Repeated Batch Fermentation Research Articles

Valorization of Biodiesel-Derived Crude Glycerol for Producing Yeast Oil as an Alternative Biodiesel Production Feedstock: Productivity Improvement in Batch and Repeated Batch Fermentation

Valorization of Biodiesel-Derived Crude Glycerol for Producing Yeast Oil as an Alternative Biodiesel Production Feedstock: Productivity Improvement in Batch and Repeated Batch Fermentation

Optimizing bioencapsulation of yeast cells by Aspergillus tubingensis TSIP9 and applications in bioethanol production through repeated-batch fermentation

This study aimed to immobilize yeast cells using filamentous fungi owing to a number of advantages including less chemical usage, spontaneous encapsulation, strong adhesion and biocompatibility. Filamentous fungus, Aspergillus tubingensis TSIP9 could form sphere-shape and packed pellets using either fungal fresh spores or those stored in liquid medium for 6 weeks at 4 °C. The fungus was used to immobilize yeast Saccharomyces cerevisiae FAI via adsorption and co-cultivation methods. Scanning electron microscopy images revealed that the bioencapsulated yeast cells via co-cultivation seemed to be more tightly adhered on fungal mycelia and surrounded by extracellular polymeric substances. The yeast biocapsules also exhibited higher stability and maintained their structural integrity during repeated-batch fermentation while the immobilized yeast cells by adsorption gradually degraded during their repeated uses. The bioethanol production from glucose by yeast biocapsules were in the range of 95–98 g/L with the bioethanol yield of 0.49–0.54 g-ethanol/g-consumed glucose. The yeast biocapsules could produce bioethanol well when using enzymatic hydrolysate of lignocellulosic palm waste, as alternative cheap carbon source, with the comparable bioethanol yield of 0.49 ± 0.17 g-ethanol/g-consumed glucose. The spontaneous and inter-species bioencapsulation show the perspectives as active biocatalysts with high cell retention for repeated uses.

Fibrous oil palm frond as a potential microbial immobilization carrier for enhanced 1,3-propanediol productivity

In line with Sustainable Development Goal 12: Responsible Consumption and Production, the biotransformation of crude glycerol into 1,3-propanediol establishes a circular bioeconomy between regional crude glycerol and value-added green polymer. However, the bioproduction of 1,3-propanediol is restricted by a slow conversion rate, which could potentially be overcome using the immobilization technique. In this work, the locally isolated Clostridium butyricum JKT 37 was immobilized on coconut shell activated carbon (CSAC), granular durian peel (GDP), or fibrous oil palm frond (FOPF) to ferment crude glycerol to produce 1,3-propanediol. Amongst these, the immobilized cell density of FOPF was four times higher than the CSAC. FOPF immobilizer had resulted in an improvement in the immobilization efficiency (45.61 %), 1,3-PDO productivity (22.93 %), and maximum instantaneous productivity (36.12 %) as compared to the CSAC. 18.99 g/L of 1,3-PDO was produced from immobilized-FOPF fermentation at the highest yield of 0.65 mol/mol. This superior cell adsorption on FOPF was supported by its high water absorption index (4.16 g/g) and morphology. GDP immobilizer had a higher immobilized cell density (9.20 g/L) and cell retention (0.18 g/g) than CSAC, with insignificant improvement in the 1,3-propanediol production. The biological activity of the immobilized cells on FOPF was validated in 12 cycles repeated batch fermentations with 1,3-PDO yield and productivity as high as 0.68 mol/mol and 1.43 g/L.h, respectively. This research has proven the high performance of fibrous immobilizers and serves as the benchmark for future investigation into the use of agricultural waste to enhance other bioprocess’s productivity.

