Lactic Fermentation Research Articles

Fermentation Analytical Technology (FAT): Monitoring industrial E. coli fermentations using absolute quantitative 1H NMR spectroscopy

BackgroundTo perform fast, reproducible, and absolute quantitative measurements in an automated manner has become of paramount importance when monitoring industrial processes, including fermentations. Due to its numerous advantages – including its inherent quantitative nature – Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy provides an ideal tool for the time-resolved monitoring of fermentations. However, analytical conditions, including non-automated sample preparation and long relaxation times (T1) of some metabolites, can significantly lengthen the experimental time and make implementation in an industrial set up unfeasible. ResultsWe present a high throughput method based on Standard Operating Procedures (SOPs) and 1H NMR, which lays the foundation for what we call Fermentation Analytical Technology (FAT). Our method was developed for the accurate absolute quantification of metabolites produced during Escherichia coli industrial fermentations. The method includes: (1) a stopped flow system for non-invasive sample collection followed by sample quenching, (2) automatic robot-assisted sample preparation, (3) fast 1H NMR measurements, (4) metabolites quantification using multivariate curve resolution (MCR), and (5) metabolites absolute quantitation using a novel correction factor (k) to compensate for the short recycle delay (D1) employed in the 1H NMR measurements. The quantification performance was tested using two sample types: buffer solutions of chemical standards and real fermentation samples. Five metabolites – glucose, acetate, alanine, phenylalanine and betaine – were quantified. Absolute quantitation ranged between 0.64 and 3.40 mM in pure buffer, and 0.71–7.76 mM in real samples. SignificanceThe proposed method is generic and can be straight forward implemented to other types of fermentations, such as lactic acid, ethanol and acetic acid fermentations. It provides a high throughput automated solution for monitoring fermentation processes and for quality control through absolute quantification of key metabolites in fermentation broth. It can be easily implemented in an at-line industrial setting, facilitating the optimization of the manufacturing process towards higher yields and more efficient and sustainable use of resources.

Impact of lactic acid bacteria starter inoculation on the Physico-chemical, microbiological, volatiles and sensory properties of natural black Gemlik (Olea europaea L., cv.) table olives

Traditionally, the fermentation of Gemlik table olives takes place spontaneously by the endogenous microbiota in Turkey, but the uncontrolled process during fermentation can result in the formation of off-flavours and a low sensory quality of the final product. In this research, the effects of inoculating naturally black Gemlik olives with Lactiplantibacillus pentosus and Lactiplantibacillus plantarum starter cultures on lactic fermentation development, volatile compounds, and sensory properties were investigated. Changes in spontaneous and culture inoculated olives were monitored during fermentation. Compared to spontaneous fermentation, rapid acidification, and sugar depletion were achieved with the use of starter cultures. Extra lactic acid bacteria and total yeast growth were detected in the inoculated trials during fermentation compared to the control trial. Volatile compounds in olives were extracted by the Purge and Trap method. 46 volatile compounds from 10 chemical groups have been identified in olives. In all three trials, volatile acids were the group of volatile compounds found in the top amount in olives, higher alcohols, phenols, aldehydes/ketones, and terpenes followed the volatile acids. Phenyl ethyl alcohol, α-farnesen, (Z)-3-hexene-1-ol and 2-hexanol, which give pleasant flavors, were determined to be in more quantity in olives with inoculated starter cultures. This study reveals that the use of starter culture provides advantages in the production of Gemlik variety table olives, positively affects aroma development, and improves sensory properties.

Kinetic analysis of lactic acid fermentation by using an extended logistic model

This study aimed to analyze the dynamics of biomass growth, lactic acid production, and glucose consumption for three microorganisms; Weissella cibaria IBUN 090–03684, Lactobacillus pantarum IBUN 090–03774, and Lactobacillus pantarum IBUN 04139 by using an extended logistic model. The microorganisms were cultivated in a 1 L bioreactor with pH control by using MRS medium at 37 ∘C for 24 h at 100 rpm. Weissella cibaria IBUN 090–03684 exhibited the highest product yield (1.26 gg−1), followed by Lactobacillus plantarum IBUN 04139 (0.96 gg−1), and Lactobacillus plantarum IBUN 090–03774 (0.90 gg−1), respectively. The results indicate that Weissella cibaria IBUN 090–03684 holds significant potential for large-scale lactic acid production. Furthermore, the reported modeling strategy fits well with experimental data for the three microorganisms evaluated.

