Fermentation — Open Access Zymology & Zymurgy Journal

Fumaric acid is a chemical building block with many applications, namely in the polymer industry. The fermentative production of fumaric acid from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. The use of existing industrial side-streams as raw-materials within biorefineries potentially enables production costs competitive against current chemical processes, while preventing the use of refined sugars competing with food and feed uses and avoiding purposely grown crops requiring large areas of arable land. However, most industrial side streams contain a diversity of molecules that will add complexity to the purification of fumaric acid from the fermentation broth. A process for the recovery and purification of fumaric acid from a complex fermentation medium containing spent sulfite liquor (SSL) as a carbon source was developed and is herein described. A simple two-stage precipitation procedure, involving separation unit operations, pH and temperature manipulation and polishing through the removal of contaminants with activated carbon, allowed for the recovery of fumaric acid with 68.3% recovery yield with specifications meeting the requirements of the polymer industry. Further, process integration opportunities were implemented that allowed minimizing the generation of waste streams containing fumaric acid, which enabled increasing the yield to 81.4% while keeping the product specifications. Full article

Potato protein liquor (PPL), a side stream from the potato starch industry, is normally used as fertilizer. However, with more than 100 g/L of sugars, 20 g/L of Kjeldahl nitrogen and Chemical Oxigen Demand (COD) of 300 g/L, it represents serious environmental challenges. The use of PPL for fungal cultivation is a promising solution to convert this waste into valuable products. In this study, PPL was characterized and used to cultivate edible zygomycete Rhizopus oryzae, which is widely used in Southeast Asian cuisine to prepare e.g., tempeh. Moreover, it can be potentially used as a protein source in animal feed worldwide. Under the best conditions, 65.47 ± 2.91 g of fungal biomass per litre of PPL was obtained in airlift bioreactors. The total Kjeldahl nitrogen content of the biomass was above 70 g/kg dry biomass. The best results showed 51% reduction of COD and 98.7% reduction in the total sugar content of PPL. Full article

The aim of this study was to evaluate the yeast diversity and physicochemical characteristics of spontaneous coffee bean fermentation conducted in the coffee-producing region in Cerrado Mineiro, Brazil. During 48 h of fermentation, yeast population increased from 6.60 to 7.89 log CFU·g⁻¹, with concomitant pulp sugar consumption and organic acids production (mainly lactic (3.35 g·L⁻¹) and acetic (1.27 g·L⁻¹) acids). According to ITS-rRNA gene sequencing, yeast population was mainly represented by Saccharomyces sp., followed by Torulaspora delbrueckii, Pichia kluyveri, Hanseniaspora uvarum, H. vineae and Meyerozyma caribbica. SPME-GC-MS analysis revealed a total of 25 volatile organic compounds with predominance of hydrocarbons (9 compounds) and higher alcohols (6 compounds). The resulting fermented, roasted coffee beans were analyzed by diverse chemical analysis methods, including Fourier Transform Infrared (FTIR) spectroscopy and mineral and thermogravimetric analysis. The thermal decomposition of the coffee beans occurred in four stages between 90 and 390 °C, with significant mass loss (68%) after the second stage at 190 °C. FTIR spectroscopy confirmed the presence of the main organic functions associated with coffee aroma, such as aromatic acids, ketones, aldehydes and aliphatic esters. The results presented in this study enrich our knowledge concerning yeast diversity and physicochemical characteristics associated with coffee bean fermentation, and can be used to promote a controlled on-farm processing. Full article