Dilute acid-assisted microbubbles-mediated ozonolysis of Eucheuma denticulatum phycocolloid for biobased L-lactic acid production

Biobased L-lactic acid (L-LA) appeals to industries; however, existing technologies are plagued by limited productivity and high energy consumption. This study established an integrated process for producing macroalgae-based L-LA from Eucheuma denticulatum phycocolloid (EDP). Dilute acid-assisted microbubbles-mediated ozonolysis (DAMMO) was selected for the ozonolysis of EDP to optimize D-galactose recovery. Through single-factor optimization of DAMMO treatment, a maximum D-galactose recovery efficiency (59.10 %) was achieved using 0.15 M H2SO4 at 80 °C for 75 min. Fermentation with 3 % (w/v) mixed microbial cells (Bacillus coagulans ATCC 7050 and Lactobacillus acidophilus-14) and fermented residues achieved a 97.67 % L-LA yield. Additionally, this culture approach was further evaluated in repeated-batch fermentation and showed an average L-LA yield of 93.30 %, providing a feasible concept for macroalgae-based L-LA production.

Optimizing Lactic Acid Production Through Dynamic Simulation in Repeated-Batch Fermentation System

In this study, we explored the lactic acid production from molasses through a repeated-batch fermentation process using dynamic simulation. Our investigation revealed that sugar concentrations ranging from 50 to 150 g/l significantly impacted the dynamic profiles of sugar consumption concentration, microbial Enterococcus faecalis RKY1 growth, and lactic acid production. Through dynamic simulation, we identified the optimal inlet sugar concentration as 68 g/l (equivalent to 130 g/l molasses), resulting in a lactic acid average productivity of 3.9 g/l h (3-repeated batch). The simulation demonstrated the viability of a sustainable lactic acid production process using Enterococcus faecalis RKY1, enabling the repeated-batch fermentation process for multiple cycles. Comparative analysis with continuous fermentation indicated that repeated-batch fermentation offers a superior alternative, characterized by higher substrate conversion to lactic acid. This study contributes valuable insights into optimizing lactic acid production processes, emphasizing the efficiency and sustainability of the repeated-batch fermentation approach.

Improvement of extracellular polysaccharides production from Cordyceps militaris immobilized alginate beads in repeated-batch fermentation

Cordyceps militaris (C. militaris) is a unique medicinal fungus of the genus Cordyceps. It has high economic value due to various biological functions from the extracellular polysaccharides (EPSs) it produces. The aim of this study was to establish a mycelium-immobilization system for EPS production in a bubble column system. First, EPS production was optimized by 90.2% (from 0.72 to 1.37 g/L) using response surface methodology under an alginate-immobilized mycelium system with 6 days of cultivation. The highest EPS production was obtained (5.3 g/L) in a flask system through elongation to 15 days of cultivation. EPS production subsequently further increased to 10.42 g/L utilizing a bubble column reactor. Furthermore, the system can be reused for at least three rounds. In the EPS characteristics analysis, the produced EPSs were mainly composed of glucose and galactose. They presented strong antibacterial activity against Staphylococcus aureus. In terms of antioxidant activity, respective the EPS concentration that caused 50% scavenging of DPPH and ABTS free radicals values (IC50) were 4.65 and 7.92 mg/mL. In summary, these findings should be helpful for establishing a suitable industrial fermentation system for C. militaris and can be studied in the food and medical industries in future work.

Lactic acid production by Lactobacillus casei using a sequence of seasonally available fruit wastes as sustainable carbon sources.

Introduction: Lactic acid (LA) production from fossil resources is unsustainable owing to their depletion and environmental concerns. Thus, this study aimed to optimize the production of LA by Lactobacillus casei in a cultured medium containing fruit wastes (FWs) from agro-industries and second cheese whey (SCW) from dairy production, supplemented with maize steep liquor (MSL, 10% v/v) as the nitrogen source. Methods: The FWs were selected based on seasonal availability [early summer (early ripening peach), full summer (melon), late summer (pear), and early autumn (apple)] and SCW as annual waste. Small-scale preliminary tests as well as controlled fermenter experiments were performed to demonstrate the potential of using various food wastes as substrates for LA fermentation, except for apple pomace. Results and discussion: A 5-cycle repeated batch fermentation was conducted to optimize waste utilization and production, resulting in a total of 180.56g/L of LA with a volumetric productivity of 0.88g/L∙h. Subsequently, mechanical filtration and enzymatic hydrolysis were attempted. The total amount of LA produced in the 5-cycle repeated batch process was 397.1g/L over 288h, achieving a volumetric productivity of 1.32g/L∙h. These findings suggest a promising biorefinery process for low-cost LA production from agri-food wastes.

