Fermentation, Volume 10, Issue 5 (May 2024) – 20 articles

Anaerobic digestion of waste-activated sludge (WAS) towards biogas recovery is constrained by the limited hydrolysis and inhibited acetogenesis steps that hinder subsequent energy recovery. This study employed Fe(VI)/S(IV) oxidation to enhance the WAS solubilization and coupled it with the syntrophic interaction of hydrogen-producing acetogen (HPA) and sulfate-reducing bacteria (SRB) to stimulate the successive procedure towards methane production. Results showed that the dosage ratio of HPA-SRB to WAS (H-S-W) with 1:1:50 outperformed with the highest methane production potential (11.63 ± 1.87 mL CH₄/(g VSS·d). Meanwhile, the efficient and sequential process from acetogenesis to methanogenesis stimulated by HPA-SRB was evidenced by a significant decrease of 30.2% in the acetate concentration. The microbial community structure further manifested the crucial role of HPA-SRB with increased abundance of Desulfobulbus (2.07%), Syntrophomonas (1.24%) and Smithella (1.63%), which stimulated acetophilic methanogen boost with Methanobacterium dominating with 77.51% in H-S-W₁₀₀. Furthermore, the positive syntrophic relationships among HPA-SRB and acetophilic methanogens towards methane production were confirmed via molecular ecological network and canonical correspondence analysis. This study highlighted the syntrophic cooperation of the mixed consortia of HPA and SRB on methane production based on Fe(VI)/S(IV) pretreatment and provided the theoretical and technical basis for the potential implementation of novel methanogenesis technology for WAS treatment. Full article

The effects of pineapple residue (PR) on fermentation quality, chemical composition, and bacterial community of corn straw (CS) silage were evaluated. CS was ensiled with 0% control group (CON), 15% (P1), 30% (P2), and 45% (P3) PR on a fresh matter (FM) basis for 45 days. P3 had lower dry matter (DM) and crude protein (CP) contents but higher ammonia-N (NH₃-N) content than the other three groups (p < 0.05). Compared with the other groups, P1 had lower a pH and higher lactic acid and acetic acid contents (p < 0.05). The lactic acid bacteria count in P1 was higher than in P2 and P3 (p < 0.05); the number of yeast in P2 was higher than in the other groups (p < 0.05). With the increasing proportion of PR addition, the relative abundance of Lactobacillus gradually increased, and the dominant genus in P3 was Acetobacter. In summary, the addition of PR can improve the quality of CS silage, and the optimum addition ratio for PR was 15% on a FM basis. Full article

The endo-1,4-β-xylanases (EC 3.2.1.8) are the largest group of hydrolytic enzymes that degrade xylan, the major component of hemicelluloses, by catalyzing the hydrolysis of glycosidic bonds β-1,4 in this polymer, releasing xylooligosaccharides of different sizes. Xylanases have considerable potential in producing bread, animal feed, food, beverages, xylitol, and bioethanol. The fungus Aspergillus tamarii Kita produced xylanases in Adams’ media supplemented with barley bagasse (brewer’s spent grains), a by-product from brewery industries. The culture extract exhibited two xylanase activities in the zymogram, identified by mass spectrometry as glycosyl hydrolase (GH) families 10 and 11 (GH 10 and GH 11). The central composite design (CCD) showed excellent predictive capacity for xylanase production (23.083 U mL⁻¹). Additionally, other enzyme activities took place during the submerged fermentation. Moreover, enzymatic saccharification based on a mixture design (MD) of three different lignocellulosic residues was helpful in the production of fermentable sugars by the A. tamarii Kita crude extract. Full article