Improvement of physicochemical properties and quercetin delivery ability of fermentation-induced soy protein isolate emulsion gel processed by ultrasound

This study aimed to investigate the effects of ultrasonic treatment at different powers on the physicochemical properties, microstructure and quercetin delivery capacity of fermentation-induced soy protein isolate emulsion gel (FSEG). The FSEG was prepared by subjecting soy protein isolate (SPI) emulsion to ultrasonic treatment at various powers (0, 100, 200, 300, and 400 W), followed by lactic acid bacteria fermentation. Compared with the control group (0 W), the FSEG treated with ultrasound had higher hardness, water holding capacity (WHC) and rheological parameters. Particularly, at an ultrasonic power of 300 W, the FSEG had the highest hardness (101.69 ± 4.67 g) and WHC (75.20 ± 1.07%) (p < 0.05). Analysis of frequency sweep and strain scanning revealed that the storage modulus (G') and yield strains of FSEG increased after 300 W ultrasonic treatment. Additionally, the recovery rate after creep recovery test significantly increased from 18.70 ± 0.49% (0 W) to 58.05 ± 0.54% (300 W) (p < 0.05). Ultrasound treatment also resulted in an increased β-sheet content and the formation of a more compact micro-network structure. This led to a more uniform distribution of oil droplets and reduced mobility of water within the gel. Moreover, ultrasonic treatment significantly enhanced the encapsulation efficiency of quercetin in FSEG from 81.25 ± 0.62 % (0 W) to 90.04 ± 1.54% (300 W). The bioaccessibility of quercetin also increased significantly from 28.90 ± 0.40% (0 W) to 42.58 ± 1.60% (300 W) (p < 0.05). This study enriches the induction method of soy protein emulsion gels and provides some references for the preparation of fermented emulsion gels loaded with active substances.

Crithmum maritimum Extract Restores Lipid Homeostasis and Metabolic Profile of Liver Cancer Cells to a Normal Phenotype

Hepatocellular carcinoma (HCC) is an alarming epidemiological clinical problem worldwide. Pharmacological approaches currently available do not provide adequate responses due to poor effectiveness, high toxicity, and serious side effects. Our previous studies have shown that the wild edible plant Crithmum maritimum L. inhibits the growth of liver cancer cells and promotes liver cell differentiation by reducing lactic acid fermentation (Warburg effect). Here, we aimed to further characterise the effects of C. maritimum on lipid metabolism and markers of cellular metabolic health, such as AMP-activated protein kinase (AMPK), Sirtuin 1 (SIRT1), and Sirtuin 3 (SIRT3), as well as the insulin signalling pathway. To better mimic the biological spectrum of HCC, we employed four HCC cell lines with different degrees of tumorigenicity and lactic acid fermentation/Warburg phenotype. Lipid accumulation was assessed by Oil Red O (ORO) staining, while gene expression was measured by real-time quantitative PCR (RT-qPCR). The activation of AMPK and insulin signalling pathways was determined by Western blotting. Results indicate that C. maritimum prevents lipid accumulation, downregulates lipid and cholesterol biosynthesis, and modulates markers of metabolic health, such as AMPK, SIRT1 and SIRT3. This modulation is different amongst HCC cell lines, revealing an important functional versatility of C. maritimum. Taken together, our findings corroborate the importance of C. maritimum as a valuable nutraceutical, reinforcing its role for the improvement of metabolic health.