Volatile fatty acids (VFAs) can be readily produced from many anaerobic microbes and subsequently utilized as precursors to renewable biofuels and biochemicals. Megasphaera elsdenii represents a promising host for production of VFAs, butyric acid (BA) and hexanoic acid (HA). However, due to the toxicity of these acids, product removal via an extractive fermentation system is required to achieve high titers and productivities. Here, we examine multiple aspects of extractive separations to produce BA and HA from glucose and lignocellulosic hydrolysate with M. elsdenii. A mixture of oleyl alcohol and 10% (v/v) trioctylamine was selected as an extraction solvent due to its insignificant inhibitory effect on the bacteria. Batch extractive fermentations were conducted in the pH range of 5.0 to 6.5 to select the best cell growth rate and extraction efficiency combination. Subsequently, fed-batch pertractive fermentations were run over 230 h, demonstrating high BA and HA concentrations in the extracted fraction (57.2 g/L from ~190 g/L glucose) and productivity (0.26 g/L/h). To our knowledge, these are the highest combined acid titers and productivity values reported for M. elsdenii and bacterial mono-cultures from sugars. Lastly, the production of BA and HA (up to 17 g/L) from lignocellulosic sugars was demonstrated. Full article

As a promising alternative of fossil fuel, ethanol has been widely used. In recent years, much attention has been devoted to bioethanol production from lignocellulosic biomass. In previous research, it is found that the pretreatment method named low-moisture anhydrous ammonia (LMAA) has the advantage of high conversion efficiency and less washing requirements. The purpose of this study was to explore the optimal conditions by employing the LMAA pretreatment method. Corn stover was treated under three levels of moisture content: 20, 50, 80 w.b.% (wet basis), and three levels of particle size: <0.09, 0.09–2, >2 mm; it was also ammoniated with a loading rate of 0.1g NH₃/g biomass (dry matter). Ammoniated corn stover was then subjected to different pretreatment times (24, 96, 168 h) and temperatures (20, 75, 130 °C). After pretreatment, compositional analysis and enzymatic digestibility were conducted to determine the highest glucose yield. As a result, the highest glucose yield was obtained under the condition of 96 h and 75 °C with 50 w.b.% and 0.09–2 mm of corn stover. The main findings of this study could improve the efficiency of bioethanol production processing in the near future. Full article

Pyruvic acid (pyruvate) is a cellular metabolite found at the biochemical junction of glycolysis and the tricarboxylic acid cycle. Pyruvate is used in food, cosmetics, pharmaceutical and agricultural applications. Microbial production of pyruvate from either yeast or bacteria relies on restricting the natural catabolism of pyruvate, while also limiting the accumulation of the numerous potential by-products. In this review we describe research to improve pyruvate formation which has targeted both strain development and process development. Strain development requires an understanding of carbohydrate metabolism and the many competing enzymes which use pyruvate as a substrate, and it often combines classical mutation/isolation approaches with modern metabolic engineering strategies. Process development requires an understanding of operational modes and their differing effects on microbial growth and product formation. Full article

The present context was investigated to optimize amylase production and cell biomass of poultry-associated Bacillus sp. using a conventional as well as statistical approach. Box-Behnken design (BBD) matrix at N = 29 was employed to optimize four independent variables, selected from one factor at a time (OFAT) technique, for maximum amylase production and biomass yield. The relative activity of crude amylase obtained from the isolate showed stability at high temperature (60 °C) and alkaline condition (pH 9) up to 4 h of incubation, thereby indicating its alkali-tolerant and thermo-stable property. The BBD resulted in enhanced amylase activity of 145.32 U/mL when the basal medium was slightly acidic (pH 6) and kept at a temperature of 35 °C with the shaking speed of 130 rpm, in addition to being incubated for 24 h. The selected factors, when employed with this statistical optimization approach, showed 1.5-fold and 2-fold enhancements in the amylase production and biomass yield respectively compared to the OFAT method. Analysis of variance (ANOVA) revealed high coefficient of determination (R²) of 0.96 to 0.99 for both the responses at significant level (p < 0.05). Three-dimensional response and 2D contour plot of the quadratic model showed interdependent interaction between the effective variables. Long-term thermo-alkali stability of amylase obtained from Bacillus sp. suggested not only its wide applications in pharmaceutical, food and biotechnological industries, but also suggested a potent replacement of existing amylases on the market. Full article

