Fermentation, Volume 3, Issue 3 (September 2017) – 19 articles

Cold-hardy grape cultivars have become popular in northern regions. Wines from these cultivars are low in tannins and lighter in color compared to Vitis vinifera. The northern regions are striving to enhance desired ″full body″ and red color qualities in the wine produced from cold-hardy grapes. The objective of this study was to compare tannin and pigment content in skins and seeds of three cold-hardy red grape cultivars, at two time points, from two locations, using the Adams-Harbertson (A-H) assay. The A-H assay is based on protein precipitation and spectrophotometry. Total tannin concentrations detected in Frontenac, Marquette, and St. Croix berries, ranged from 0.29 to 0.66 mg/berry catechin equivalents (CE). Bitter seed tannins were most abundant in Marquette berries (0.54 ± 0.66 mg/berry CE). Softer skin tannins were most abundant in St. Croix berries (0.24 ± 0.19 mg/berry CE). Monomeric anthocyanins contributed to over 60% of the total color at pH 4.9 and were highest in St. Croix skins (74.21% of the total color at pH 4.9). Varying amounts of short polymeric pigments and long polymeric pigments were present in grape skins, indicating that pigmented tannins had already formed by harvest. This is the first evaluation of tannins and pigments in Frontenac, Marquette, and St. Croix berries. Full article

Yeast β-glucan polysaccharide is a proven immunostimulant molecule for human and animal health. In recent years, interest in β-glucan industrial production has been increasing. The yeast cell wall is modified during the fermentation process for biomass production. The impact of environmental conditions on cell wall remodelling has not been extensively investigated. The aim of this research work was to study the impact of glucose and NaCl stress on β-glucan formation in the yeast cell wall during alcoholic fermentation and the assessment of the optimum fermentation phase at which the highest β-glucan yield is obtained. VIN 13 Saccharomyces cerevisiae (S. cerevisiae) strain was pre-cultured for 24 h with 0% and 6% NaCl and inoculated in a medium consisting of 200, 300, or 400 g/L glucose. During fermentation, 50 mL of fermented medium were taken periodically for the determination of Optical Density (OD), cell count, cell viability, cell dry weight, β-glucan concentration and β-glucan yield. Next, dry yeast cell biomass was treated with lytic enzyme and sonication. At the early stationary phase, the highest β-glucan concentration and yield was observed for non-NaCl pre-cultured cells grown in a medium containing 200 g/L glucose; these cells, when treated with enzyme and sonication, appeared to be the most resistant. Stationary is the optimum phase for cell harvesting for β-glucan isolation. NaCl and glucose stress impact negatively on β-glucan formation during alcoholic fermentation. The results of this work could comprise a model study for yeast β-glucan production on an industrial scale and offer new perspectives on yeast physiology for the development of antifungal drugs. Full article

Previous investigations of the sponge-derived fungus Stachylidium bicolor (S. bicolor) 293K04 led to the isolation of the biosynthetically unusual polyketides marilines A-C and the bioactive tetrapeptides endolides A-B, identified as potential neuropathic drug leads. Furthermore, prior extended solid cultivation of S. bicolor 293K04 for 60 days resulted in a significant increase of polyketide yield, and the isolation of seven new polyketides. Due to the interest in endolide activity, unusual biosynthetic diversity, and the late stage polyketide production, we studied the cultivation conditions for determining the production time distribution and yields of these secondary metabolites. Results indicated a first production phase of secondary metabolite dominated by peptides, after 21–23 days. Polyketide mariline A1/A2 only started at day 35 of growth, an unusually late period for secondary metabolite expression. This unusual bimodal sequential expression of different families of secondary metabolites suggests value in exploring extended cultivation times to identify novel bioactive fungal compounds. Full article

Ayeast isolate, selected for its lipolytic activity from a meat product, was characterized as Pichia anomala. Lipolytic activity, determined on p-NPA as esterase, was maximum at 28 °C, pH 6.5, and induced by the short chain triglyceride tributyrin. Fermentations in 2 L and 10 L stirred tank bioreactors, with 20 and 60 g/L glucose respectively, showed that in the second case lipolytic activity increased 1.74-fold, while the biomass increased 1.57-fold. Under otherwise identical aeration conditions, improved mixing in the 10 L reactor maintained higher dissolved oxygen levels which, along with the elevated glucose concentration, resulted in significant increase of specific rates of lipolytic activity (51 vs. 7 U/g/L), while specific rates of growth and glucose consumption maintained lower. The Crabtree-negative yeast (glucose insensitive growth) exhibited a Pasteur effect at lower dissolved oxygen concentrations while elevated glucose prevented ethanol formation under oxygen saturation. The particular physiological traits can be exploited to obtain significant lipolytic activity in a scalable aerobic process. Full article