A key goal in current research on industrial curdlan production is the expansion of carbon sources for fermentation. In this study, a recombinant bacterial strain, sp-AmyAXCC, capable of fermenting and synthesizing curdlan using dextrin as a carbon source, was produced via heterologous expression of IPTG-inducible α-amylase from Xanthomonas campestris NRRL B-1459 in Agrobacterium tumefaciens ATCC31749. External expression of the enzyme was confirmed by western blotting, and the expression levels of exogenous proteins during the fermentation process were monitored. Additionally, the properties of the curdlan product were characterized using attenuated total reflectance-Fourier transform infrared spectroscopy and X-ray diffraction. The recombinant strain produced curdlan at a titer of 30.40 ± 0.14 g/L, gel strength of 703.5 ± 34.2 g/cm², and a molecular weight of 3.58 × 10⁶ Da, which is 33% greater than the molecular weight of native curdlan (2.69 × 10⁶ Da). In the batch fermentation of sp-AmyAXCC with 12% dextrin as a carbon source, the titer of curdlan was 66.7 g/L with a yield of 0.56 g/g, and a productivity rate of 0.62 g/L/h at 108 h. The results of this study expand the substrate spectrum for Agrobacterium fermentation in curdlan production and provides guidance for further industrialization of curdlan production. Full article

Polyamide is a thermoplastic polymer widely used for several applications, including cables in offshore oil and gas operations. Due to its growing annual production worldwide, this poorly biodegradable material has been a source of pollution. Given this scenario, the need has arisen to develop environmentally friendly techniques to degrade this waste, and biotechnology has emerged as a possible solution to mitigate this problem. This study aimed to investigate the potential of Yarrowia lipolytica to biodepolymerize polyamide fibers (PAF). Microbial cultures were grown in shaken flasks containing different concentrations of PAF (0.5 and 2 g·L⁻¹) and in a bioreactor with and without pH adjustment. PAF mass loss was up to 16.8%, achieved after 96 h of cultivation in a bioreactor without pH adjustment. Additionally, NMR analyses revealed that the amorphous regions of PAF, which are more susceptible to depolymerization, were reduced by 6% during cultivation. These preliminary results indicate the biotechnological potential of Y. lipolytica to depolymerize PAF. Full article

This research investigates how different fermentation techniques using non-Saccharomyces yeast (Candida ethanolica Ce, Hanseniaspora guilliermondii Hg, Hanseniaspora thailandica Ht) and Saccharomyces cerevisiae (Sc) affect the synthesis of hesperidin, nobiletin, and other flavonoid and aromatic substances, which play a vital role in improving the overall quality of fruit wines due to their various biological properties. The combination of Sc:(Ce.Ht)-1:100 (Ce 0.5 × 10⁷ CFU/mL, Ht 0.5 × 10⁷ CFU/mL, Sc 1 × 10⁵ CFU/mL) yielded the highest hesperidin content at 4.12 ± 0.08 mg/L, followed by the Sc:(Ce.Hg)-1:1 (Ce 0.5 × 10⁷ CFU/mL, Hg 0.5 × 10⁷ CFU/mL, Sc 1 × 10⁷ CFU/mL) combination at 4.08 ± 0.06 mg/L. The highest nobiletin content was achieved by the (Hg.Ht)-10-Sc (Hg 0.5 × 10⁷ CFU/mL, Ht 0.5 × 10⁷ CFU/mL, Sc 1 × 10⁷ CFU/mL) combination, reaching 1.04 ± 0.05 mg/L, which was significantly higher than other multi-strain combinations. Additionally, the hesperidin content produced by the (Hg.Ht)-10-Sc combination was relatively high at 4.04 ± 0.02 mg/L, demonstrating a richness and complexity of aroma superior to that of fermentation with commercial yeast strains alone. The findings suggest that the (Hg.Ht)-10-Sc combination is the most effective multi-strain combination for increasing the levels of nobiletin and hesperidin in citrus wine, thereby enhancing the overall quality of the wine. These experimental results offer a promising approach for enhancing the quality of citrus wines and other fruit wines. Full article

