Semi-continuous Fermentation Research Articles

Optimization and semi-continuous fermentation of gluco-oligosaccharide production with Weissella cibaria YRK005

Optimization and semi-continuous fermentation of gluco-oligosaccharide production with Weissella cibaria YRK005

Anaerobic valorization of sewage sludge pretreated through hydrothermal carbonization: Volatile fatty acids and biomethane production

Hydrothermal carbonization (HTC) emerged as an effective technology for the treatment of various types of wet biomass and organic residues, including sewage sludge, offering the potential for sludge reduction and resource recovery. HTC pretreatment impact on downstream sludge fermentation is investigated. Results obtained at optimal conditions for HTC pretreatment (170 °C for 30 min) indicated that soluble carbon was significantly increased in the liquid fraction, enhancing feedstock availability for fermentation. Semi-continuous fermentation of HTC-treated sludge resulted in a stable process in which a mixed microbial community produced volatile fatty acids (VFAs) with longer chain acids content, acidification yield of 0.59 ± 0.05 g COD-VFA g−1 CODin and volumetric productivity of 1.6 ± 0.5 g COD-VFA L−1 d−1. Biomethane Potential tests evidenced high values for hydrochar. Overall, the HTC pretreatment enables improved conversion efficiencies, in the view of valorizing the liquid for VFA synthesis and the hydrochar for biomethane production.

A Semi-Continuous Fermentation Process for Pulque Production Using Microfiltration-Sterilized Aguamiel and Aseptic Conditions to Standardize the Overall Quality of the Beverage

Despite the current appreciation of pulque as a probiotic fermented beverage, pulque has been also regarded as a poor-quality product, particularly due to the lack of sanitary control during its elaboration. To address this problem, a semi-continuous fermentation system was established, emulating the artisanal production process. Microfiltration-sterilized aguamiel was employed as the substrate, whereas a good-quality pulque was used as the fermentation inoculum. During the fermentation, the physicochemical, microbiological (lactic acid and Leuconostoc-type bacteria and yeasts) and sensory characteristics of the must were monitored. The isolated microorganisms were identified by molecular biology and MALDI-MS techniques. The sterilization of aguamiel by microfiltration did not negatively affect its physicochemical attributes. After 6–8 days of operation of the semi-continuous bioreactor, the fermentation reached a quasi-stationary state considering most of the parameters monitored during the experiment. The final fermentation product presented similar physicochemical, microbial and sensory properties to those of the pulque inoculum. The genera identified were Leuconostoc, Lentilactobacillus, Lactobacillus, Liquorilactobacillus, Fructilactobacillus and Saccharomyces. The strains Lentilactobacillus diolivorans and Liquorilactobacillus capillatus and uvarum have not been previously isolated from pulque. In conclusion, the fermentation system developed in this work was effective to standardize the quality of pulque while preserving the positive attributes of the artisanal process, thus harnessing the probiotic properties of pulque.

Airlift bioreactor–based strategies for prolonged semi-continuous cultivation of edible Agaricomycetes