The volume and market share loss for classical beer types such as pils beer and wheat beer has been declining for several years, but the overall beer market remains almost unchanged as a result of the increasing interest in beer specialties Due to high biodiversity, the diversity of the strains, and the different flavor profiles, reliable and practical information regarding the characteristics of individual brewing strains is required to help brewers to find the right strain for their brewing purposes. This paper presents a comparison of 10 commercially available Technical University of Munich (TUM) brewing yeast strains. The strains were screened for genetic and phenotypic characteristics. After confirming the genetic distinctiveness by using species-specific real-time polymerase chain reaction (RT-PCR) systems and a strain typing method based on PCR-capillary electrophoresis of the partial intergenic spacer 2 (IGS2) fragment (IGS2-314 PCR-capillary electrophoresis), the strains were tested regarding phenotypic characteristics under controlled and identical fermentation conditions in small-scale brewing trials. Besides the fermentation performance, flocculation behavior, sugar metabolism and other phenotypic characteristics, the main focus was on the flavor and aroma profile of each investigated TUM yeast strain. Full article

This proof-of-concept study establishes Yarrowia lipolytica (Y. lipolytica) as a whole cell factory for the de novo production of long chain dicarboxylic acid (LCDCA-16 and 18) using glycerol as the sole source of carbon. Modification of the fatty acid metabolism pathway enabled creating a pool of fatty acids in a β-oxidation deficient strain. We then selectively upregulated the native fatty acid ω-oxidation pathway for the enhanced terminal oxidation of the endogenous fatty acid precursors. Nitrogen-limiting conditions and leucine supplementation were employed to induce fatty acid biosynthesis in an engineered Leu⁻ modified strain. Our genetic engineering strategy allowed a minimum production of 330 mg/L LCDCAs in shake flask. Scale up to a 1-L bioreactor increased the titer to 3.49 g/L. Our engineered yeast also produced citric acid as a major by-product at a titer of 39.2 g/L. These results provide basis for developing Y. lipolytica as a safe biorefinery platform for the sustainable production of high-value LCDCAs from non-oily feedstock. Full article

Broad and increasing interest in sustainable wastewater treatment has led a paradigm shift towards more efficient means of treatment system operation. A key aspect of improving overall sustainability is the potential for direct wastewater effluent reuse. Anaerobic membrane bioreactors (AnMBRs) have been identified as an attractive option for producing high quality and nutrient-rich effluents during the treatment of municipal wastewaters. The introduction of direct effluent reuse does, however, raise several safety concerns related to its application. Among those concerns are the microbial threats associated with pathogenic bacteria as well as the emerging issues associated with antibiotic-resistant bacteria and the potential for proliferation of antibiotic resistance genes. Although there is substantial research evaluating these topics from the perspectives of anaerobic digestion and membrane bioreactors separately, little is known regarding how AnMBR systems can contribute to pathogen and antibiotic resistance removal and propagation in wastewater effluents. The aim of this review is to provide a current assessment of existing literature on anaerobic and membrane-based treatment systems as they relate to these microbial safety issues and utilize this assessment to identify areas of potential future research to evaluate the suitability of AnMBRs for direct effluent reuse. Full article

Among other fermentation processes, lactic acid fermentation is a valuable process which enhances the safety, nutritional and sensory properties of food. The use of starters is recommended compared to spontaneous fermentation, from a safety point of view but also to ensure a better control of product functional and sensory properties. Starters are used for dairy products, sourdough, wine, meat, sauerkraut and homemade foods and beverages from dairy or vegetal origin. Among lactic acid bacteria, Lactobacillus, Lactococcus, Leuconostoc, Streptococcus and Pediococcus are the majors genera used as starters whereas Weissella is not. Weissella spp. are frequently isolated from spontaneous fermented foods and participate to the characteristics of the fermented product. They possess a large set of functional and technological properties, which can enhance safety, nutritional and sensory characteristics of food. Particularly, Weissella cibaria and Weissella confusa have been described as high producers of exo-polysaccharides, which exhibit texturizing properties. Numerous bacteriocins have been purified from Weissella hellenica strains and may be used as bio-preservative. Some Weissella strains are able to decarboxylate polymeric phenolic compounds resulting in a better bioavailability. Other Weissella strains showed resistance to low pH and bile salts and were isolated from healthy human feces, suggesting their potential as probiotics. Despite all these features, the use of Weissella spp. as commercial starters remained non-investigated. Potential biogenic amine production, antibiotic resistance pattern or infection hazard partly explains this neglecting. Besides, Weissella spp. are not recognized as GRAS (Generally Recognized As Safe). However, Weissella spp. are potential powerful starters for food fermentation as well as Lactococcus, Leuconostoc or Lactobacillus species. Full article