We designed a mini tower fermentor that is suitable to perform adaptive laboratory evolution (ALE) with gravity imposed as selective pressure, and suitable to evolve a weak flocculating industrial brewers’ strain towards a strain with a more extended aggregation phenotype. This phenotype is of particular interest in the brewing industry, since it simplifies yeast removal at the end of the fermentation, and many industrial strains are still not sufficiently flocculent. The flow of particles (yeast cells and flocs) was simulated, and the theoretical retainment advantage of aggregating cells over single cells in the tower fermentor was demonstrated. A desktop stereolithography (SLA) printer was used to construct the mini reactor from transparent methacrylic acid esters resin. The printed structures were biocompatible for yeast growth, and could be sterilised by autoclaving. The flexibility of 3D printing allowed the design to be optimized quickly. During the ALE experiment, yeast flocs were observed within two weeks after the start of the continuous cultivation. The flocs showed a “snowflake” morphology, and were not the result of flocculin interactions, but probably the result of (a) mutation(s) in gene(s) that are involved in the mother/daughter separation process. Full article

Acetone-butanol-ethanol (ABE) fermentation using Clostridium acetobutylicum is a process that can be used to produce butanol, which can be utilized as an alternative to petroleum-based fuels. Immobilization of the bacteria using three different fibrous materials was studied in order to see how to improve the ABE fermentation process. The results were compared to those of non-immobilized bacteria. Modal and charcoal fibers had OD levels below one at 72 h with the butanol concentration reaching 11.0 ± 0.5 and 10.7 ± 0.6 g/L, respectively, each of which were close to the free cell concentration at 11.1 ± 0.4 g/L. This suggests that bacteria can be efficiently immobilized in these fibrous materials. Although an extended lag phase was found in the fermentation time course, this can be easily solved by pre-treating fibrous materials with 3.5% HCl for 12 h. From comparisons with previous studies, data in this study suggests that a hydrophilic surface facilitates the adsorption of C. acetobutylicum. Full article

Aspartic proteases are of significant importance for medicine and biotechnology. In spite of sufficient evidence that many non-Saccharomyces yeasts produce extracellular proteases, previous research has focused on the enzymes of Candida species because of their role as virulence factors. Nowadays, there is also increasing interest for their applications in industrial processes, mainly because of their activities at low pH values. Here, we report the features of new acid proteases isolated from wine-relevant yeasts Metschnikovia pulcherrima and Wickerhamomyces anomalus. To our knowledge, this is the first detailed description of such an enzyme derived from strains of W. anomalus. Deviating to most former studies, we could demonstrate that the yeasts produce these enzymes in a natural substrate (grape juice) during the active growth phase. The enzymes were purified from concentrated grape juice by preparative isoelectric focusing. Biochemical data (maximum activity at ≈ pH 3.0, inhibition by pepstatin A) classify them as aspartic proteases. For W. anomalus 227, this assumption was confirmed by the protein sequence of WaAPR1 determined by LC-MS/MS. The sequence revealed a signal peptide for secretion, as well as a peptidase A1 domain with two aspartate residues in the active site. The enzyme has a calculated molecular mass of 47 kDa and an isolelectric point of 4.11. Full article

Many studies have been published lately verifying the probiotic character of kefir grains. Most of them focused on the benefits to human health through the consumption of fermented food with kefir grains. However, the challenge is to characterize and isolate specific probiotic microorganisms involved in the kefir microbiota. The main reason for this is that the food industry prefers to apply isolated probiotic microorganisms from kefir grains rather than kefir grains in order to produce respective fermented products with added value. Thus, modern molecular techniques such as polymerase chain reaction (PCR)-based amplification, new generation sequencing (NGS) or denaturing gradient gel electrophoresis (DGGE) analyses have been applied. Furthermore, this review emphasizes the latest outcomes regarding the health benefits of the consumption of foods fermented with kefir grains and particularly the isolation of microorganisms from kefir grains worldwide, some of them exhibiting probiotic properties. Full article