Recovery of organic acids from lactic acid-enriched fermentation broth via salting-out assisted solvent extraction

The organic acids (OAs) market has been expanding due to their versatility and wide range of uses in several industrial applications, thus prompting a transition from chemical to biological approaches to generate such high-value products. Nevertheless, downstream processes to separate OAs from the fermentation broth represent, to date, the main cost in biological OAs manufacturing. With the goal of limiting this issue, the present study investigated OAs separation from a lactic acid (LA)-rich fermentation broth through physical, i.e., using ethyl acetate (EA), and physico-chemical, i.e., using tributyl phosphate (TBP), solvent extraction. In addition, the salting-out effect of different salts on OAs recovery was investigated. Among the investigated conditions, a combination of pure TBP and 40% ammonium sulfate ensured the highest LA extraction efficiency of 65%. Besides, mixing TBP with EA (50:50) enabled to maintain the same LA extraction efficiency while reducing the process costs, limiting the toxicity of the chemicals involved, and obtaining an extracted OAs mix with lower co-metabolites, i.e. volatile fatty acids, compared to using pure TBP. Overall, solvent extraction assisted by salting-out agents were shown to be a promising method to separate OAs from a fermentation broth in liquid form, especially for fermentation processes operated at low pH.

Gut Microbiome and Cytokine Profiles in Post-COVID Syndrome.

Recent studies highlight the crucial role of the gut microbiome in post-infectious complications, especially in patients recovering from severe COVID-19. Our research aimed to explore the connection between gut microbiome changes and the cytokine profile of patients with post-COVID syndrome. Using 16S rRNA amplicon sequencing, we analyzed the composition of the gut microbiome in 60 COVID-19 patients over the course of one year. We also measured the levels of serum cytokines and chemokines using the Milliplex system. Our results showed that severe SARS-CoV-2 infection cases, especially those complicated by pneumonia, induce a pro-inflammatory microbial milieu with heightened presence of Bacteroides, Faecalibacterium, and Prevotella_9. Furthermore, we found that post-COVID syndrome is characterized by a cross-correlation of various cytokines and chemokines MDC, IL-1b, Fractalkine, TNFa, FGF-2, EGF, IL-1RA, IFN-a2, IL-10, sCD40L, IL-8, Eotaxin, IL-12p40, and MIP-1b as well as a shift in the gut microbiome towards a pro-inflammatory profile. At the functional level, our analysis revealed associations with post-COVID-19 in homolactic fermentation, pentose phosphate, NAD salvage, and flavin biosynthesis. These findings highlight the intricate interplay between the gut microbiota, their metabolites, and systemic cytokines in shaping post-COVID symptoms. Unraveling the gut microbiome's role in post-infectious complications opens avenues for new treatments for those patients with prolonged symptoms.

Impact of Lactic Acid Bacteria Fermentation on (Poly)Phenolic Profile and In Vitro Antioxidant and Anti-Inflammatory Properties of Herbal Infusions.

Recently, the development of functional beverages has been enhanced to promote health and nutritional well-being. Thus, the fermentation of plant foods with lactic acid bacteria can enhance their antioxidant capacity and others like anti-inflammatory activity, which may depend on the variations in the total content and profile of (poly)phenols. The present study aimed to investigate the impact of fermentation with two strains of Lactiplantibacillus plantarum of several herbal infusions from thyme, rosemary, echinacea, and pomegranate peel on the (poly)phenolic composition and whether lacto-fermentation can contribute to enhance their in vitro antioxidant and anti-inflammatory effects on human colon myofibroblast CCD18-Co cells. HPLC-MS/MS analyses revealed that fermentation increased the content of the phenolics present in all herbal infusions. In vitro analyses indicated that pomegranate infusion showed higher antioxidant and anti-inflammatory effects, followed by thyme, echinacea, and rosemary, based on the total phenolic content. After fermentation, despite increasing the content of phenolics, the antioxidant and anti-inflammatory effects via reduction pro-inflammatory markers (IL-6, IL-8 and PGE2) were similar to those of their corresponding non-fermented infusions, with the exception of a greater reduction in lacto-fermented thyme. Overall, the findings suggest that the consumption of lacto-fermented herbal infusions could be beneficial in alleviating intestinal inflammatory disorders.

Mutualistic interactions of lactate-producing lactobacilli and lactate-utilizing Veillonella dispar: Lactate and glutamate cross-feeding for the enhanced growth and short-chain fatty acid production.