Proteins are an abundant biopolymer in organic waste feedstocks for biorefining. When degraded, amino acids are released, but their fate in non-methanogenic microbiomes is not well understood. The ability of a microbiome obtained from an anaerobic digester to produce volatile fatty acids from the twenty proteinogenic amino acids was tested using batch experiments. Batch tests were conducted using an initial concentration of each amino acid of 9000 mg COD L⁻¹ along with 9000 mg COD L⁻¹ acetate. Butyrate production was observed from lysine, glutamate, and serine fermentation. Lesser amounts of propionate, iso-butyrate, and iso-valerate were also observed from individual amino acids. Based on 16S rRNA gene amplicon sequencing, Anaerostignum, Intestimonas, Aminipila, and Oscillibacter all likely play a role in the conversion of amino acids to butyrate. The specific roles of other abundant taxa, including Coprothermobacter, Fervidobacterium, Desulfovibrio, and Wolinella, remain unknown, but these genera should be studied for their role in fermentation of amino acids and proteins to VFAs. Full article

Sorghum (Sorghum bicolour (L.) Moench) is an emerging reliable alternative for mahewu production. The aim of this study was to evaluate the health-promoting compounds, physicochemical and microbiological properties of sorghum-based mahewu produced by different traditional brewers in Thohoyandou, South Africa. A total of 18 mahewu samples produced by six traditional brewers (TB1–TB6) were collected and compared for antioxidant, physicochemical, and microbiological properties. Commercial sorghum mahewu was used as a control sample. The total phenolic content of the mahewu samples varied from 27.37 to 65.89 GAE /g, with commercial mahewu having a lower value. The flavonoid content ranged from 0.18 to 0.30 GAE/g, and commercial mahewu had a higher value. The DPPH scavenging activity and FRAP of mahewu samples ranged from 44.62% to 49% and 1.47 to 2.36 mg GAE/g, respectively. Commercial mahewu had a higher DPPH value but a lower FRAP value. The pH of mahewu varied significantly, ranging from 3.38 to 3.66, but was within the acceptable range. The °Brix values varied from 9.68 to 17.49, with traditional mahewu samples having higher values than commercial mahewu. Total titratable acidity ranged from 0.63 to 1.17%. The viscosity ranged from 444.33 to 1297.00 cP, with commercial mahewu having a higher value. There was a significant variation in the color of the mahewu samples with respect to L*, a*, b*, C, Hue, and ΔΕ. The growth of yeasts and molds varied from 7.95 log₁₀ to 8.99 log₁₀ (cfu/mL) in traditional mahewu samples, and coliforms ranged from 3.68 to 5.96 log₁₀ (cfu/mL) and were not isolated in commercial mahewu. The total plate count ranged from 7.914 to 8.978 log₁₀ (cfu/mL). The microbiological results show that traditional brewers are meeting the legal limit and can increase their products for commercialization. Full article

The specific sensory properties attributed to distillates from different plum varieties are intricately linked to aromatic substances, fruit quality, and technology employed during production. This study compares four plum brandies, each made from a renowned plum variety: Presenta, Valjevka, Čačanská lepotica, and Čačanská rodná on a production scale. Analytical and sensory profiles were assessed using GC-FID, an available analytical method advantageous for monitoring industrial fruit distillate production. Between 71 and 85 analytes were detected in the distillates, with the Presenta plum distillate containing the highest number of substances. Statistically significant differences in analyte concentration between plum varieties (p < 0.05) were observed for 11 analytes. The comparison of analytical profiles and sensory evaluation revealed that a high concentration of 1-propanol, despite its negative sensory perception, significantly impacts the overall perception of a distillate, contrasting with substances like acetaldehyde and propyl acetate, which have positive sensory evaluations but lesser significance in content. Our work identified key compounds and procedures that can be used as benchmarks for production of plum brandy with positive sensory evaluation. These findings demonstrate the broad application potential of GC-FID in fruit distillate production as an independent tool for aromatic profile assessment and quality control. Full article

