Fermentation of Dairy-Relevant Sugars by Saccharomyces, Kluyveromyces, and Brettanomyces: An Exploratory Study with Implications for the Utilization of Acid Whey, Part I

Acid whey from Greek-style yogurt (YAW) is an underutilized byproduct and a challenge for the dairy industry. One alternative is the fermentation of YAW by yeasts such as Saccharomyces, Brettanomyces, and Kluyveromyces spp., to produce new styles of fermented beverages. Previous research in our group suggested that the sugar profiles of the dairy coproducts impacted the fermentation profiles produced by B. claussenii. The present work aims to describe the fermentation of dairy sugars by S. cerevisiae, K. marxianus, and B. claussenii, under conditions comparable to those of YAW. For this purpose, four preparations of yeast nitrogen base, each containing 40 g/L of either lactose (LAC), glucose (GLU), galactose (GAL), or a 1:1 mixture of glucose and galactose (GLU:GAL), all at pH 4.20, were used as fermentation media. The fermentation was performed independently by each organism at 25 °C under anoxic conditions, while density, pH, cell count, ethanol, and organic acids were monitored. Non-linear modeling was used to characterize density curves, and Analysis of Variance and Tukey’s Honest Significant Difference tests were used to compare fermentation products. K. marxianus and S. cerevisiae displayed rapid sugar consumption with consistent ethanol yields in all media, as opposed to B. claussenii, which showed more variable results. The latter organism exhibited what appears to be a selective glucose fermentation in GLU:GAL, which will be explored in the future. These results provide a deeper understanding of dairy sugar utilization by relevant yeasts, allowing for future work to optimize fermentations to improve value-added beverage and ingredient production from YAW.

Insulin Regulation of Escherichia coli Abiotic Biofilm Formation: Effect of Nutrients and Growth Conditions

Escherichia coli plays an important role in biofilm formation across a wide array of disease and ecological settings. Insulin can function as an adjuvant in the regulation of biofilm levels. The modulation of insulin-regulated biofilm formation by environmental conditions has not been previously described. In the present study, the effects that various environmental growth conditions and nutrients have on insulin-modulated levels of biofilm production were measured. Micropipette tips were incubated with E. coli ATCC® 25922™ in a Mueller Hinton broth (MH), or a yeast nitrogen base with 1% peptone (YNBP), which was supplemented with glucose, lactose, galactose and/or insulin (Humulin®-R). The incubation conditions included a shaking or static culture, at 23 °C or 37 °C. After incubation, the biofilm production was calculated per CFU. At 23 °C, the presence of insulin increased biofilm formation. The amount of biofilm formation was highest in glucose > galactose >> lactose, while the biofilm levels decreased in shaking cultures, except for galactose (3-fold increase; 0.1% galactose and 20 μU insulin). At 37 °C, regardless of condition, there was more biofilm formation/CFU under static conditions in YNBP than in MH, except for the MH containing galactose. E. coli biofilm formation is influenced by aeration, temperature, and insulin concentration in combination with the available sugars.

Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and β-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, β-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding β-carotene hydroxylase from different organisms were individually introduced into β-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)–rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and β-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Counter-Acting Candida albicans-Staphylococcus aureus Mixed Biofilm on Titanium Implants Using Microbial Biosurfactants

This study aimed to grow a fungal-bacterial mixed biofilm on medical-grade titanium and assess the ability of the biosurfactant R89 (R89BS) coating to inhibit biofilm formation. Coated titanium discs (TDs) were obtained by physical absorption of R89BS. Candida albicans-Staphylococcus aureus biofilm on TDs was grown in Yeast Nitrogen Base, supplemented with dextrose and fetal bovine serum, renewing growth medium every 24 h and incubating at 37 °C under agitation. The anti-biofilm activity was evaluated by quantifying total biomass, microbial metabolic activity and microbial viability at 24, 48, and 72 h on coated and uncoated TDs. Scanning electron microscopy was used to evaluate biofilm architecture. R89BS cytotoxicity on human primary osteoblasts was assayed on solutions at concentrations from 0 to 200 μg/mL and using eluates from coated TDs. Mixed biofilm was significantly inhibited by R89BS coating, with similar effects on biofilm biomass, cell metabolic activity and cell viability. A biofilm inhibition >90% was observed at 24 h. A lower but significant inhibition was still present at 48 h of incubation. Viability tests on primary osteoblasts showed no cytotoxicity of coated TDs. R89BS coating was effective in reducing C. albicans-S. aureus mixed biofilm on titanium surfaces and is a promising strategy to prevent dental implants microbial colonization.