The human gut hosts numerous ecological niches for microbe-microbe and host-microbe interactions. Gut lactate homeostasis in humans is crucial and relies on various bacteria. Veillonella spp., gut lactate-utilizing bacteria, and lactate-producing bacteria were frequently co-isolated. A recent clinical trial has revealed that lactate-producing bacteria in humans cross-feed lactate to Veillonella spp.; however, their interspecies interaction mechanisms remain unclear. Veillonella dispar, an obligate anaerobe commonly found in the human gut and oral cavity, ferments lactate into acetate and propionate. In our study, we investigated the interaction between V. dispar ATCC 17748T and three representative phylogenetically distant strains of lactic acid bacteria, Lactobacillus acidophilus ATCC 4356T, Lacticaseibacillus paracasei subsp. paracasei ATCC 27216T, and Lactiplantibacillus plantarum ATCC 10241. Bacterial growth, viability, metabolism and gene level adaptations during bacterial interaction were examined. V. dispar exhibited the highest degree of mutualism with L. acidophilus. During co-culture of V. dispar with L. acidophilus, both bacteria exhibited enhanced growth and increased viability. V. dispar demonstrated an upregulation of amino acid biosynthesis pathways and the aspartate catabolic pathway. L. acidophilus also showed a considerable number of upregulated genes related to growth and lactate fermentation. Our results support that V. dispar is able to enhance the fermentative capability of L. acidophilus by presumably consuming the produced lactate, and that L. acidophilus cross-feed not only lactate, but also glutamate, to V. dispar during co-culture. The cross-fed glutamate enters the central carbon metabolism in V. dispar. These findings highlight an intricate metabolic relationship characterized by cross-feeding of lactate and glutamate in parallel with considerable gene regulation within both L. acidophilus (lactate-producing) and V. dispar (lactate-utilizing). The mechanisms of mutualistic interactions between a traditional probiotic bacterium and a potential next-generation probiotic bacterium were elucidated in the production of short-chain fatty acids.

The Probiotic Paradox: Thriving in High-Hopped Sour Beer

The global craft beer market is witnessing substantial growth, with a particular spotlight on sour beers. These unique brews are created through lactic fermentation initiated by lactic acid bacteria (LAB). However, the presence of hops (Humulus lupulus L.), renowned for their antimicrobial properties, can pose a limitation within the realm of sour beer production. This research aims to explore and assess the potential of sour beer as a probiotic beverage. It involves a comparative analysis of the viability of Lacticaseibacillus paracasei subsp. paracasei F19 (F19) and 431 (L431) in a sour beer with an International Bitterness Unit (IBU) value of 22, alongside examining the expression of genes such as horA, horB, horC, hitA, bsrA, and recA, which are associated with hop resistance. The study encompasses the development of two formulations of sour beers characterized by both high hop content and a robust probiotic count (8.44–8.77 log CFU/mL). The findings suggest that both probiotic strains of F19 and L431, are well-suited to produce sour beers with elevated hop levels, demonstrating excellent stability. Notably, the expression of genes responsible for hop resistance exhibited distinct modulation patterns among the two strains. It appears that a higher concentration of bsrA, as observed in strain L431, may be more effective in mitigating the effects of hop-related stress, potentially surpassing the combined expression of horA and bsrA. Nonetheless, further research is essential to validate this observation in gene expression. F19 and L431 emerge as promising candidates for the development of sour beers with increased hop intensity.

Producción de fertilizante orgánico líquido utilizando levadura (Saccharomyces cerevisiae) residual del proceso de elaboración de cerveza

El objetivo del estudio fue revalorar la levadura (Saccharomyces cerevisiae), del proceso de elaboración de cerveza, para obtener fertilizante orgánico líquido. El experimento se realizó en dos etapas: en campo, la recolección de materia prima y en el laboratorio, la determinación del porcentaje de humedad y los análisis microbiológicos, fisicoquímicos y agronómicos. Se utilizó un diseño completo al azar (DCA) con arreglo factorial de 4x4 con tres repeticiones. La caracterización de la levadura fue pH: 6,52, CE: 3,60 dS/m, HR relativa: 84,94% y materia orgánica en solución: 66,64%. Mientras que, la caracterización del fertilizante orgánico líquido estuvo en un pH: 3,96, CE: 21,40 dS/m, sólidos totales: 183,30 g/L y materia orgánica en solución: 146,40 g/L. La fermentación láctica realizada por las bacterias ácido-lácticas del consorcio microbiano (B-lac), es una opción para la producción de fertilizante orgánico líquido utilizando levadura residual del proceso de elaboración de cerveza. Mediante la caracterización de levadura, se demostró que la concentración de materia orgánica, el contenido de macronutrientes y micronutrientes de la levadura residual es alto en determinados elementos, tales como; nitrógeno (9681 mg/L), fósforo (2203,70 mg/L) y potasio (3850 mg/L); resultando ser un producto de calidad agronómica.