The use of machine learning and deep learning has become prominent within various fields of bioprocessing for countless modeling and prediction tasks. Previous reviews have emphasized machine learning applications in various fields of bioprocessing, including biomanufacturing. This comprehensive review highlights many of the different machine learning and multivariate analysis techniques that have been utilized within Chinese hamster ovary cell biomanufacturing, specifically due to their rising significance in the industry. Applications of machine and deep learning within other bioprocessing industries are also briefly discussed. Full article

Silage is the most important component of a ruminant diet and has important production and health significance in ruminant production. The aim of the research was to investigate how the mixed silage of Chinese cabbage waste and rice straw (mixed silage) impacts the fecal microorganisms and metabolites in Hu sheep using Illumina sequencing and metabolomic analysis. A total of 16 Hu sheep (8 rams and 8 ewes) weighing about 39 kg and 5.5 months old were used as experimental sheep and divided into two groups (4 rams and 4 ewes, n = 8) using the principle of randomized trials: the control group with peanut sprouts, corn husks, and sorghum husks as roughage and the silage group with the mixed silage as roughage. There were no significant differences in the average daily gain (ADG), dry matter intake (DMI), or feed conversion rate (FCR) between the control group and the mixed silage groups (p > 0.05). Microbiome results showed that 15 microorganisms such as Ruminococcaceae UCG 010, Breznakia, Erysipelothrix, Desulfovibrio, Succiniclasticum, and Shuttleworthia were significantly different between the two groups. In addition, metabolomics showed that the mixed silage modulated the concentrations and metabolic pathways of metabolites in the manure. Significantly different metabolites were mainly enriched in amino acid anabolism (“glycine, serine, and threonine metabolism”, “valine, leucine, and isoleucine biosynthesis”, “arginine biosynthesis”, etc.), nucleic acid metabolism (pyrimidine metabolism). In conclusion, the addition of mixed silage to the diet of Hu sheep can alter the structure of the hindgut microflora and regulate the metabolism of amino acids and nucleotides, which affects health performance. Full article

The discovery and biotechnological application of new antimicrobial peptides are impeded by a lack of sensitive methods for peptide quantification. Paenibacillin is an emerging antimicrobial lantibiotic that was discovered in Paenibacillus polymyxa OSY-DF ATCC PTA-7852, isolated from the fermented vegetable Kimchee. This lantibiotic has potency against many foodborne pathogenic and spoilage bacteria. To advance the research and application of paenibacillin, a rapid, specific, and sensitive detection and quantification immunoassay was developed. After anti-paenibacillin polyclonal antibodies (pAbs) were generated and purified, a competitive enzyme-linked immunosorbent assay (cELISA) was developed and optimized for paenibacillin quantification. The dynamic range of the cELISA was determined by using a three-parameter nonlinear regression model, achieving a correlation (R²) value of 0.95. The cELISA displayed high sensitivity, with the ability to detect paenibacillin at levels as low as 15.6 ng/mL, which is significantly lower than the limit of detection of the conventional antimicrobial assay (20 µg/mL paenibacillin). The cELISA successfully differentiated paenibacillin concentrations in cell-free crude supernatants of P. polymyxa wild type and its mutant strain when grown at 30 °C and 37 °C; higher paenibacillin levels were found in the mutant (0.248–0.276 µg/mL) than in the wild type (0.122–0.212 µg/mL) culture. These findings were validated by the transcriptional analysis of 11 paenibacillin biosynthetic genes, which were significantly upregulated (≥2-fold increase) in the mutant compared with the wild strain. Additionally, the cELISA exhibited high sensitivity by recovery of paenibacillin titers spiked at 2.5 and 10 µg/mL in de Man, Rogosa, and Sharpe (MRS) broth and diluted skim milk. These results suggest that the anti-paenibacillin pAbs and the developed cELISA could be valuable in quantifying paenibacillin in complex matrices and in aiding the discovery of paenibacillin-producing natural microbiota. Full article