Submerged cultivation of edible filamentous fungi (Agaricomycetes) in bioreactors enables maximum mass transfer of nutrients and has the potential to increase the volumetric productivity of fungal biomass compared to solid state cultivation. These aspects are paramount if one wants to increase the range of bioactives (e.g. glucans) in convenient time frames. In this study, Trametes versicolor (M9911) outperformed four other Agaricomycetes tested strains (during batch cultivations in an airlift bioreactor). This strain was therefore further tested in semi-continuous cultivation. Continuous and semi-continuous cultivations (driven by the dilution rate, D) are the preferred bioprocess strategies for biomass production. We examined the semi-continuous cultivation of T. versicolor at dilution rates between 0.02 and 0.1 h−1. A maximum volumetric productivity of 0.87 g/L/h was obtained with a D of 0.1 h−1 but with a lower total biomass production (cell dry weight, CDW 8.7 g/L) than the one obtained at lower dilution rates (12.3 g/L at D of 0.04 and vs 13.4 g/L, at a D of 0.02 h−1). However, growth at a D of 0.1 h−1 resulted in a very short fermentation (18 h) which terminated due to washout (the specific D exceeded the maximum growth rate of the fungal biomass). At a D of 0.04 h−1, a CDW of 12.3 g/L was achieved without compromising the total residence time (184 h) of the fermentation. While the D of 0.04 h−1 and 0.07 h−1 achieved comparable volumetric productivities (0.5 g/L/h), the total duration of the fermentation at D of 0.07 h−1 was only 85 h. The highest glucan content of cells (27.8 as percentage of CDW) was obtained at a D of 0.07 h−1, while the lowest glucan content was observed in T. versicolor cells grown at a D of 0.02 h−1.Key points• The highest reported volumetric productivity for fungal biomass was 0.87 g/L/h.• Semi-continuous fermentation at D of 0.02 h−1 resulted in 13.4 g/L of fungal biomass.• Semi-continuous fermentation at D of 0.07 h−1 resulted in fungal biomass with 28% of total glucans.

Time-dependent interference of surfactants and CeO2/Fe2O3 nanoparticles co-occurrence on the volatile fatty acids biosynthesis during semi-continuous sludge fermentation

Various exogenous contaminants typically coexist in waste activated sludge (WAS), and the long-term impacts of these co-occurring contaminants on WAS anaerobic fermentation and associated mechanisms remain largely unknown. This study reveals that the co-occurrence of surfactants and nanoparticles (NPs, i.e., Fe2O3 and CeO2, frequently detected in sludge) exhibited time-dependent impacts on the volatile fatty acids (VFAs) biosynthesis. Surfactants triggered WAS decomposition and enhanced NPs dispersion, leading to increased exposure of functional anaerobes to NPs toxicity, negatively affecting them. Consequently, key fermentation processes, acidogenic bacterial abundance, and metabolic functions were inhibited in co-occurrence reactors compared to those containing only surfactants in the early stage (before 56 d). Surprisingly, the fermentation systems containing surfactants collapsed subsequently, with VFAs yield at 72 d decreasing by 48.59–71.27 % compared to 56 d. The keystone microbes (i.e., Acidobacteria (16 d) vs Patescibacteria (56 d)) were reshaped, and metabolic traits (i.e., proB involved in intracellular metabolism) were downregulated by 0.05–78.02 % due to reduced microbial adaptive capacity (i.e., quorum sensing (QS)). Partial least squares path modeling (PLS-PM) analysis suggests that the microbial community was the predominant factor influencing VFAs generation. This study provides new insights into the long-term effects of co-contaminants on the biological treatment of WAS.

Medium-chain carboxylates production from plant waste: kinetic study and effect of an enriched microbiome

BackgroundThe need for addition of external electron donors such as ethanol or lactate impairs the economic viability of chain elongation (CE) processes for the production of medium-chain carboxylates (MCC). However, using feedstocks with inherent electron donors such as silages of waste biomass can improve the economics. Moreover, the use of an appropriate inoculum is critical to the overall efficiency of the CE process, as the production of a desired MCC can significantly be influenced by the presence or absence of specific microorganisms and their metabolic interactions. Beyond, it is necessary to generate data that can be used for reactor design, simulation and optimization of a given CE process. Such data can be obtained using appropriate mathematical models to predict the dynamics of the CE process.ResultsIn batch experiments using silages of sugar beet leaves, cassava leaves, and Elodea/wheat straw as substrates, caproate was the only MCC produced with maximum yields of 1.97, 3.48, and 0.88 g/kgVS, respectively. The MCC concentrations were accurately predicted with the modified Gompertz model. In a semi-continuous fermentation with ensiled sugar beet leaves as substrate and digestate from a biogas reactor as the sole inoculum, a prolonged lag phase of 7 days was observed for the production of MCC (C6–C8). The lag phase was significantly shortened by at least 4 days when an enriched inoculum was added to the system. With the enriched inoculum, an MCC yield of 93.67 g/kgVS and a productivity of 2.05 gMCC/L/d were achieved. Without the enriched inoculum, MCC yield and productivity were 43.30 g/kgVS and 0.95 gMCC/L/d, respectively. The higher MCC production was accompanied by higher relative abundances of Lachnospiraceae and Eubacteriaceae.ConclusionsEnsiled waste biomass is a suitable substrate for MCC production using CE. For an enhanced production of MCC from ensiled sugar beet leaves, the use of an enriched inoculum is recommended for a fast process start and high production performance.