EVALUATION OF ORGANOLEPTIC &amp;IN-VITRO BIOAVAILABILITY PROPERTIES OF IRON FORTIFIED YOGURT

Yogurt is a fermented dairy product formed by the action of Lactobacillus spp. with the help of lactic acid fermentation procedure.In India major cause of anaemia is dietary iron deficiency.Iron deficiency causes poor cognitive performance, behavioural abnormalities, poor physical growth of children, disturbed immune function, lack of physical activity and poor work performance in all age groups. Worldwide dairy foods are most popular nutritious product so dairy foods may be used as a vehicle of food fortification. The iron compound which are used as a food fortificant should contain highly bioavailable iron and the physical chemical characteristics of the fortified food product should be similar to non-fortified product. In the present study yogurt was fortified with ammonium ferrous sulphate, ferrous sulphate, iron-casein complex in three different concentrations (20mg, 30mg, 40 mg iron) /kg milk sample. Sensory evaluation and In-vitro bioavailability study of yogurt samples were analyzed at 1st, 4th, 7th day of storage. Statistical analysis of the study shows ferrous sulphate and iron-casein complex fortified yogurt enhances the iron absorption compared to ammonium ferrous sulphate fortified yogurt.

L-Lactate production from carbon dioxide in the red alga Cyanidioschyzon merolae

Lactate/lactic acid is a commodity chemical used in various products such as bioplastics. The optical isomers of lactate are l- and d-lactate. Both the lactate enantiomers are required to manufacture lactate derived bioplastics. Eukaryotic microalgae and cyanobacteria can produce l-lactate or d-lactate using carbon dioxide as their sole carbon source. However, eukaryotic microalgae and cyanobacteria with high l-lactate productivity have not yet been identified. Cyanidioschyzon merolae is a hot-spring red alga preferring an acidic pH and high temperature. The entire genomic sequence was determined. C. merolae contains five l-lactate dehydrogenases that catalyze l-lactate-generating reactions during lactate fermentation. This study revealed that C. merolae produces l-lactate with high productivity during dark anaerobic cultivation. Nitrogen starvation before dark anaerobic cultivation increased the l-lactate productivity in C. merolae. l-Lactate accounted for over 90% of the total organic acids excreted by C. merolae cells. C. merolae exhibited a high l-lactate productivity under cultivation at a neutral pH. The l-lactate productivity in C. merolae at 16.0–19.4 mg/L/h was higher than that in a unicellular cyanobacterium at 2.7 mg/L/h. These findings indicate that C. merolae is one of the candidates for a host of l-lactate production from carbon dioxide.

Integration of lactic acid biorefinery with treatment of red mud from alumina refinery: win–win paradigm for waste valorization

The cost of detoxification and neutralization poses certain challenges to the development of an economically viable lactic acid biorefinery with lignocellulosic biomass as feedstock. Herein, red mud, an alkaline waste, was explored as both a detoxifying agent and a neutralizer. Red mud treatment of lignocellulosic hydrolysate effectively removed the inhibitors generated in dilute acid pretreatment, improving the lactic acid productivity from 1.0 g/L·h−1 to 1.9 g/L·h−1 in later fermentation. In addition, red mud could replace CaCO3 as a neutralizer in lactic acid fermentation, which in turn enabled simultaneous bioleaching of valuable metals (Sc, Y, Nd, and Al) from red mud. The neutralization of alkali in red mud by acids retained in lignocellulosic hydrolysate and lactic acid produced from fermentation led to effective dealkalization, rendering a maximum alkali removal efficiency of 92.2 %. Overall, this study offered a win–win strategy for the valorization of both lignocellulosic biomass and red mud.

A Novel Bio-Purification Process Employing an Engineered E. coli Strain for Downstream Processing of Lactic Acid Solutions from the Fermentation of Agro-Industrial by-Products.