Orange juice production generates a very high quantity of residues (Orange Peel Waste or OPW-50–60% of total weight) that can be used for cattle feed as well as feedstock for the extraction or production of essential oils, pectin and nutraceutics and several monosaccharides by saccharification, inversion and enzyme-aided extraction. As in all solid wastes, simple pretreatments can enhance these processes. In this study, hydrothermal pretreatments and knife milling have been analyzed with enzyme saccharification at different dry solid contents as the selection test: simple knife milling seemed more appropriate, as no added pretreatment resulted in better final glucose yields. A Taguchi optimization study on dry solid to liquid content and the composition of the enzymatic cocktail was undertaken. The amounts of enzymatic preparations were set to reduce their impact on the economy of the process; however, as expected, the highest amounts resulted in the best yields to glucose and other monomers. Interestingly, the highest content in solid to liquid (11.5% on dry basis) rendered the best yields. Additionally, in search for process economy with high yields, operational conditions were set: medium amounts of hemicellulases, polygalacturonases and β-glucosidases. Finally, a fractal kinetic modelling of results for all products from the saccharification process indicated very high activities resulting in the liberation of glucose, fructose and xylose, and very low activities to arabinose and galactose. High activity on pectin was also observed, but, for all monomers liberated initially at a fast rate, high hindrances appeared during the saccharification process. Full article

The exact and reliable detection of sugar monomers and fructans provides important information for the evaluation of carbohydrate metabolism in plants and animals. Using the HPLC method; a large number of samples and single sugars; with both high sensitivity and selectivity; may be analysed. It was shown that the described method—using a Nucleosil column loaded with Pb²⁺ ions; a refractive index detector (RID); and HPLC-grade water as the eluent—gives precise and reproducible results regarding the detection of individual sugars in extracts of plants and feed materials. The method can be applied for the detection of sucrose; maltose; lactose; xylose; glucose; galactose; arabinose; fructose; ribose; and mannitol. Furthermore; depending on the plant material; the sugars verbascose; stachyose; and raffinose can be separated. The peaks were well resolved and the reproducibility of the analysis; with 94–108% of recovery (RC) and relative standard deviation (RSD) of up to 5%; was very good. The method was successfully applied to a variety of green forages and samples of sugar beet pulp silages. It is also possible to determine fructan with inulin as a standard; together with the other sugars; or alone by a different protocol and column. Full article

Disposal of biodegradable waste has become a stringent waste management and environmental issue. As a result, anaerobic digestion has become one of the best alternative technology to treat the organic fraction of municipal solid wastes and can be an important source of bioenergy. This study focuses on the evaluation of biogas and methane yields from the digestion and co-digestion of mixtures of waste untreated sludge and the organic fraction of municipal solid wastes. These are compared with the results obtained from the digestion and codigestion of mixtures containing waste active sludge and the organic fraction of municipal solid wastes. The two types of substrates were used to perform biomethanation potential tests, in mesophilic conditions (35 °C) at lab scale. It was observed a maximum biogas yield for 100% of untreated sewage sludge, corresponding to 0.644 Nm ${}^3$ /kg VS and 0.499 Nm ${}^3$ /kg VS of biogas and methane production respectively. The study also demonstrates the possibility of increasing biogas production up to 36% and methane content up to 94% using waste untreated sludge substrate in both digestion and codigestion, compared to waste active sludge substrate. Full article

Increasing demand for plant oil for food, feed, and fuel production has led to food-fuel competition, higher plant lipid cost, and more need for agricultural land. On the other hand, the growing global production of biodiesel has increased the production of glycerol as a by-product. Efficient utilization of this by-product can reduce biodiesel production costs. We engineered Yarrowia lipolytica (Y. lipolytica) at various metabolic levels of lipid biosynthesis, degradation, and regulation for enhanced lipid and citric acid production. We used a one-step double gene knock-in and site-specific gene knock-out strategy. The resulting final strain combines the overexpression of homologous DGA1 and DGA2 in a POX-deleted background, and deletion of the SNF1 lipid regulator. This increased lipid and citric acid production in the strain under nitrogen-limiting conditions (C/N molar ratio of 60). The engineered strain constitutively accumulated lipid at a titer of more than 4.8 g/L with a lipid content of 53% of dry cell weight (DCW). The secreted citric acid reached a yield of 0.75 g/g (up to ~45 g/L) from pure glycerol in 3 days of batch fermentation using a 1-L bioreactor. This yeast cell factory was capable of simultaneous lipid accumulation and citric acid secretion. It can be used in fed-batch or continuous bioprocessing for citric acid recovery from the supernatant, along with lipid extraction from the harvested biomass. Full article