Co-digestion of filter cake, biogas effluent, and anaerobic sludge for hydrogen and methane production: Optimizing energy recovery through two-stage anaerobic digestion

This study assesses a two-stage anaerobic digestion process designed to efficiently recover energy through hydrogen and methane production by co-digesting filter cake (FC), biogas effluent (BE), and anaerobic sludge (AS) from the sugar and ethanol industry. The optimal proportions of FC, BE, and AS were determined in batch fermentation experiments using Design Expert software, which identified a suitable ratio of 31.05:28.95:0.00 (g VS/L), respectively. The results indicated that hydrogen production in the first stage could occur solely through the hydrogenic bacteria present in the BE and FC mixture, without the need for AS as an inoculum. This optimized FC:BE ratio was then applied in a semi-continuous fermentation system, achieving a hydrogen production rate of 193.6 mL H2/L.d and a hydrogen yield of 9.8 mL H2/g VS at an optimal hydraulic retention time (HRT) of 3 d. In the subsequent second stage, the effluent from the hydrogen reactor was used for methane production. This stage achieved a methane production rate of 422.0 mL CH4/L·d and a methane yield of 140.2 mL CH4/g VS, with an HRT of 20 d. Overall, the two-stage process exhibited an impressive energy output, peaking at 5.17 kJ/g VS. This suggests the potential for an annual electricity generation of 194,655 MWh and an estimated reduction of 85,668 tCO2eq/year in greenhouse gas emissions. This study highlights the efficiency of the two-stage anaerobic digestion process for harnessing energy through the co-digestion of FC and BE, without the need for additional AS inoculum. The findings demonstrate the potential for sustainable energy recovery from industrial waste streams while mitigating environmental impacts.

Development of a hybrid bio-purification process of lactic acid solutions employing an engineered E. coli strain in a membrane bioreactor

BackgroundA potential alternative to lactic acid production through sugar fermentation is its recovery from grass silage leachate. The separation and purification of lactic acid from fermentation broths remain a key issue, as it amounts to up to 80% of its industrial production cost. In this study, a genetically engineered E. coli strain (A1:ldhA), that cannot catabolize lactic acid, has been used to selectively remove impurities from a synthetic medium comprising typical components (i.e., glucose and acetic acid) of green grass silage leachate. A systematic approach has been followed to provide a proof-of-concept for a bio-purification process of lactic acid solutions in a membrane bioreactor operating in semi-continuous mode.ResultsThe synthetic medium composition was initially optimized in shake-flasks experiments, followed by scale-up in bench-scale bioreactor. Complete (i.e., 100%) and 60.4% removal for glucose and acetic acid, respectively, has been achieved in batch bioreactor experiments with a synthetic medium comprising 0.5 g/L glucose and 0.5 g/L acetic acid as carbon sources, and 10 g/L lactic acid; no lactic acid catabolism was observed in all batch fermentation tests. Afterwards, a hybrid biotechnological process combining semi-continuous bioreactor fermentation and ultrafiltration membrane separation (membrane bioreactor) was applied to in-situ separate purified medium from the active cells. The process was assessed under different semi-continuous operating conditions, resulting in a bacteria-free effluent and 100% glucose and acetic acid depletion, with no lactic acid catabolism, thus increasing the purity of the synthetic lactic acid solution.ConclusionsThe study clearly demonstrated that a bio-purification process for lactic acid employing the engineered E. coli strain cultivated in a membrane bioreactor is a technically feasible concept, paving the way for further technological advancement.