This study aims to integrate a novel bio-purification process employing an engineered E. coli strain in the downstream processing of lactic acid (LA) fermentation broths from low-cost renewable biological feedstocks. Fermentation broth of candy waste and digestate mixture was used as a real biological feedstock. An engineered E. coli strain that selectively catabolize impurities without catabolizing LA was initially adapted on the biological feedstock, followed by shake flask experiments to prove the bio-purification concept. Scale-up and validation in a bench-scale bioreactor followed, before developing a semi-continuous membrane bioreactor (MBR) bio-purification process. The MBR bio-purification was assessed with biological feedstocks which simulated ultrafiltration or nanofiltration permeates. Incomplete removal of impurities and increased fouling was observed in the case of the ultrafiltration permeate. Contrarily, the nanofiltration permeate was successfully treated with MBR bio-purification, since low membrane fouling, 100% maltose and acetic acid removal, and no LA catabolism was achieved. MBR bio-purification as a post-treatment step in the downstream processing of LA was demonstrated as a promising technology for increasing the purity of LA solutions.

Inoculated fermentation of cv. Conservolea natural black olives with multifunctional starter cultures in reduced‐sodium brines

SummaryThe purpose of this research was to investigate the fermentation of Conservolea variety natural black olives using two lactic acid bacteria (LAB) with multifunctional potential as starter cultures. Moreover, the effect of 50% substitution of NaCl by KCl in the brines was examined to produce a reduced‐sodium product with multifunctional potential. For this purpose, Lactiplantibacillus pentosus B363 and Lactiplantibacillus plantarum B380, previously isolated from table olive fermentations and characterised for technological and probiotic potential, were inoculated at the onset of fermentation in 6% (w/v) NaCl and 3% (w/v) NaCl/3% (w/v) KCl salt brines. The microbial populations (LAB, yeasts, and Enterobacteriaceae), pH, acidity, salt content, organic acids, and ethanol were measured in the brine for 146 days. Additionally, the recovery of each starter culture from the olive drupes at the beginning (5 days) and final (146 days) stages of fermentation was determined by plating, genotyping, and identification through RAPD‐PCR and 16S region amplicon sequencing, respectively, and subsequent strain differentiation through rep‐GTG5 fingerprint analysis. The results revealed the dominance of LAB in populations from 5.5 to 8.1 log CFU/mL. The final recovery rate of L. pentosus B363 on the olives was approximately 80% in both 6% (w/v) NaCl and reduced (3% (w/v) NaCl/3% (w/v) KCl) salt brines. Similarly, L. plantarum B380 recovery rates were 89% and 85%, respectively, for the same fermentations. Yeasts coexisted with LAB in counts ranging from 2.3 to 5.8 log CFU/mL, whereas no enterobacteria were enumerated in the brines and olives at the end of fermentation. The obtained fermentation profile and the sensory analysis of the olives revealed a typical lactic acid fermentation in both control and reduced‐sodium brines. The current results support the feasibility of the production of reduced‐sodium natural black olives with probiotic potential. The proposed fermentation process, including the selected starters, can be readily employed by the table olive industry to meet consumers' needs for low‐sodium dietary intake while enhancing the functional potential of table olives.

The effect of kweni mango and randu honey addition on the characteristics of synbiotic etawa goat milk

Abstract. Rizal S, Suharyono AS, Astuti S, Dewanti AS, Nugroho YB. 2024. The effect of kweni mango and randu honey addition on the characteristics of synbiotic etawa goat milk. Biodiversitas 25: 1580-1587. The addition of kweni mango and randu honey as ingredients for producing synbiotic etawa goat milk is widely known to have a significant effect on the characteristics of the product. Therefore, this study aimed to identify the best formulation of kweni mango and randu honey in producing synbiotic etawa goat milk. The experiment was conducted using a completely randomized block design with 4 replications. A single factor with six levels of formulations of kweni mango extract and randu honey was used in producing synbiotic etawa goat milk. These formulations included F1 (0%:25%), F2 (5%:20%), F3 (10%:15%), F4 (15%:10%), F5 (20%:5%), and F6 (25%:0%). Subsequently, the microbiological, chemical, and sensory properties, as well as antimicrobial activity of the product were evaluated. The data obtained were statistically analyzed using ANOVA and the Least Significant Difference (LSD) test at a 5% significance level. The results showed that formulation (F4) containing 15% kweni mango extract and 10% randu honey produced the best synbiotic Etawa goat milk beverage. In addition, F4 formulation showed higher antibacterial activity against Staphylococcus aureus than Escherichia coli. The optimal beverage met the standards for lactic fermentation products from a chemical perspective and was favored by the panelists.