The sparkling wine market has expanded in recent years, boosted by the increasing demand of the global market. As for other fermented beverages, technological yeasts and bacteria selected to design commercial starter cultures represent key levers to maximize product quality and safety. The increasing economic interest in the sector of sparkling wine has also implied a renewed interest in microbial resource management. In this review, after a brief introduction, we report an overview of the main characterization criteria in order to select Saccharomyces cerevisiae strains suitable for use as starter cultures for the production of base wines and to drive re-fermentation of base wines to obtain sparkling wines. Particular attention has been reserved to the technological characterization aspects of re-fermenting phenotypes. We also analysed the possible uses of selected non-Saccharomyces and malolactic strains in order to differentiate specific productions. Finally, we highlighted the main safety aspects related to microbes of enological interest and underlined some microbial-based biotechnological applications helpful to pursue product and process innovations. Overall, the sparkling wine industry may find a relevant benefit from the exploitation of the wide resources associated with vineyard/wine microbial diversity. Full article

(1) Background: Probiotication is an important method in the food industry, and the use of probiotic dairy products has prevented lactose intolerant patients and vegetarians from their consumption. Hence, there is a need to incorporate probiotics in fruit juice without lactose; (2) Method: Probiotic viability, and physicochemical and sensory evaluation of stored probioticated pineapple juice using lactic acid bacteria (LAB) (Pediococcus pentosaceus LaG1, Lactobacillus rhamnosus GG, Pediococcus pentosaceus LBF2) as a single and mixed starter was investigated; (3) Results: There was an increase in the lactic acid production, and reduction in pH, vitamin C content, and colour during storage. At weeks 3 and 4, Propp2 and Pcontrol samples had the highest lactic acid content (317.9 mg/L and 160.34 mg/L). The vitamin C content ranged from 2.91–7.10 mg/100 g. There was a general reduction in total soluble solids during storage. The probiotic LAB were viable throughout the storage time (1.05–1.10 × 10⁹ cfu/mL) in the juice samples. There was no significant difference in terms of taste, aroma, colour, or appearance during the time of storage; (4) Conclusion: The pineapple juice supported the viability, lactic acid production, vitamin C development, and the antagonistic potential of the probiotic candidate. This result is useful for the development of probiotic fruit juice as functional foods and nutraceuticals with health beneficial effect. Full article

Pretreatment is the requisite step for the bioconversion of lignocellulosics. Since most of the pretreatment strategies are cost/energy intensive and environmentally hazardous, there is a need for the development of an environment-friendly pretreatment process. An ionic liquid (IL) based pretreatment approach has recently emerged as the most appropriate one as it can be accomplished under ambient process conditions. However, IL-pretreated biomass needs extensive washing prior to enzymatic saccharification as the enzymes may be inhibited by the residual IL. This necessitated the exploration of IL-stable saccharification enzymes (cellulases). Current study aims at optimizing the bioprocess variables viz. carbon/nitrogen sources, medium pH and fermentation time, by using a Design of Experiments approach for achieving enhanced production of ionic liquid tolerant cellulase from a bacterial isolate Bacillus subtilis SV1. The cellulase production was increased by 1.41-fold as compared to that under unoptimized conditions. IL-stable cellulase was employed for saccharification of IL (1-ethyl-3-methylimidazolium methanesulfonate) pretreated pine needle biomass in a newly designed bioprocess named as “one pot consolidated bioprocess” (OPCB), and a saccharification efficiency of 65.9% was obtained. Consolidated bioprocesses, i.e., OPCB, offer numerous techno-economic advantages over conventional multistep processes, and may potentially pave the way for successful biorefining of biomass to biofuel, and other commercial products. Full article

Yeast nanobiotechnology is a recent field where nanotechniques are used to manipulate and analyse yeast cells and cell constituents at the nanoscale. The aim of this review is to give an overview and discuss nanobiotechnological analysis and manipulation techniques that have been particularly applied to yeast cells. These techniques have mostly been applied to the model yeasts Saccharomyces cerevisiae and Schizosaccaromyces pombe, and the pathogenic model yeast Candida albicans. Nanoscale imaging techniques, such as Atomic Force Microscopy (AFM), super-resolution fluorescence microscopy, and electron microscopy (scanning electron microscopy (SEM), transmission electron microscopy (TEM), including electron tomography) are reviewed and discussed. Other nano-analysis methods include single-molecule and single-cell force spectroscopy and the AFM-cantilever-based nanomotion analysis of living cells. Next, an overview is given on nano/microtechniques to pattern and manipulate yeast cells. Finally, direct contact cell manipulation methods, such as AFM-based single cell manipulation and micropipette manipulation of yeast cells, as well as non-contact cell manipulation techniques, such as optical, electrical, and magnetic cells manipulation methods are reviewed. Full article

