Fermentation, Volume 9, Issue 12 (December 2023) – 37 articles

Amino acids have relevance in biorefining as fermentation nutrients but also as valued coproducts obtainable from plant biomass. Soy flour was studied as a representative low-cost protein source requiring hydrolysis to free primary amino acids for utilization. Within the context of biorefining, process schemes, reactant concentrations, times, and temperatures were varied to explore the efficiency of dilute sulfuric acid hydrolysis of soy flour to release amino acids. Two process strategies were optimized. Either soy flour was co-processed with switchgrass biomass using a dilute-acid pretreatment, or it was hydrolyzed alone with dilute acid. Significant improvement to hydrolysate fermentability was accomplished by adding 2.5–10 g/L soy flour to switchgrass pretreatment with dilute sulfuric acid (0.936% v/v) for 15 min at 160 °C. This practice optimized accumulation of neutral sugars and resulted in a 25% reduction in furfural while boosting xylose 7% and up to doubling primary amino nitrogen (PAN), as compared to no soy flour addition to switchgrass pretreatment. When soy flour was hydrolyzed alone, PAN titers were optimized to 1588 mg N/L (9.9 g amino acids/L) and yield to 0.0529 g PAN/g flour (61% of theoretical) using a 10% (v/v) (1.8 M) sulfuric acid hydrolysis 30 min at 160 °C. Full article

The natural fermentation of antibiotics, along with semi-synthetic and synthetic approaches, is one of the most important methods for their production. The majority of the antibiotic market comes from the fermentation of high-yielding (HY) fungal strains. These strains have been obtained since the 1950s from wild-type (WT) isolates as a result of classical strain improvement (CSI) programs primarily involving multi-round random mutagenesis and screening. However, the molecular basis leading to high-yield production was unknown. In recent years, due to the application of multiomic approaches, key changes that occur in CSI programs, with WT strains that become HY industrial producers of a particular antibiotic, have begun to be understood. It becomes obvious that, during CSI, certain universal events are selected, which lead both to a direct increase in the production of the target metabolite and affect other vital processes of the cell (side mutations). These key events include: the upregulation of the target biosynthetic gene cluster (BGC), changes in the system of global regulation, disruption of alternative BGCs, the rearrangement of energy fluxes in favor of the target SM (secondary metabolite), changes in the regulation of the response to stress, and the redirection of primary metabolic pathways to obtain more precursors for target production. This knowledge opens up the possibility of both introducing targeted changes using genetic engineering methods when creating new producers and increasing the production of CSI strains as a result of fermentation with low-molecular compounds, targeted to compensate for the effects of side mutations. Full article

Succinic acid (SA), a C4 tricarboxylic acid cycle intermediate, is used as raw material for bulk chemicals and specialty chemicals, such as tetrahydrofuran and 1,4-butanediol, as well as also being used to synthesize the biodegradable biopolymers PBS (polymer poly (butylene succinate)). Actinobacillus succinogenes, which is facultative anaerobic and gram-negative, is one of the most promising natural SA-producing organisms, but genetic engineering of A. succinogenes is rare so far. In this study, a series of engineered strains was constructed using the pLGZ922 expression vector and a cytosine base editor (CBE) based on CRIPSR/Cas9; we found that phosphoenolpyruvate carboxylase (PEPC) was more important for the CO₂ fixation pathway than pyruvate carboxylase (PYC) in A. succinogenes, and the annotated oxaloacetic acid decarboxylase (Asuc_0301 and Asuc_0302) had little correlation with the SA synthesis pathway. The by-product pathway was closely related to cell growth, and overexpression of FDH was beneficial to growth, while the knockout of the ackA gene reduced the growth. For the first time, the hypothetic sugars and SA transporters were mined and identified in A. succinogenes, of which Asuc_0914 was responsible for glucose uptake, and Asuc_0715 and Asuc_0716 constituted SA exporters. This deepens the understanding of SA biosynthesis in A. succinogenes and is also valuable for SA production by fermentation. Full article

We investigated the effects of the moisture content and silage starter preparation on the fermentation quality, nutritional value, and in vitro digestibility of waxy corn processing byproducts and rice bran (WRB) mixed silage and waxy corn processing byproducts and rice polished powder (WRPP) mixed silage. Two mixed silages with 55%, 60%, and 65% moisture content (MC) were set up without any additives (control) or with former Lactobacillus (L), and opened on the 60th day after storage the fermentation quality, nutritive value, and in vitro digestibility of the silages in each treatment. The optimal formulation of high-quality waxy corn processing byproduct (WCPP) silage was screened to provide a reference and theoretical basis for the further development and utilisation of WCPPs. The results showed that the proportions of ammonia nitrogen to total nitrogen (AN/TN) and acid detergent lignin (ADL) significantly decreased with a decreasing MC, whereas the levels of lactic acid (LA), crude protein (CP), dry matter (DM), and in vitro crude protein digestibility (IVCPD) significantly increased (p < 0.05) for both mixed silages with L. After treatment with 60% MC, the content of neutral detergent fibre (NDF) was significantly lower, and the CP content was significantly greater in the WRB mixed silage treated with L (p < 0.05). With 55% MC, the addition of L not only reduced the pH and AN/TN ratio of the two mixed silages but also significantly improved their in vitro digestibility (p < 0.05). Studies have shown that reducing the MC of silage raw materials and adding L allows for the preparation of high-quality silage. Full article