The required processes and steps for making bread include technological and innovative concepts. The current trend is the use of less toxic compounds and green methods. Besides lactic acid bacteria and yeast, other microorganisms with unique properties, such as enzymes, new aromas and flavors, exopolysaccharides, and vitamins, among other compounds with beneficial properties, could be added to bread manufacture, improving bread quality and health effects for the consumers. The preservation of microbial cultures and starters is crucial in bread-making. New encapsulation methods, cryoprotectants, spray-drying, fluidized bed drying, and vacuum drying are employed for microorganism cultures that will be used as starters or biological additives in fermentation. A development is observed in the antimicrobial methods used as bread preservatives, and studies with plant extracts and essential oils have been proposed and introduced, replacing chemical agents, such as propionate, within the clean-label bread formulations concept. Baking science is a growing research line that incorporates innovative methods, biological additives, new methods, and processes focusing on microbiological protection. Full article

The enzyme-based degradation of lignocellulose for bioenergy production is an eco-friendly and sustainable approach. This study aimed to elucidate the enzymatic characteristics of endoglucanase (EGL), β-glucosidase (BGL), and xylanase (XYN) from Trichoderma guizhouence NJAU4742, and to explore the potential mechanisms underlying their synergistic degradation of different natural substrates. The results demonstrated that the three enzymes possessed remarkable high-temperature catalytic activity, broad pH adaptability, and responsiveness to different metal ions. The functional group absorption peaks of different substrates were shifted and altered after the synergistic action, particularly for C=O and O-H. Simultaneously, the crystallinity index of wheat straw, soybean straw, rice straw, and corn straw decreased by 7.40%, 2.37%, 20.60%, and 7.67%, respectively, compared to CK (natural straw). Additionally, the dense structure of different substrates was destroyed, and the inner parenchyma began to be exposed after the synergistic action, as observed by SEM. These findings offer valuable theoretical guidance for the development of lignocellulase applications. Full article

To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca²⁺, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca²⁺ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca²⁺ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca²⁺ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency. Full article

Cocoa is the third most important global agricultural export commodity. However, because it is a crop sensitive to climatic change, there has been an active search for cocoa substitutes worldwide. Roasted jackfruit seeds were previously described as having a chocolate aroma and are affordable and accessible. In this study, we characterized and identified by SPME-GC-O and SPME-GC-MS the aroma profile of cocoa-based beverages formulated with jackfruit seed flour as a natural cocoa substitute. Our analysis tentatively identified 71 odor-active aroma descriptors with some similarities between formulations. Overall, 15 odor-active aromas were present in all beverages. The formulation containing only cocoa/chocolate showed the following aroma descriptors: cocoa, hazelnut, peanut butter, earthy, and roast, which are mostly related to the presence of 2,3-dimethylpyrazine and 2,3-diethyl-5-methylpyrazine. The fermented beverage had a content of complex pyrazines such as 2,3,5-trimethyl-6-isopentylpyrazine and methylpropylpyrazine. Our data indicated that both the control and fermented beverages showed a similar aromatic profile, mainly earthy, pyrazine, and chocolate. Qualitative similarities in the pyrazine content were observed between the fermented jackfruit seed flour and cocoa beverages. In conclusion, fermented jackfruit seed flour can be incorporated into cocoa-based beverages as a natural chocolate substitute, offering the potential to elevate the chocolate aroma. Full article

The abundance of fruit waste from the food industry and wineries, particularly peels, seeds, and other fruit pomace throughout the year, could lead to health and environmental hazards if not channelled into productive areas. Improving or transforming these waste products for better use in other vital sectors could be achieved via solid-state fermentation (SSF) since most waste products are solid. One such productive and important area is the feeding of livestock, which will guarantee millennium food security goals for many nations of the world. The nutritional and antioxidant composition of abundantly available fruit pomace and agro-industrial byproducts could be improved via solid-state fermentation for overall livestock productivity. They contain substantial dietary fibre, protein, and phenolic compounds; hence, improving them via fermentation could serve the livestock industry in dual capacities, including nutraceutical and conventional feedstuff. This review seeks to provide reinforcing evidence on the applicability and impact of fruit pomaces on livestock nutrition. The significant nutrient improvements, beneficial outcomes in feeding trials, and inconsistencies or areas of research gap were also explored. Full article