Succinic acid production by Actinobacillus succinogenes using acid and enzymatic hydrolysates of potato and beer wastes and repeated batch operation

The replacement of fossil resources by biomass is one of the key strategies for the development of bioeconomy. Here we have focused on the bioproduction of succinic acid, a biorefinery platform of great versatility and importance. In this study, the anaerobic bacterium Actinobacillus succinogenes was selected as the biocatalyst, choosing discarded potatoes and spent brewer's yeast hydrolysates served as carbon and nitrogen sources, respectively. The use of food residues is critical to reduce operating expenses in this type of processes. Furthermore, to analyze and improve the process performance, repeated batch fermentations were carried out in several conditions: with potato waste hydrolysate or 40 g L−1 of pure glucose as carbon sources and spent brewer's yeast hydrolysate or commercial yeast extract as nitrogen sources. Very promising results were obtained with the residue preparations: 35.8 g L−1 of succinic acid were produced, with a yield of 0.84 g g−1 and an average productivity of 1.19 g L−1 h −1, increasing the selectivity towards the target acid in comparison to pure glucose and yeast extract. Finally, a previously developed unstructured non-segregated global kinetic model is successfully applied, obtaining the parameters values in all conditions.

Improvement of ɣ-Aminobutyric Acid Production and Cell Viability of Lactiplantibacillus plantarum B7 via Whole-Cell Immobilisation in Repeated Batch Fermentation System

Improvement of ɣ-Aminobutyric Acid Production and Cell Viability of Lactiplantibacillus plantarum B7 via Whole-Cell Immobilisation in Repeated Batch Fermentation System

Immobilization of rough morphotype Mycolicibacterium neoaurum R for androstadienedione production.

Enhance the androstadienedione (Androst-1,4-diene-3,17-dione, ADD) production of rough morphotype Mycolicibacterium neoaurum R by repeated-batch fermentation of immobilized cells. M. neoaurum R was a rough colony morphotype variant, obtained from the routine plating of smooth M. neoaurum strain CICC 21097. M. neoaurum R showed rougher cell surface and aggregated in broth. The ADD production of M. neoaurum R was notably lower than that of M. neoaurum CICC 21097 during the free cell fermentation, but the yield gap could be erased after proper cell immobilization. Subsequently, repeated-batch fermentation of immobilized M. neoaurum R was performed to shorten the production cycle and enhance the bio-production efficiency of ADD. Through the optimization of the immobilization carriers and the co-solvents for phytosterols, the ADD productivity of M. neoaurum R immobilized by semi-expanded perlite reached 0.075g/L/h during the repeated-batch fermentation for 40days. The ADD production of the rough-type M. neoaurum R was notably enhanced by the immobilization onto semi-expanded perlite. Moreover, the ADD batch yields of M. neoaurum R immobilized by semi-expanded perlite were maintained at high levels during the repeated-batch fermentation.

Capability of immobilized Clostridium beijerinckii for batch and repeated-batch butanol fermentation from sweet sorghum stem juice in various bioreactors

Mixing in bioreactors is important for butanol fermentations. The type of impeller (6-blade disc, 2-pitch blade and 3-pitch blade turbines) in a stirred-tank bioreactor (STR) did not affect butanol production by immobilized Clostridium beijerinckii TISTR 1461 on bamboo chopstick pieces. Using a spin-filter in the STR could reduce the fermentation time from 48 h to 36 h. Butanol production from sweet sorghum stem juice (butanol concentration, PB, ∼11 g/L and butanol productivity, QB, 0.30 g/L·h) by the immobilized cells in an internal loop gas-lift bioreactor using a carrier inside the draft tube were similar to those using an STR with a 6-blade disc turbine integrated with a spin-filter. Repeated-batch butanol fermentation in the internal loop gas-lift bioreactor could be operated for four cycles. However, high levels of butanol production could be maintained for only two cycles. Simultaneous immobilization and fermentation was a suitable process for repeated-batch fermentation by immobilized cells. An internal loop gas-lift bioreactor is a potential low-cost bioreactor for batch and repeated-batch butanol fermentation by the immobilized cells.