Soy (tofu) whey is one of the two side-streams from tofu processing, which has been shown to be suitable for microbial growth. In this work, probiotic Bifidobacterium animalis subsp. lactis Bl-04 and B94 were used to ferment soy whey with different supplements to explore the possibility of harnessing Bifidobacterium fermentation to upcycle soy whey. Soy whey was supplemented with different supplements (control, CN; glucose, G; glucose + cysteine, GC; glucose + cysteine + yeast extract, GCY) and inoculated with either B. lactis Bl-04 or B94. Growth, substrate utilization, and metabolic products were monitored before and after fermentation. Bl-04 managed to grow in all four media, while B94 needed cysteine to grow. The contents of sucrose, cysteine, methionine, and succinic acid decreased in the fermented samples. Acetic and lactic acids were produced in fermented soy whey ranging from 0.49–2.66 g/L and 0.58–2.88 g/L, respectively, with vitamin B₁₂ at 2.06–4.56 μg/L. Increases in isoflavone aglycones (0.19–25.05 mg/L) and iron (0.03–0.12 mg/L) were observed. The PCA analysis of volatiles showed a distinct clustering due to short-chain fatty acids (acetic, butyric, and isovaleric acid), 2,3-butanedione (diacetyl), H₂S, and 3-methylthiophene. Overall, the selection of suitable bifidobacterial strains and supplements for soy whey fermentation can open avenues to upcycle soy whey. Full article

This work focuses on the establishment of analytical and sensory differences between young wines obtained from the same grape must fermented using different strategies. The main purpose is to provide winemakers with objective criteria to help them to choose the best fermentation method to obtain the desired wine characteristics. The effects of four strategies were tested: a spontaneous fermentation with wild yeasts (WYs) and the addition of starter cultures of Lachancea thermotolerans (LT), Metschnikowia pulcherrima (MP), and a Saccharomyces cerevisiae strain that is an overproducer of glutathione (SC) in different batches of the same must of the Pedro Ximénez white grape. The analytical results obtained show as the LT wine has the highest glutathione content, while the lowest ethanol concentrations. The use of chemometric tools applied to the volatile compounds allowed to differentiate the four wines by a principal component analysis (PCA) and the identification of 27 key compounds. The four wines did not show statistical differences in their smell or taste attributes and only the LT wine was visually differentiated from the rest. Full article

Metal chloride salt pretreatment was performed to isolate and convert cellulose to glucose from poplar. A glucose yield of 82.0% ± 0.7 was achieved after 0.05 mol/L AlCl₃ pretreatment conducted at 180 °C for 20 min, ascribing to the removal of hemicellulose, the alteration of crystallinity, surface morphology, and the retention of the majority of cellulose. Then, the influence of different additives on glucose yield was assessed, generating the highest glucose yield of 88.5 ± 0.06 with the addition of PEG 8000. Meanwhile, a similar glucose yield of 82.8% ± 0.3 could be obtained with PEG 8000 when hydrolysis time was reduced by a quarter and enzyme dosage by three-quarters. It can be seen that AlCl₃ pretreatment is a viable and efficient pretreatment method for poplar, while the addition of PEG 8000 can enhance the enzymatic efficiency and reduce cellulase loading, ascribing to the reservation of free enzyme and enzyme activity in the supernatant and the reduction in surface tension, which provide an idea to improve the economics of the enzymatic conversion of poplar. Full article