Cervical cancer is caused by a persistent and high-grade infection. It is caused by the Human Papillomavirus (HPV), which, when entering cervical cells, alters their physiology and generates serious lesions. HPV 18 is among those most involved in carcinogenesis in this region, but there are still no drug treatments that cause cure or total remission of lesions caused by HPV. It is known that L-asparaginase is an amidohydrolase, which plays a significant role in the pharmaceutical industry, particularly in the treatment of specific cancers. Due to its antitumor properties, some studies have demonstrated its cytotoxic effect against cervical cancer cells. However, the commercial version of this enzyme has side effects, such as hypersensitivity, allergic reactions, and silent inactivation due to the formation of antibodies. To mitigate these adverse effects, several alternatives have been explored, including the use of L-asparaginase from other microbiological sources, which is the case with the use of the fungus Aspergillus niger, a high producer of L-asparaginase. The study investigated the influence of the type of fermentation, precipitant, purification, characterization, and in vitro cytotoxicity of L-asparaginase. The results revealed that semisolid fermentation produced higher enzymatic activity and protein concentration of A. niger. The characterized enzyme showed excellent stability at pH 9.0, temperature of 50 °C, resistance to surfactants and metallic ions, and an increase in enzymatic activity with the organic solvent ethanol. Furthermore, it exhibited low cytotoxicity in GM and RAW cells and significant cytotoxicity in HeLa cells. These findings indicate that L-asparaginase derived from A. niger may be a promising alternative for pharmaceutical production. Its attributes, including stability, activity, and low toxicity in healthy cells, suggest that this modified enzyme could overcome challenges associated with antitumor therapy. Full article

Innovation in the beer market focuses on research into the different ingredients that make up this popular and sought-after beverage. Some of these innovations have focused on the use of autochthonous ingredients, which bring added value to products, reduce production costs and environmental impact, and provide beers with special organoleptic characteristics. This study focuses on the use of autochthonous ingredients from the Community of Madrid and the design of four recipes that are suited to these ingredients and consumer demand. In terms of the ingredients used, the micromalting of barley grown in the CM was carried out, while hops (Cascade variety for bittering hops and wild hops Rayo verde Tajuña and Torreta Tajuña for aroma) and yeast strains (Saccharomyces cerevisiae G 520 and G 354) were selected from previous studies, which were also sourced from the CM. For the barley malt, parameters such as protein content were determined, which was at the maximum limit but did not affect the final quality of the beers. The content of α- and β-acids in the hops was also determined, being higher in the Cascade variety. Different physicochemical analyses (GC, aromatic compounds, bitterness, colour, SO₂, polyphenols, and antioxidant capacity) were carried out to determine the quality of the beers obtained from the four recipes designed, as well as tastings by a panel of experts and consumers. Finally, two recipes were rated the best by a panel of experts and consumers, where the cereal, hop, and fruit flavours stood out for their aromatic profile. Full article

The use of whole-cell biocatalysts in microbial cell factories is of great interest to produce added-value compounds. Through large-scale fermentative processes, which use secondary raw materials as substrates, it is possible to recycle and upgrade agro-industrial by-products. This review addresses the main fermentative processes and bioreactors currently used for the valorization of orange peel, a by-product of the Citrus processing industry. Among the main added-value products, bioethanol, organic acids, enzymes, single cell proteins (SCPs), dyes and aromatic compounds have been industrially produced using orange peel via solid state fermentation and submerged fermentation. This approach fits within the circular economy goals in terms of clean technology and renewable energy, valorization and recycling, upgrade of industrial by-products and sustainability. Full article