Fumaric acid is a platform chemical with many applications in bio-based chemical and polymer production. Fungal cell morphology is an important factor that affects fumaric acid production via fermentation. In the present study, pellet and dispersed mycelia morphology of Rhizopus arrhizus NRRL 2582 was analysed using image analysis software and the impact on fumaric acid production was evaluated. Batch experiments were carried out in shake flasks using glucose as carbon source. The highest fumaric acid yield of 0.84 g/g total sugars was achieved in the case of dispersed mycelia with a final fumaric acid concentration of 19.7 g/L. The fumaric acid production was also evaluated using a nutrient rich feedstock obtained from soybean cake, as substitute of the commercial nitrogen sources. Solid state fermentation was performed in order to produce proteolytic enzymes, which were utilised for soybean cake hydrolysis. Batch fermentations were conducted using 50 g/L glucose and soybean cake hydrolysate achieving up to 33 g/L fumaric acid concentration. To the best of our knowledge the influence of R. arrhizus morphology on fumaric acid production has not been reported previously. The results indicated that dispersed clumps were more effective in fumaric acid production than pellets and renewable resources could be alternatively valorised for the biotechnological production of platform chemicals. Full article

An experimental design based on Response Surface Methodology (RSM) was used for the formulation of a growth medium based on sugar beet and wheat processing coproducts adapted to the cultivation of Lactobacillus reuteri (L. reuteri) DSM17938. The strain was cultivated on 30 different media varying by the proportions of sugar beet and wheat processing coproducts, and the concentration of yeast extract, tween 80 and vitamin B12. The media were used in a two-step process consisting of L. reuteri cultivation followed by the bioconversion of glycerol into 3-hydroxypropionic acid by resting cells. The efficiency of the formulations was evaluated according to the maximal optical density at the end of the growth phase (ΔOD_620nm) and the ability of the resting cells to convert glycerol into 3-hydroxypropionic acid, a platform molecule of interest for the plastic industry. De Man, Rogosa, and Sharpe medium (MRS), commonly used for the cultivation of lactic bacteria, was used as the control medium. The optimized formulation allowed increasing the 3-HP production. Full article

Phenolic compounds are important inhibitors of the microorganisms used in the Acetone-Butanol-Ethanol (ABE) fermentation. The degradation of phenolic compounds in a wood pre-hydrolysate, a potential substrate for the production of ABE, was studied in this article. First, physicochemical methods for detoxification such as nanofiltration and flocculation were applied and the best combination was selected. With a flocculated sample, the concentration of phenolic compounds decreases from 1.20 to 0.28 g/L with the addition of a solid laccase at optimum conditions, which is below the phenolic compounds limit of inhibition. This results in an increase in butanol production, more than double, compared to a pre-hydrolysate non-treated with laccase enzymes. Full article

Conventional bio-based succinic acid production involves anaerobic bacterial fermentation of pure sugars. This study explored a new route for directly producing succinic acid from minimally-pretreated lignocellulosic biomass via a consolidated bioprocessing technology employing a mixed lignocellulolytic and acidogenic fungal co-culture. The process involved a solid-state pre-fermentation stage followed by a two-phase slurry fermentation stage. During the solid-state pre-fermentation stage, Aspergillus niger and Trichoderma reesei were co-cultured in a nitrogen-rich substrate (e.g., soybean hull) to induce cellulolytic enzyme activity. The ligninolytic fungus Phanerochaete chrysosporium was grown separately on carbon-rich birch wood chips to induce ligninolytic enzymes, rendering the biomass more susceptible to cellulase attack. The solid-state pre-cultures were then combined in a slurry fermentation culture to achieve simultaneous enzymatic cellulolysis and succinic acid production. This approach generated succinic acid at maximum titers of 32.43 g/L after 72 h of batch slurry fermentation (~10 g/L production), and 61.12 g/L after 36 h of addition of fresh birch wood chips at the onset of the slurry fermentation stage (~26 g/L production). Based on this result, this approach is a promising alternative to current bacterial succinic acid production due to its minimal substrate pretreatment requirements, which could reduce production costs. Full article

Rotundone is a key aromatic compound for cool-climate Shiraz. This compound is produced in the skin of grape berries and extracted into wine during fermentation. This project investigated the influence of fermentation techniques on the concentration of rotundone in the resultant wine. Wine was fortified with ethanol and sucrose on the 1st and 5th days of fermentation and rotundone, volatile aroma compounds and colour were assessed in the resultant wine. The relationship between the concentration of rotundone and alcoholic strength during fermentation process was also investigated. Wine alcoholic strength and skin–wine contact time were two factors affecting rotundone extraction rate from grapes into wine. Fortification significantly enhanced rotundone extraction rate, and improved wine colour and phenolics and affects the concentration of ethyl acetate, 3-methylbutyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, methyl nonanoate, isopentanol and phenylethyl alcohol in the resultant wine. Understanding how ethanol produced during fermentation can change the extraction of skin-bound aroma compounds and the colour and flavour of wine allows greater control of fermentation parameters to produce quality wine. Full article