D-Fructose Assimilation and Fermentation by Yeasts Belonging to Saccharomycetes: Rediscovery of Universal Phenotypes and Elucidation of Fructophilic Behaviors in Ambrosiozyma platypodis and Cyberlindnera americana

The purpose of this study was to investigate the ability of ascomycetous yeasts to assimilate/ferment d-fructose. This ability of the vast majority of yeasts has long been neglected since the standardization of the methodology around 1950, wherein fructose was excluded from the standard set of physiological properties for characterizing yeast species, despite the ubiquitous presence of fructose in the natural environment. In this study, we examined 388 strains of yeast, mainly belonging to the Saccharomycetes (Saccharomycotina, Ascomycota), to determine whether they can assimilate/ferment d-fructose. Conventional methods, using liquid medium containing yeast nitrogen base +0.5% (w/v) of d-fructose solution for assimilation and yeast extract-peptone +2% (w/v) fructose solution with an inverted Durham tube for fermentation, were used. All strains examined (n = 388, 100%) assimilated d-fructose, whereas 302 (77.8%) of them fermented d-fructose. In addition, almost all strains capable of fermenting d-glucose could also ferment d-fructose. These results strongly suggest that the ability to assimilate/ferment d-fructose is a universal phenotype among yeasts in the Saccharomycetes. Furthermore, the fructophilic behavior of Ambrosiozyma platypodis JCM 1843 and Cyberlindnera americana JCM 3592 was characterized by sugar consumption profiles during fermentation.

Neonatal sepsis due to non-albicans Candida species and their susceptibility to antifungal agents: first report from Bangladesh

Background and objectives: Frequency of neonatal sepsis in Neonatal Intensive Care Units (NICU) has been increasing worldwide over the last decades. The emergence of non-albicans Candida (NAC) species and their resistance to common antifungal agents become an important preventive and therapeutic issue. The present study was undertaken to find out the role of NAC species in neonatal sepsis/candidemia in the NICUs of hospitals of Dhaka city. The susceptibility pattern of NAC species to antifungal agents was also determined. Materials and methods: Suspected cases of neonatal sepsis admitted in NICU of four tertiary care hospitals of Dhaka city, from March to December 2018 were enrolled. In this cross sectional study, blood samples were collected from neonates with suspected sepsis for culture. Identification of Candida species was done by carbohydrate (CHO) assimilation tests using swab auxanographic technique, CHO impregnated yeast nitrogen base plate method (YNB), microtiter plate based miniaturized method and by HiCromeTM Candida Differential Media. Susceptibility of the isolated Candida species to antifungal agents was determined by disk diffusion (DD) and by minimum inhibitory concentration (MIC) methods. MIC was determined by broth microdilution method using RPMI 1640 and trypticase soy broth (TSB). Results: In the present study, NAC species were isolated from 39.7% neonates. C. tropicalis was the predominant species (81.0%) followed by C. parapsilosis (12.1%), C. auris (5.2%) and C. dubliniensis (1.7%). Isolated NAC species were 98.3% sensitive to voriconazole. Sensitivity to fluconazole, ketoconazole, itraconazole, and clotrimazole was 3.5%, 15.5%, 86.2% and 56.9% respectively by DD method. All the isolates (100%) were sensitive to miconazole and nystatin. All the C. tropicalis, C. auris and C. dubliniensis were sensitive to amphotericin B and anidulafungin. One and four C. parapsilosis were found resistant to amphotericin B and anidulafungin respectively. The MIC results obtained by using RPMI 1640 and TSB as growth medium were concordant suggesting that TSB media was a good alternative to expensive RPMI 1640. Conclusion: The advent of NAC species merits attention as they are highly resistant to most of the azoles. Therefore, speciation of Candida in neonatal candidemia is essential to institute appropriate antifungal therapy. Ibrahim Med. Coll. J. 2020; 14(2): 19-26