This study investigates the impact of inoculating Torulaspora delbrueckii (Td) strains during the initial phase of carbonic maceration (CM) vinification, aiming to enhance the fermentative process and unique characteristics of CM wines. CM is a winemaking technique where whole bunches (without destemming and crushing) are enclosed in tanks filled with carbon dioxide, inducing intracellular fermentation. This study compares the effects of two Td strains on the MLF performance and sensory characteristics of CM wines using both inoculated and spontaneous MLF strategies. Although general physicochemical parameters remained consistent across conditions, organoleptic attributes showed significant differences due to T. delbrueckii presence. T. delbrueckii introduction during CM resulted in wines with increased anthocyanin content and a particular volatile profile. Isoamyl acetate, a key aroma in CM wines, was notably elevated, especially in the TdP strain. Sensory evaluations also revealed distinctions, with TdV wines displaying more pronounced aromas of red fruit, banana, and grass. Regarding MLF, T. delbrueckii presence notably enhanced performance, particularly in spontaneous MLF cases, accelerating fermentation completion. Inoculating the Oenococcus oeni strain OoVP41 also shortened MLF duration. These findings highlight the potential of Td strains to improve MLF efficiency and sensory attributes in CM wines. Using T. delbrueckii strains strategically enables winemakers to optimize MLF and improve sensory profiles, offering an opportunity to produce higher-quality CM wines. Full article

The extracted olive pomace (EOP) is an industrial lignocellulosic by-product of olive pomace oil extraction, currently mainly used for energy production through combustion. In this work, the hemicellulosic fraction of EOP was selectively hydrolyzed by diluted acid hydrolysis to obtain pentose-rich hydrolysates that can potentially be upgraded by Debaryomyces hansenii, targeting xylitol production. The monosaccharides and degradation by-products released along the pre-treatment were quantified and several detoxification methods for the removal of potentially toxic compounds were evaluated, including pH adjustment to 5.5, the use of anion-exchange resins, adsorption into activated charcoal, concentration by evaporation, and membrane techniques, i.e., nanofiltration. The latter approach was shown to be the best method allowing the full removal of furfural, 41% of 5-hydroxymethylfurfural, 54% of acetic acid, and 67% of the phenolic compounds present in the hydrolysate. The effects of the supplementation of both non-detoxified and detoxified hydrolysates were also assessed. The non-detoxified hydrolysate, under aerobic conditions, supported the yeast growth and xylitol production at low levels. Supplementation with the low-cost corn steep liquor of the nanofiltration detoxified hydrolysate showed a higher xylitol yield (0.57 g/g) compared to the non-detoxified hydrolysate. The highest xylitol productivity was found in hydrolysate detoxified with anionic resins (0.30 g/L·h), which was 80% higher than in the non-detoxified culture medium. Overall, the results showed that EOP dilute acid hydrolysates can efficiently be used for xylitol production by D. hansenii if detoxification, and supplementation, even with low-cost supplements, are performed. Full article

Lactic acid fermentation is one of the oldest and most commonly used methods of bioconservation. This process is widely used for food preservation and also for a production technique that relies on the metabolism of lactic acid bacteria (LAB) to convert carbohydrates into lactic acid. This fermentation imparts unique flavors and texture of foods, extends their shelf life, and can offer health benefits. There are both traditional and new microorganisms involved in the lactic acid fermentation of food. The current review outlines the issues of fermented foods. Based on traditional fermentation methods, a broad panorama of various food products is presented, with the microorganisms involved. The methods of both traditional fermentation (spontaneous and back-slopping) as well as the importance and application of starter cultures in mass food production are presented. Currently, based on the results of scientific research, the health-promoting effect of fermented foods is becoming more and more important. This is due to the presence of probiotic microorganisms that are naturally presented or may be added to them, as starter cultures or additives, and from the presence of prebiotics and postbiotics. New innovative methods of using probiotic microorganisms open up new and broad perspectives for fermented functional foods. Full article

During the production of nattokinase (NK) by Bacillus species, certain by-products such as poly-γ-glutamic acid (γ-PGA) are simultaneously synthesized. The impact of γ-PGA synthesis on NK production remains unclear. In this study, we knocked out the pgsC gene, a component of the γ-PGA synthetase cluster (pgsBCA), and constructed a novel recombinant strain, Bacillus licheniformis BL11. Next, we compared the fed-batch fermentation profiles of BL11 and its parental strain BL10, conducted transcriptional analysis, and measured intracellular ATP content. We also optimized glucose-feeding strategies under varying oxygen supply conditions. Our results indicated that the utilization rates of glucose and soybean meal were both improved in the pgsC-deficient strain BL11, and NK activity was enhanced. Furthermore, the transcriptional levels of genes involved in glycolysis and the TCA cycle were relatively upregulated in BL11. The maximal NK activity reached 2522.2 FU/mL at 54 h of fermentation using a constant glucose-feeding rate of 5.0 g/(L·h) under high oxygen supply conditions. The newly developed recombinant strain B. licheniformis BL11, along with the optimized feeding strategy, shows promise for large-scale NK production. Full article