Co-production of lactate and volatile fatty acids through repeated-batch fermentation of fruit and vegetable waste: Effect of cycle time and replacement ratio

In this study, repeated-batch fermentation was used to convert fruit and vegetable waste to lactate and volatile fatty acids (VFAs), which are essential carbon sources for medium-chain fatty acids (MCFAs) production. The effect of cycle time and replacement ratio on acidification in long-term fermentation was investigated. The results showed that they had a significant impact on product yield, productivity, and type of products. Considering the yield, productivity, and lactate/VFAs ratio, a replacement ratio of 30% and a cycle time of 2 d may be more suitable for further production of MCFAs. Its productivity and lactate/VFAs ratio were 4.07 ± 0.24 g/(L·d) and 5 ± 0.6, respectively. The lactic acid bacteria, such as Enterococcus (63%) and Lactobacillus (33%), stabilized in the reactor, resulting in the generation of both lactate and VFAs by heterolactic fermentation. The present study demonstrated a new strategy with the potential to recover high-value products from organic waste streams.

Enhanced exopolysaccharide production of Cordyceps militaris via mycelial cell immobilization on plastic composite support in repeated-batch fermentation

Repeated-batch fermentation with fungal mycelia immobilized in plastic composite support (PCS) eliminates the lag phase during fermentation and improves metabolite productivity. The strategy is implemented herein, and a novel modified PCS is developed to enhance exopolysaccharide (EPS) production from the medicinal fungus Cordyceps militaris. A modified PCS (SYE + PCS) was made by compositing polypropylene (PP) with a nutrient mixture containing soybean hull, peptone, yeast extract, and minerals (SYE+). The use of SYE + PCS has consistent cell productivity throughout the multiple fermentation cycles, which resulted in a more higher cell productivity after second batch compared to unmodified PCS. The cell grown on SYE + PCS also generates a higher yield of EPS (3.36, 6.93, and 5.72 g/L in the first, second, and third fermentation cycles, respectively) up to three-fold higher than the cell immobilized on unmodified PCS. It is also worth noting that the EPS from mycelium grown on SYE + PCS contains up to 2.3-fold higher cordycepin than those on unmodified PCS. The presence of nutrients in SYE + PCS also affects the hydrophobicity and surface roughness of the PC, improving mycelial cell adhesion. This study also provides a preliminary antioxidant activity assessment of EPS from immobilized C. militaris grown with SYE + PCS.

Development of Biochar-Based Whole-Cell Biocatalysts for the Production of l-Tryptophan and l-Phenylalanine

Microbial cell immobilization by adsorption is a simple and effective method to obtain sustainable whole-cell biocatalysts that provide unique advantages and are widely used for the biosynthesis of chemicals. Adsorption between cells and biochar allows direct diffusion of substrates and products but does not have high immobilization efficiency. In this study, a certain amount of negatively charged amino acids were displayed on the surface of engineered Escherichia coli cells using truncated ice-nucleation proteins (INPs) as anchoring motifs. An optimized FeCl3 solution modification was performed to obtain modified biochar MBC-Fe with a positively charged surface, facilitating enhanced adsorption between the cells and MBC-Fe. The strategy to enhance adsorption was applied to the previously developed E. coli strains for producing l-tryptophan and l-phenylalanine. Results showed that charged amino acids can be anchored on the cell surface by INaA, and the fusion proteins successfully provided corresponding charges to the cells. The constructed whole-cell biocatalysts Mp-trpAN45@MBC-Fe and Cc-pheAN45@MBC-Fe carrying the most negative charges yielded good results in terms of production of L-Trp and L-Phe and reusability in all five repeated batch fermentations. The novel strategy developed in this study was effective in reusability of whole-cell immobilization for improving the production of target compounds.

