribute aroma, six for mouthfeel, five for appearance, four for taste and one for aftertaste. The unblanched pickled agave flowers were characterized by crunchy, fibrous and chewy mouthfeel, bitter taste, green pepper colour and cactus appearance, and a cucumber odour. For the blanched pickled agave flowers, taste descriptors were prominent, namely sweet, sour and sweet-sour, followed by a sweet aftertaste, combined with a moist appearance. For all the unblanched flowers, regardless of treatment, some of the descriptors had negative connections, like fibrous, bitter, cactus and rancid. Descriptors for the blanched flowers, again regardless of treatment, were more favourable and included sweet, sour and sweet-sour taste, and green bean and nutty odour. Three panels of 50 members each participated in the consumer acceptance tests, one each for the steamed breads and chocolate cakes, and one for the stew and deep fried flowers. Apart from acceptability, aroma, taste and texture were also evaluated for the baked products. The breads, cakes and stews were defrosted at 4 ºC overnight. The breads and cakes were left at room temperature (22ºC) before serving, while the stews were served heated. The breads, fritters and stews were prepared with blanched flowers, while the cake was made with unblanched flour. All the products were acceptable, but in different degrees. The scores were lower for aroma and final acceptability of the bread, due to unfamiliar texture experienced by the consumer panel. The agave cake scored lower for aroma and taste, but higher than the bread on texture. The stews and battered agave fritters were liked by the consumer panels and scored between 6.92 and 7.26 on the hedonic scale.

It has previously been implicated in literature that intracellular gas bubbles cannot form in yeast cells even under high gas supersaturation conditions. Furthermore, not even protein-coated gas vesicles found in Cyanobacteria are expected in yeasts. The lack of intracellular gas bubbles has been attributed to the increased structuring of water and lack of water with nucleation properties. This, however, is considered a missing link since yeasts, the workhorses of the baking and brewing industry, are known to produce and vigorously release carbon dioxide (CO2) gas during fermentation. Here we resolve the missing link between CO2 production by glycolysis and the eventual release of CO2 from the cells, and show that yeasts are capable of producing intracellular gas bubbles which were found to occupy a significant part of the cell. These gas bubbles do not contain a membrane that surrounds them. Furthermore, addition of zinc to the growth medium resulted in the âgalvanizationâ of the bubbles suggesting that the gas bubbles may possibly contain CO2. These findings should pave way for future research on CO2 behaviour under pressurized conditions that may have an impact on fermentation biotechnology. Furthermore we show that these intracellular gas bubbles deform cell organelles such as the nucleus. The skin surrounding the gas bubbles is able to withstand tension as they do not disintegrate when they come in contact with organelle membranes. Further research should now be performed on the mechanical effects of the gas bubbles on metabolic and coding functions of yeasts as gas bubbles deform and contort cell organelles. From these findings careful consideration is required during optimization of fermentation parameters to prevent CO2 toxicity effects on fermentation performance and flavor formation in practical brewing.