This study investigated the synergistic effects of combining Saccharomyces cerevisiae and Oenococcus oeni during the alcoholic fermentation of a low-acidity cider. The initial population of indigenous wild lactic acid bacteria (LAB) in the apple must was 10⁴ CFU/mL. Alcoholic fermentations were carried out without (Cider I) and with (Cider II) the O. oeni inoculation at 10⁵ CFU/mL. As S. cerevisiae grows, a declining trend was observed in indigenous and inoculated LAB populations. While the wild LAB exhibited higher sensitivity than O. oeni, they were not eliminated during alcoholic fermentation. The addition of O. oeni impacted the growth and metabolic activity of S. cerevisiae. The bioconversion of malic acid into lactic acid predominantly occurred during the growth phase (43–66%) and stationary phase (4–27%). The resurgence of O. oeni following alcoholic fermentation significantly impacted the production of volatile compounds. After 20 days of fermentation, Cider II displayed a twofold increase in these compounds, resulting in a more favorable sensory profile according to evaluators. Consequently, malolactic fermentation (MLF) coincided with alcoholic fermentation, leading to a reduction in malic acid content. Furthermore, post alcoholic fermentation, MLF positively enhanced the aromatic quality of low-acid cider made from apples with low acidity. Full article

Phosphatidylserine (PS) is a natural phospholipid with particular importance in the food, cosmetic, and pharmaceutical industries. Recently, the synthesis of PS mediated by phospholipase D (PLD) has drawn great attention. But the application of free PLD is limited by various drawbacks, including its instability under extreme conditions, difficulties in reuse and recovery, and high costs. In this work, saPLD-inorganic hybrid nanoflowers (saPLD@NFs) were synthesized with PLD from Streptomyces antibioticus (saPLD) as the organic component and Ca₃(PO₄)₂ as the inorganic component. The saPLD@NFs demonstrated outstanding immobilization capability and achieved a 119% enzyme activity recovery rate. Furthermore, the saPLD@NFs exhibited better thermostability and pH stability in comparison to free saPLD. The PS yield of saPLD@NFs was about 57.4% in the first cycles and still reached 60.4% of its initial PS yield after four cycles. After 25 d storage at 4 °C, saPLD@NFs retained 66.5% of its original activity, but free saPLD only retained 38.3%, indicating that saPLD@NFs have excellent storage stability. Thus, this study established a new method of preparing PLD nanoflowers for effective PS synthesis, which might accelerate the practical utilization of this biocatalyst. Full article

A model predictive control (MPC) method was investigated as a route to optimize and control the growth of E. coli BL21 (DE3) and biosynthesis of two different recombinant proteins (nerve growth factor NGF and coat protein of bacteriophage Qβ (Qβ-CP)). To determine the target trajectory for the E. coli cultivation process and estimate the model parameters, the off-line run-to-run optimization method was used. The proven method allowed us to successfully control the growth of microbial biomass, with a deviation of 6–12% from the target trajectory. It was proven that it is possible to obtain a “Golden Batch” profile for the implementation of MPC using datasets from only four to eight fermentation runs. The method showed its robustness when the cultivation of E. coli was carried out with two different titrant supply control systems—volumetric and gravimetric. Furthermore, the MPC method exhibited high adaptability, reliability, and resistance to various types of disturbances. MPC proved to be a reliable and effective method for controlling the cultivation and recombinant protein biosynthesis of fast-growing microorganisms such as E. coli. Full article

In the present investigation, the potential of various newly isolated strains which belong to the Enterobacteriaceae family to produce 2,3-butanediol (BDO), an important bio-based compound, was studied. The most interesting strain, namely Klebsiella oxytoca FMCC-197, was selected for further investigation. Commercial (raw) sucrose or molasses, which are important agro-industrial surpluses, were employed as carbon sources for most of the trials performed. Different fermentation parameters (viz. incubation te4mperature, utilization of different carbon sources, substrate inhibition, aeration) were tested to optimize the process. Fermentations under non-aseptic conditions were also conducted to investigate the potential of growth of the strain K. oxytoca FMCC-197 to surpass the growth of other microorganisms in the culture medium and produce BDO. Besides BDO production, in trials in which molasses was employed as the sole carbon source, significant color removal was observed simultaneously with the production of microbial metabolites. The very high BDO concentration ≈115 g L⁻¹ was reported in approximately 64 h during a fed-batch bioreactor experiment, using sucrose and molasses as carbon sources at 30 °C, reaching a conversion yield (Y_BDO) of 0.40 g g⁻¹ and a productivity rate (P_BDO) of 1.80 g L⁻¹ h⁻¹, while similar results were also obtained at 37 °C. The strain demonstrated remarkable results in non-previously sterilized media, as it produced 58.0 g L⁻¹ in 62 h during a fed-batch bioreactor experiment, while the potential to decolorize molasses-based substrates over 40% was also recorded. From the results obtained it is shown that this wild-type strain can be used in large-scale microbial BDO production using various raw materials as fermentative substrates. The wastewater derived after BDO fermentation by K. oxytoca FMCC-197 can be disposed relatively safely into the environment. Full article