Enhanced lactic acid production by Lactobacillus rhamnosus ATCC 7469 through simultaneous saccharification and fermentation from household food waste

AbstractA large amount of household food waste (HFW) is generated yearly and may negatively impact the environment and human health. The recovery of value‐added chemicals lactic acid (LA) from HFW through anaerobic treatment is a promising approach. However, it remains a challenge to increase the LA yield. In this paper, the LA production from HFW was enhanced by Lactobacillus rhamnosus ATCC 7469 through simultaneous saccharification and fermentation (SSF). The results showed that the mixed commercial enzymes of plant enzymes (E1), animal proteases (E2), and glucoamylase (E3) were more conducive to LA production than those used separately. Compared to the separate hydrolysis and fermentation (SHF), the LA production increased by 14% in the SSF, and the glucose concentration showed different change rules. Additionally, an average LA concentration of 47.7 g L–1 was obtained by repeated batch fermentation through SSF. However, the maximum LA concentration decreased with the fermentation ongoing. The trend of decreasing LA production could be improved by repeated batch fermentation with supplementary inoculation.

Genome and fermentation analyses of Enterococcus faecalis DB-5 isolated from Japanese Mandarin orange: An assessment of potential application in lactic acid production

Enterococcus faecalis strain DB-5 is a lactic acid bacterium newly isolated from the Japanese mandarin orange (mikan). The DB-5 strain produces organic acid from various carbohydrate sources including glycerol and starch. To gain deeper insights into its potential application in lactic acid fermentation (LAF), the genome and fermentation analyses of E.faecalis DB-5 were performed. Whole genome sequencing was carried out using the DNBSEQ platform. After trimming and assembly, the total size of the assembled genome was revealed to be 3,048,630 bp, distributed into 63 contigs with an N50 value of 203,673. The genome has 37.2% GC content, 2928 coding DNA sequences, and 54 putative RNA genes. The DB-5 strain harbored two l-lactate dehydrogenases (L-LDHs), both of which conserved the catalytic domain sequences. The optical purity measurement showed that strain DB-5 is homofermentative and produced only l-lactic acid (LA), which correlated with genome-based pathway analysis. To confirm its LA productivity at high temperatures, open repeated batch fermentation was performed at 45°C using sucrose as a carbon source. The volumetric LA productivity of DB-5 was averaged at 3.66gL-1h-1 for 24h during the 3rd to 11th fermentation cycles. E.faecalis DB-5 could efficiently convert around 94% of sucrose to LA throughout the fermentation cycles at 45°C. These genomic characteristics and fermentation properties of E.faecalis DB-5 provide beneficial information for a deeper understanding of the functional properties of future high-temperature LAFs from biomass resources.

Evaluation of Fermentative Xylitol Production Potential of Adapted Strains of Meyerozyma caribbica and Candida tropicalis from Rice Straw Hemicellulosic Hydrolysate

Dilute acid hydrolysis of lignocellulosic biomass generates inhibitors in the hydrolysate which hamper yeast metabolism and the fermentation process. Therefore, understanding the effect of these compounds on the performance of microorganisms becomes essential to achieve improved product yields. In this study, the effect of acetic acid, furfural, and hydroxymethylfurfural was evaluated on yeast growth and fermentation efficiency. Various parameters for the pretreatment of rice straw, such as an acid catalyst, and its concentration and residence time, were optimized for the maximum liberation of sugars in the hydrolysate. Further, the yeast strains Candida tropicalis and Meyerozyma caribbica were adapted for the tolerance of inhibitors at higher concentrations. A comparative analysis was carried out using un-adapted and adapted strains of Candida tropicalis and Meyerozyma caribbica for xylitol production. The findings of this study revealed that sulfuric acid (1.25% v/v) at 121 ரC for 30 min can efficiently convert rice straw xylan to xylose, with the release of 16.07 g/L xylose in the hydrolysate. Further, the adaptation results showed an increase of 76.42% and 69.33% in xylose assimilation by C. tropicalis and M. caribbica, respectively. The xylitol production with the adapted C. tropicalis was increased by 7.54% to 28.03 g/L xylitol. However, the xylitol production with the adapted M. caribbica was increased by 8.33%, yielding 26.02 g/L xylitol in the non-detoxified hydrolysate when compared to the un-adapted strains. Repeated batch fermentation was carried out for seven batches, and xylitol was found to be efficiently produced by the yeasts during five successive batches without any significant loss in the xylitol yield. Moreover, the results suggest that M. caribbica is a promising microorganism for the transformation of rice straw-derived xylose to xylitol.