Specific vitamin requirements of the wine spoilage yeast, Brettanomyces bruxellensis, were evaluated. Previous studies had not taken into influences of ethanol or nutrient carry-over between sequential transfers into vitamin-omitted media. Knowing nutritional needs, limiting growth of the yeast in wine by selective removal of important vitamins was investigated. Six strains of B. bruxellensis were grown and sequentially transferred into single vitamin-omitted media. None of the strains required p-aminobenzoic acid, folic acid, nicotinic acid, myo-inositol, pantothenic acid, or riboflavin. While some needed thiamin depending on the absence/presence of ethanol, growth of all strains was greatly affected by biotin. Here, concentrations ≥0.2 µg/L were required to achieve yeast populations >10⁶ cfu/mL for high (10⁴ cfu/mL) or low (10² cfu/mL) initial inoculums. At concentrations <0.2 µg/L, culturabilities either remained unchanged or increased less than two logs after 40 days. Since the protein avidin binds irreversibly to biotin, egg whites containing avidin or the purified protein were added to a wine to diminish bioavailability of the vitamin. While biotin concentrations were reduced, populations of B. bruxellensis achieved were decreased by one to three logs, thereby supporting further development of biotin depletion strategies in winemaking. Full article

This study evaluated batch fermentation modes, namely, separate hydrolysis and fermentation (SHF), quasi-simultaneous saccharification and fermentation (Q-SSF), and simultaneous saccharification and fermentation (SSF), and fermentation conditions, i.e., enzyme and yeast loadings, nutrient supplementation and sterilization, on high titer bioethanol production from SPORL-pretreated Douglas-fir forest residue without detoxification. The results indicated that Q-SSF and SSF were obviously superior to SHF operation in terms of ethanol yield. Enzyme loading had a strong positive correlation with ethanol yield in the range studied. Nutrient supplementation and sterility were not necessary for ethanol production from SPORL-pretreated Douglas-fir. Yeast loading had no substantial influence on ethanol yield for typical SSF conditions. After 96 h fermentation at 38 °C on shake flask at 150 rpm, terminal ethanol titer of 43.2 g/L, or 75.1% theoretical based on untreated feedstock glucan, mannan, and xylan content was achieved, when SSF was conducted at whole slurry solids loading of 15% with enzyme and yeast loading of 20 FPU/g glucan and 1.8 g/kg (wet), respectively, without nutrition supplementation and sterilization. It is believed that with mechanical mixing, enzyme loading can be reduced without reducing ethanol yield with extended fermentation duration. Full article

Fermentative biohydrogen production has been flagged as a future alternative energy source due to its various socio-economical benefits. Currently, its production is hindered by the low yield. In this work, modelling and optimization of fermentative biohydrogen producing operational setpoint conditions was carried out. A box-behnken design was used to generate twenty-nine batch experiments. The experimental data were used to produce a quadratic polynomial model which was subjected to analysis of variance (ANOVA) to evaluate its statistical significance. The quadratic polynomial model had a coefficient of determination (R²) of 0.7895. The optimum setpoint obtained were potato-waste concentration 39.56 g/L, pH 5.56, temperature 37.87 °C, and fermentation time 82.58 h, predicting a biohydrogen production response of 537.5 mL H₂/g TVS. A validation experiment gave 603.5 mL H₂/g TVS resulting to a 12% increase. The R² was above 0.7 implying the model was adequate to navigate the optimization space. Therefore, these findings demonstrated the feasibility of conducting optimized biohydrogen fermentation processes using response surface methodology. Full article