The world climate crisis has triggered the search for renewable energy sources. Oleaginous yeasts are a potential renewable source of biofuels. However, the yeast-derived biofuels cost is still non-competitive with the fossil fuel prices. To improve the sustainability of yeast-derived biofuels, it is necessary to valorize all yeast biomass fractions, an approach based on the biorefinery concept. This review describes the present situation of the oleaginous yeast biorefinery research, emphasizing the feedstock, processes and techniques involved in this concept, as well as on potential bioproducts that can be obtained from oleaginous yeast biomass. Full article

This study focuses on improving the second fermentation and the in-bottle ageing over lees process for 9 and 21 months of a red sparkling wine. The aim of the study was to enhance wine quality and try to make it more pleasant for consumers. For this purpose, four different yeast derivatives (yeast walls, yeast walls with tannins, inactivated yeasts and mannoproteins) and β-glucanases were added to a red base sparkling wine and were aged over lees during two different periods: 9 and 21 months. Oenological parameters, total polysaccharides, total proteins, free amino nitrogen, phenolic composition, foaming properties, and volatile compounds were analysed in conjunction with a sensory evaluation. Results show the different incidences of the studied adjuvants on the final parameters, highlighting the importance of the interactions of the studied adjuvants over time. In fact, time itself turned out to be one of the main factors affecting the final characteristics of the wine, so influencing consumers’ opinions and modifying the wine’s acceptability and quality. Full article

The cultivation and overall quality of Litchi, a fruit of significant commercial value in China, are hindered by the presence of the oomycetes pathogen Peronophythora litchii. This pathogen is responsible for the occurrence of litchi downy blight, resulting in substantial economic losses during the storage and transportation of the fruit, and affects nutritional growth. Effective and environmentally safe methods to control litchi downy blight are urgently needed. The application of biocontrol agents such as Streptomyces bacteria has proven effective for controlling plant diseases. Our present study isolated the Streptomyces strain TJGA-19, identified as S. abikoensis, with potent inhibitory activity against P. litchii. The antifungal active substances are mainly in the aqueous phase of TJGA-19 fermentation filtrate extraction. The fermentation filtrate of TJGA-19 not only suppressed the pathogen growth, sporulation, and sporangia germination, but also delayed the disease development of litchi downy blight. In addition, the stability of the TJGA-19 fermentation filtrate was not sensitive to the proteinase K, temperature, white-flourescence light, or ultraviolet treatment. Furthermore, the morphology and ultrastructure of P. litchii treated with fermentation filtrate was characterized by marked shrinking and deformation, with serious disruption of plasma membrane permeabilization and the organelles. Hence, S. abikoensis TJGA-19 and its metabolites demonstrated marked efficiency against the phytopathogenic pathogen P. litchii and provide a potential candidate for controlling litchi downy blight. Full article

In this study, an algal–bacterial symbiotic consortium was integrated with the sediment microbial fuel cell (SMFC) to construct an algal–bacterial cathode SMFC (AC-SMFC) for excess sewage sludge treatment and electricity generation. A bacterial cathode SMFC (BC-SMFC) and a static settling system (SS-system) were used as controls. Electrochemical analysis confirmed that the algal–bacterial biofilm on the cathode improved electricity production. The maximum power density of AC-SMFC was 75.21 mW/m², which was 65.70% higher than that of the BC-SMFC (45.39 mW/m²). After 60 days of treatment, AC-SMFC achieved much higher removal efficiencies of the total chemical oxygen demand (TCOD) (59.60%), suspended solids (SS) (62.42%), and volatile suspended solids (VSS) (71.44%) in the sediment, compared to BC-SMFC and the SS-system, exhibiting an effective degradation of the organic matter in the sediment sludge. Moreover, the lower concentration of total nitrogen (TN) and total phosphorus (TP) in the overlying water of AC-SMFC demonstrated that the algae on the cathode could inhibit the accumulation of nitrogen and phosphorus released from the sediments. The three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy revealed that the tryptophan protein and aromatic protein in the loosely bound extracellular polymeric substances (LB-EPS) of the sediment sludge in the AC-SMFC were significantly decreased. Additionally, the abundance of functional microbiota in the AC-SMFC increased, such as Trichococcus, Alphaproteobacteria, and Clostridia, which contributed to electricity generation and sludge degradation. The combined application of microalgae and the SMFC provided a promising approach for excess sludge reduction and energy recovery. Full article