Elevated nutrient availability enhances chondrocyte metabolism and biosynthesis in tissue-engineered cartilage

ObjectiveChondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in HIF-1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. MethodsPrimary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4 – 16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). ResultsIncreasing media volumes resulted in higher accumulation of cartilaginous extracellular matrix (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the TCA cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. ConclusionsElevated media availability upregulates extracellular matrix synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in extracellular matrix accumulation.

Whole genome sequencing analysis of Komagataeibacter nataicola reveals its potential in food waste valorisation for cellulose production.

Komagataeibacter nataicola (K. nataicola) is a gram-negative acetic acid bacterium that produces natural bacterial cellulose (BC) as a fermentation product under acidic conditions. The goal of this work was to study the complete genome of K. nataicola and gain insight into the functional genes in K. nataicola that are responsible for BC synthesis in acidic environments. The pure culture of K. nataicola was obtained from yeast-glucose-calcium carbonate (YGC) agar, followed by genomic DNA extraction, and subjected to whole genome sequencing on a Nanopore flongle flow cell. The genome of K. nataicola consists of a 3,767,936bp chromosome with six contigs and 4,557 protein coding sequences. The maximum likelihood phylogenetic tree and average nucleotide identity analysis confirmed that the bacterial isolate was K. nataicola. The gene annotation via RAST server discovered the presence of cellulose synthase, along with three genes associated with lactate utilization and eight genes involved in lactate fermentation that could potentially contribute to the increase in acid concentration during BC synthesis. A more comprehensive genome study of K. nataicola may shed light into biological pathway in BC productivity as well as benefit the analysis of metabolites generated and understanding of biological and chemical interactions in BC production later.

Valorisation of cotton post-industrial textile waste into lactic acid: chemo-mechanical pretreatment, separate hydrolysis and fermentation using engineered yeast

BackgroundThe textile industry has several negative impacts, mainly because it is based on a linear business model that depletes natural resources and produces excessive amounts of waste. Globally, about 75% of textile waste is disposed of in landfills and only 25% is reused or recycled, while less than 1% is recycled back into new garments. In this study, we explored the valorisation of cotton fabric waste from an apparel textile manufacturing company as valuable biomass to produce lactic acid, a versatile chemical building block.ResultsPost-industrial cotton patches were pre-treated with the aim of developing a methodology applicable to the industrial site involved. First, a mechanical shredding machine reduced the fabric into individual fibres of maximum 35 mm in length. Afterwards, an alkaline treatment was performed, using NaOH at different concentrations, including a 16% (w/v) NaOH enriched waste stream from the mercerisation of cotton fabrics. The combination of chemo-mechanical pre-treatment and enzymatic hydrolysis led to the maximum recovery yield of 90.46 ± 3.46%, corresponding to 74.96 ± 2.76 g/L of glucose released, which represents a novel valorisation of two different side products (NaOH enriched wastewater and cotton textile waste) of the textile industry. The Saccharomyces cerevisiae strain CEN.PK m850, engineered for redirecting the natural alcoholic fermentation towards a homolactic fermentation, was then used to valorise the glucose-enriched hydrolysate into lactic acid. Overall, the process produced 53.04 g/L ± 0.34 of l-lactic acid, with a yield of 82.7%, being the first example of second-generation biomass valorised with this yeast strain, to the best of our knowledge. Remarkably, the fermentation performances were comparable with the ones obtained in the control medium.ConclusionThis study validates the exploitation of cotton post–industrial waste as a possible feedstock for the production of commodity chemicals in microbial cell-based biorefineries. The presented strategy demonstrates the possibility of implementing a circular bioeconomy approach in manufacturing textile industries.Graphical