Evaluation of Biofilm Formation in Candida tropicalis Using a Silicone-Based Platform with Synthetic Urine Medium

Molecular mechanisms of biofilm formation in Candida tropicalis and current methods for biofilm analyses in this fungal pathogen are limited. (2) Methods: Biofilm biomass and crystal violet staining of the wild-type and each gene mutant strain of C. tropicalis were evaluated on silicone under synthetic urine culture conditions. (3) Results: Seven media were tested to compare the effects on biofilm growth with or without silicone. Results showed that biofilm cells of C. tropicalis were unable to form firm biofilms on the bottom of 12-well polystyrene plates. However, on a silicone-based platform, Roswell Park Memorial Institute 1640 (RPMI 1640), yeast nitrogen base (YNB) + 1% glucose, and synthetic urine media were able to induce strong biofilm growth. In particular, replacement of Spider medium with synthetic urine in the adherence step and the developmental stage is necessary to gain remarkably increased biofilms. Interestingly, unlike Candida albicans, the C. tropicalis ROB1 deletion strain but not the other five biofilm-associated mutants did not cause a significant reduction in biofilm formation, suggesting that the biofilm regulatory circuits of the two species are divergent. (4) Conclusions: This system for C. tropicalis biofilm analyses will become a useful tool to unveil the biofilm regulatory network in C. tropicalis.

Saccharide sources do not influence the biofilm formation in Scedosporium/Lomentospora species

Scedosporium and Lomentospora species are ubiquitous saprophytic filamentous fungi that emerged as human pathogens with impressive multidrug-resistance profile. The ability to form biofilm over several biotic and abiotic surfaces is one of the characteristics that contributes to their resistance patterns against almost all currently available antifungals. Herein, we have demonstrated that Scedosporium apiospermum, Scedosporium minutisporum, Scedosporium aurantiacum and Lomentospora prolificans were able to form biofilm, in similar amounts, when conidial cells were incubated in a polystyrene substrate containing Sabouraud medium supplemented or not with different concentrations (2%, 5% and 10%) of glucose, fructose, sucrose and lactose. Likewise, the glucose supplementation of culture media primarily composed of amino acids (SCFM, synthetic cystic fibrosis medium) and salts (YNB, yeast nitrogen base) did not modulate the biofilm formation of Scedosporium/Lomentospora species. Collectively, the present data reinforce the ability of these opportunistic fungi to colonize and to build biofilm structures under different environmental conditions.

Assessment of acid phosphatase enzyme and influence of potassium iodide on its production in the yeast form of Sporothrix schenckii

Background: Sporotrichosis is caused by a dimorphic fungal species, Sporothrix schenckii (S. schenckii). The enzyme acid phosphatase is pervasive among yeast and yeast like fungi. It has been studied in various fungi like Aspergillus oryzae, Candida albicans etc. but in S. schenckii little is known about enzyme acid phosphatase. The present study depicts the in-vitro influence of Potassium Iodide (KI) on the enzyme acid phosphatase produced by the S. schenckii (yeast form).Methods: A master culture was prepared by incorporating the standard strain of S. schenckii in YNB (Yeast Nitrogen Base) medium and was incubated at 37ºC. After preparing the increasing concentrations with KI in YNB medium, 1.0 mL suspension of master culture was inoculated into each bottle and incubated at 37ºC for different time period 6th, 12th, 18th day (early, mid, peak of log period) respectively. After centrifuging, a 5% homogenate was prepared, which was used for acid phosphatase enzyme assay.Results: The mean acid phosphatase level of control specimen was 20.9±2.01, 50.0±2.25, 45.0±5.10 μg and test specimens was ranged from 14.9±4.89 to 20.2±3.49, 10.2±4.19 to 40.0±6.39 and 10.0±1.81 to 34.7±6.08 μg on day 6, 12 and 18 respectively. The mean value was lower significantly for all the test concentrations as compared to control (p<0.05).Conclusions: The low activity of the enzyme acid phosphatase indicates that KI has inhibitory effect on the growth of S. schenckii that has led to decrease in the activity of the enzyme.