With the addition of Tween 80, potassium hydroxide pretreatment and enzymatic hydrolysis were statistically optimized to maximize sugar yields from corn cob (CC). The results indicated that the sugar yields from CC could be influenced significantly by the potassium hydroxide concentration, temperature and time during pretreatment. The optimized pretreatment conditions were as follows: potassium hydroxide, 46 g·L⁻¹; Tween 80, 3.0 g·L⁻¹; solid dose, 200 g·L⁻¹; temperature, 78 °C; and time, 50 min. After optimization, the lignin reduction and recoveries of cellulose and hemicellulose were 89.7%, 97.8% and 68.0%, respectively. In addition, sugar production could also be influenced by the biomass loading, enzyme loading and reaction time. A maximal glucose production (518.48 mg·g_ds⁻¹, milligrams per gram of dry substrate) and xylose production (351.14 mg·g_ds⁻¹), 97.2% cellulose conversion and 82.9% hemicellulose conversion from CC could be obtained when the biomass loading was 195 g·L⁻¹ and the enzyme loading was 8.9 FPU·g_ds⁻¹ (filter paper activity units per gram of dry substrate) and when the Tween 80 concentration was 3.0 g·L⁻¹ at 50 °C for 30.4 h during hydrolysis. This is the first systematic study of combined Tween 80 pretreatment of CC by potassium hydroxide and hydrolysis of CC by cellulase preparation to increase sugar production. Full article

Brewers’ spent grain (BSG) constitutes the primary by-product of the brewing industry. The valorization of BSG from a circular economy perspective is of high industrial interest. The objective of this study was the exploitation of BSG for the microbial production of branched-chain fatty acids (BCFAs) and polyunsaturated fatty acids (PUFAs), representing two different classes of high-value fatty acids (FAs). In the present study, this waste material underwent treatment with hot water in an autoclave and the resultant extract was utilized for the preparation of a novel liquid medium (BSG medium) to be employed for microbial fermentation. Screening and subsequent scaling-up experiments confirmed the suitability of the BSG medium to support the microbial production of various high-value FAs. In particular, Streptomyces jeddahensis and Conidiobolus heterosporus could be employed for BCFAs production, Pythium ultimum and Mortierella alpina could be used to provide cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and arachidonic acid (ARA), whereas Mucor circinelloides, when grown in a BSG medium, was able to accumulate γ-linolenic acid (GLA). Full article

A continuing interest of scientists regarding the development of new β-carotene production technologies is due to the high biological activity of this compound and its wide application range. Bacteria are considered among the possible β-carotene producers convenient for industrial use. The purpose of this study was to develop a Mycolicibacterium neoaurum strain with an enhanced ability for β-carotene production and to optimize the fermentation conditions to improve the final yield of the target compound. Using chemical mutagenesis with N-nitroso-N-methylurea along with further strain selection, a M. neoaurum strain Ac-501/22, whose productivity was 2.7-fold higher than that of the parental strain Ac-501, was developed. The effect of nitrogen and carbon sources as well as cultivation conditions on the growth of M. neoaurum Ac-501/22 and β-carotene production were studied to select the optimal fermentation regime. Due to an increase in the temperature of cultivation from 30 to 35 °C, replacement of glucose with glycerin (20.0 g/L) and degreased soybean flour with powdered milk (10.0 g/L), and increase in the urea content from 0.5 to 1.0 g/L, the level of β-carotene production was improved to 183.0 mg/kg that was 35% higher than in the control. Further strain fermentation in a 3 L bioreactor using an optimized medium with the pH level maintained at 7.0–7.2 and 50% pO₂ provided the maximum output of the target compound (262.4 mg/kg of dry biomass) that confirmed the prospects of the developed strain as an industrial β-carotene producer. Full article

Traditional dairy products, especially cheeses, represent part of the cultural food heritage of many countries. In addition, these cheeses constitute microbiological “reservoirs”, of which many have been lost due to the introduction of the pasteurization of milk in the dairy industry. Increased awareness of the importance of microorganisms that make up the biodiversity of traditional cheeses, as well as the development of molecular methods in recent decades, have enabled efforts to identify and preserve them. Traditional Livno cheese is a full-fat hard cheese, considered one of the most famous traditional cheeses of Bosnia and Herzegovina and is seasonally produced from a mixture of raw sheep’s milk supplemented with cow’s milk. Often, Livno cheese has variable quality, due to microbial contamination and poor milk quality. In this study, traditional Livno cheese was studied during the ripening of cheeses produced by different producers during two seasons. Culture-dependent analyses were made during ripening using microbiological plating on suitable media. Likewise, culture-independent methods Denaturing Gradient Gel Electrophoresis (DGGE) and Automated Ribosomal Intergenic Spacer Analysis (ARISA) were used to elucidate the cheese microbiota. Results of analysis showed Lactococcus spp., Streptococcus spp., Lactobacillus spp., Pediococcus spp. and Leuconostoc spp. to be dominant species in traditional Livno cheese. However, when comparing the use of culture-dependent and culture-independent methods in the evaluation of Livno cheese microbiota, Enterococcus was not detected by culture-independent DGGE methods. The microbial population of both the milk and the environment determines the fermentation processes during cheese production and ripening, and thereby defines the quality of this cheese. The numbers of bacteria in the cheese were shown to be dependent on the manufacturer, the degree of ripening and the production season. Full article