Production of Pigments under Submerged Culture through Repeated Batch Fermentation of Immobilized Talaromyces atroroseus GH2

Pigments of natural origin have become a research trend, and fungi provide a readily available alternative source. Moreover, developing novel processes that increase yields, reduce process time and simplify downstream processing is of increased interest. In this sense, this work proposes an alternative for Talaromyces atroroseus GH2 biomass re-utilization to produce pigments through consecutive batches using immobilized mycelium. Different support materials were evaluated for pigment production and immobilization capacity. Then, Taguchi’s method was applied to determine the effect of four factors related to fungal immobilization and pigment production (inoculum concentration, support density, working volume and support volume). Afterward, process kinetics for pigment production using immobilized cells of T. atroroseus GH2 in consecutive batches were evaluated. All evaluated factors were significant and affected pigment production and microorganism growth differently. At improved conditions, immobilization capacity reached 99.01 ± 0.37% and the pigment production was 30% higher than using free cells. Process kinetics showed that the production could continue for three batches and was limited by excessive microorganism growth. Indeed, more studies are still needed, but the immobilization of Talaromyces atroroseus GH2 represents a promising strategy for allowing downstream-processing intensification since immobilized biomass is easily removed from the fermentation media, thus paving the way for the further development of a continuous process.

Repeated-Batch Ethanol Fermentation from Sweet Sorghum Stem Juice under a Very High Gravity Condition Using a Stirred Tank Bioreactor Coupled with a Column Bioreactor by Immobilized Saccharomyces cerevisiae

The ethanol fermentation efficiency of sweet sorghum stem juice (SSJ) under a very high gravity (VHG) condition (250 g/L of sugar) was improved by immobilized Saccharomyces cerevisiae SSJKKU01, using a stirred tank bioreactor (STR) coupled with a column bioreactor (CR). Dried rattan pieces (as carriers for cell immobilization) at 50% of the working volume of the CR were suitable for use in a batch ethanol fermentation. The average ethanol concentration (PE) and ethanol productivity (QP) of repeated-batch fermentation in the CR for eight successive cycles were 109.85 g/L and 1.88 g/L⋅h, respectively. Then an STR coupled with a CR was applied for repeated-batch ethanol fermentation in two systems. System I was an STR (1.8 L working volume), and System II was an STR (1 L) coupled with a CR, referred to as a CR-F (0.8 L). Both systems were connected to a new CR, called CR-I, containing sterile dried rattan pieces at 50% of its working volume. Active yeast cells were inoculated only into the STR, and the medium circulation rate between bioreactors was 5.2 mL/min. The results showed that at least eight successive cycles could be operated with an average PE of 108.51 g/L for System I and 109.44 g/L for System II. The average QP and SC values of both systems were also similar, with values of 1.87 to 1.88 g/L⋅h and 93 to 94%, respectively. The morphology of the carriers with and without immobilized cells before and after the fermentation was investigated. The obtained results demonstrated that a repeated-batch fermentation by immobilized cells on rattan pieces, using an STR coupled with a CR, was successfully used to produce high levels of ethanol from SSJ under a VHG condition.

Propionic acid production via two-step sequential repeated batch fermentations on whey and flour

In this study, two-stage process was developed for propionic acid production via repeated batch and fed-batch fermentations. In the first step, either whey lactose or flour hydrolysate was used as carbon source to produce lactic acid using Lactobacillus plantarum in repeated batch and fed-batch fermentations. Then, produced lactic acid was used as a carbon source for propionic acid fermentation using Propionibacterium freudenreichii in repeated batch fermentations. Maximum lactic acid concentrations were achieved as 100 g/L at 250th hour of incubation (corresponding to 6th cycle of repeated batch fermentation) on whey lactose and 97 g/L at 500th hour (1st cycle) on flour hydrolysate as substrate. For propionic acid production, maximum propionic acid concentrations were obtained as 28 g/L at 700th hour (13th cycle) on lactic acid obtained from whey and 35 g/L at 3600th hour (10th cycle) on lactic acid obtained from flour hydrolysate. Lactic acid and propionic acid productivities were 0.80 g/Lh and 0.050 g/Lh on whey lactose higher than the values 0.12 g/Lh and 0.025 g/Lh obtained on flour hydrolysate respectively.