Bamei pigs, an eximious local breed reared on the Tibetan Plateau of China, are facing problems such as feed shortages, weaning stress, and antibiotic abuse. This study aimed to improve the quality of feed, growth performance, intestinal microbiota, and immunity of Bamei pigs through feeding with potentially probiotic-fermented feed. Different feeds were administered to weaned Bamei piglets for 60 days, creating the following five experimental treatment groups: basal feed group; Lactiplantibacillus plantarum-fermented-feed group; Bacillus subtilis-fermented-feed group; mixed-fermented-feed group; and antibiotic-supplemented-feed group. The results showed that the pH, neutral detergent fiber, and acid detergent fiber of the potentially probiotic-fermented feeds were significantly reduced; organic acids were produced; and Coliform bacteria, Clostridium, and aerobic bacteria were effectively inhibited. Feeding with potentially probiotic-fermented feed not only reduced the feed-conversion ratio but also improved immunity by increasing the production of anti-inflammatory cytokines, as well as decreasing pro-inflammatory cytokines and typical inflammatory pathways. The 16s rDNA high-throughput-sequencing results showed that probiotic-fermented feed improved the diversity of intestinal microbiota, inhibited the growth of the opportunistic pathogens Clostridium and Streptococcus, increased the relative abundance of Lactobacillus and Prevotella, and promoted gut health, demonstrating the promising application prospects of potentially probiotic-fermented feed. Full article

Recently, the extensive spread of some algae along coastlines has surged into unmanageable thick decomposition layers. This study aimed to demonstrate the use of Sargassum horneri as a biomass resource for ethanol production through the continuous hydrolysis, enzymatic saccharification, and fermentation process. Sugars from S. horneri were obtained using a combination of thermal acid hydrolysis and enzymatic saccharification. The optimal conditions for thermal acid hydrolysis involved a 10% (w/v) S. horneri slurry treated with 100 mM H₂SO₄ at 121 °C for 60 min; enzymatic saccharification using 16 U/mL Cellic CTec2 further boosted the monosaccharide concentration to 23.53 g/L. Fermentation experiments were conducted with mannitol-adapted Saccharomyces cerevisiae BY4741 using S. horneri hydrolysate. Enhanced ethanol production was observed in the hydrolysate, particularly with mannitol-adapted S. cerevisiae BY4741, which yielded 10.06 g/L ethanol. Non-adapted S. cerevisiae produced 8.12 g/L ethanol, as it primarily utilized glucose and not mannitol. Regarding ethanol fermentation using 5 L- and 500 L-scale fermenters, the ethanol concentrations reached 10.56 g/L and 7.88 g/L with yields of 0.51 and 0.45, respectively, at 48 h. This study confirmed the economic viability of ethanol production using waste seaweed with optimized pretreatment conditions and the adaptive evolution of S. cerevisiae to mannitol. Full article

High production cost is one of the major factors that limit the market growth of polyhydroxyalkanoates (PHAs) as a biopolymer. Improving PHA synthesis performance and utilizing low-grade feedstocks are two logical strategies for reducing costs. As an oleaginous yeast, Y. lipolytica has a high carbon flux through acetyl-CoA (the main PHB precursor), which makes it a desired cell factory for PHB biosynthesis. In the current study, two different metabolic pathways (NBC and ABC) were introduced into Y. lipolytica PO1f for synthesizing PHB. Compared to the ABC pathway, the NBC pathway, which includes NphT7 to redirect the lipogenesis pathway and catalyze acetoacetyl-CoA synthesis in a more energy-favored reaction, led to PHB accumulation of up to 11% of cell dry weight (CDW), whereas the ABC pathway resulted in non-detectable accumulations of PHB. Further modifications of the strain with the NBC pathway through peroxisomal compartmentalization and gene dose overexpression reached 41% PHB of CDW and a growth rate of 0.227 h⁻¹. A low growth rate was observed with acetate as the sole source of carbon and energy or glucose as the sole substrate at high concentrations. Using a co-substrate strategy helped overcoming the inhibitory and toxic effects of both substrates. Cultivating the engineered strain in the optimal co-substrate condition predicted by response surface methodology (RSM) led to 83.4 g/L of biomass concentration and 31.7 g/L of PHB. These results offer insight into a more cost-effective production of PHB with engineered Y. lipolytica. Full article

In the evolving landscape of sustainable biopharmaceutical process development, the utilization of bacteria in the production of various compounds via fermentation has attracted extensive attention from scientists. A successful fermentation process and the release of its associated products hinge on the synergy between an efficient bacterial strain and the formulation of a suitable growth medium. Balancing all nutrient levels of a growth medium to maximize microbial growth and the product quality is quite an intricate task. In this context, significant advancements have been achieved via the strategic implementation of design-of-experiment (DOE) methodologies and the utilization of parallel microbioreactor systems. This work presents a case study of the fermentation growth medium optimization of a Gram-negative bacterium of the Neisseriaceae family that releases outer membrane vesicles (OMVs), which represent a potential vaccine platform. To achieve this, the ability of Sartorius MODDE^®13 DOE software to explore multiple variables and their interactions was combined with the functionality of a Sartorius Ambr^® 15F parallel microbioreactor system. The findings reported in this study have led to the design of a well-suited fermentation medium for a Gram-negative bacterium and an improvement in the quality of the OMVs produced from it. Full article

Trans-2-decenoic acid has a wide range of applications, including those in medicine, food, and health care. Therefore, the industrial production of trans-2-decenoic acid is particularly important. However, few studies have focused on medium-chain unsaturated fatty acids. Therefore, we aimed to optimize the fermentation process of decanoic acid biocatalysis to synthesize trans-2-decenoic acid using an engineered Escherichia coli constructed in the laboratory. Early-stage culture and the effect of the seed liquid culture time, culture temperature, inoculum amount, induction temperature, dissolution effects of the substrate solvent, metal ions, and substrate loading on the titer of trans-2-decenoic acid were evaluated. Based on a single-factor experimental optimization, a Box–Behnken design (BBD) was used for response surface testing using the substrate feeding concentration, inducer concentration, and MnCl₂ concentration as response variables and trans-2-decenoic acid production as the response value. The optimal fermentation process was as follows: Seed culture time of 20 h, culture temperature of 37 °C, inoculation amount of 1%, induction temperature of 30 °C, substrate flow of 0.15 g/L, inducer concentration of 5.60 g/L, and MnCl₂ concentration of 0.10 mM. Under these conditions, the average production of trans-2-decenoic acid was 1.982 ± 0.110 g/L, which was 1.042 g/L higher than that obtained in the basic LB medium. Compared with that of the previous period, the titer of the trans-2-decenoic acid studied increased by 1.501 ± 0.110 g/L, providing a basis for further research on the fermentation process of the biocatalytic decanoic acid synthesis of trans-2-decenoic acid. Full article

Vitamin C, a water-soluble vitamin with strong reducing power, cannot be synthesized by the human body and participates in a variety of important biochemical reactions. Vitamin C is widely used in the pharmaceutical, food, health care, beverage, cosmetics, and feed industries, with a huge market demand. The classical two-step fermentation method is the mainstream technology for vitamin C production. D-sorbitol is transformed into L-sorbose by Gluconobacter oxydans in the first step of fermentation; then, L-sorbose is transformed into 2-keto-L-gulonic acid (2-KGA) by a coculture system composed of Ketogulonicigenium vulgare and associated bacteria; and finally, 2-KGA is transformed into vitamin C through chemical transformation. The conversion of L-sorbose into 2-KGA in the second fermentation step is performed by K. vulgare. However, considering the slow growth and low 2-KGA production of K. vulgare when cultured alone, it is necessary to add an associated bacteria to stimulate K. vulgare growth and 2-KGA production. Although the mechanism by which the associated bacteria promote K. vulgare growth and 2-KGA production has extensively been studied, this remains a hot topic in related fields. Based on the latest achievements and research, this review summarizes the metabolic characteristics of K. vulgare and associated bacteria and elucidates the mechanism by which the associated bacteria promote the growth and 2-KGA production of K. vulgare. Full article

Several emerging mosquito control technologies require mass releases of adult male mosquitoes. Previous studies resulted in the generation of a laboratory female-specific larvicidal yeast strain targeting the GGT gene, which facilitated the laboratory sex separation of male Culex quinquefasciatus mosquitoes. Global deployment of this yeast-based sex-separation technology requires engineering second generation yeast strains which can be used in industrial-scale fermentations to support global mosquito control programs. In this study, the RNA-guided Cas-CLOVER system was used in combination with piggyBac transposase to generate robust Saccharomyces cerevisiae strains with multiple integrated copies of the insecticidal GGT shRNA expression cassette. Top expressing Cas-CLOVER strains killed Culex quinquefasciatus female larvae which consumed the yeast, facilitating male sex separation. Scaled fermentation resulted in kilogram-scale production of the yeast, which can be heat-killed and dried for global deployment to mosquito mass-rearing facilities. Full article