Efficient semi-continuous fermentation production of nuclease P1 by Penicillium citrinum TKZY02

Efficient semi-continuous fermentation production of nuclease P1 by Penicillium citrinum TKZY02

Valorization of organic carbon in primary sludge via semi-continuous dark fermentation: First step to establish a wastewater biorefinery

In this study, lab-scale, bench-scale, and pilot-scale experiments were carried out to optimize short-chain fatty acids production from primary sludge. Batch tests showed the requirement of short retention times and semi-continuous operation mode showed a plateau of maximum daily productivity at 36-hours hydraulic retention time with minimal methanation. Optimization from pH 5 to pH 10 at 36 h-hydraulic retention time under long-term semi-continuous operating mode revealed that production of short-chain fatty acids was pH dependent and highest yields could be achieved at pH 7 by establishing optimum redox conditions for fermentation. Pilot-scale experiments at 32 °C showed that daily productivity (3.1 g∙Lreactor-1∙dHRT-1) and yields (150 mg∙gVS-1; OLR = 21 gVS∙Lreactor-1∙dHRT-1; pH 7) of short-chain fatty acids could be significantly improved, specifically for acetic and propionic acids. From these results, a robust dark fermentation step for recovery of valuable products from the solids treatment step in a biorefinery can be achieved.

Thermally enhanced solid-liquid separation process in food waste biorefinery: modelling the anaerobic digestion of solid residues.

The biochemical valorization potential of food waste (FW) could be exploited by extracting decreasing added-value bio-based products and converting the final residues into energy. In this context, multi-purpose and versatile schemes integrating thermal and biochemical conversion processes will play a key role. An upstream thermal pretreatment + solid-liquid separation unit was here proposed to optimize the conversion of the liquid fraction of FW into valuable chemicals through semi-continuous fermentation process, and the conversion of the residual solid fraction into biomethane through anaerobic digestion. The solid residues obtained after thermal pretreatment presented a higher soluble COD fraction, which resulted in higher methane production with respect to the raw residues (0.33 vs. 0.29 Nm3CH4 kg-1VSfed) and higher risk of acidification and failure of methanogenesis when operating at lower HRT (20d). On the contrary, at HRT = 40 d, the pretreatment did not affect the methane conversion rates and both tests evidenced similar methane productions of 0.33 Nm3CH4 kg-1VSfed. In the reactor fed with pretreated residue, the association of hydrogenotrophic methanogens with syntrophic bacteria prevented the acidification of the system. Modelling proved the eligibility of the FW solid residues as substrates for anaerobic digestion, given their small inert fractions that ranged between 0% and 30% of the total COD content.

Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences

Itaconic acid is an important bio-based chemical. The present study aims to evaluate the applicability of semi-continuous fermentation technique for itaconic acid production by Aspergillus terreus. The fermentation is planned to be connected with bipolar membrane electrodialysis unit for acid recovery. This process allows the reuse of residual glucose from the effluent. Our particular attention was focused on the effect of glucose concentration. Two different glucose supplementation strategies were tested: constant glucose concentration in the refilling medium and adjusted glucose concentration in order to maintain a continuously high – 120 g/L – glucose concentration in the fermentor. The itaconic acid titre, yield and productivity for the 24 h time periods between draining/refilling interventions were investigated. The constantly high glucose concentration in the fermentor resulted in doubled biomass formation. The average itaconic acid titre was 32.9 ± 2.7 g/L. The producing strain formed numerous spores during semi-continuous fermentation that germinated continuously. Yield and volumetric productivity showed a periodic pattern during the procedure.

Iterative optimization of exogenous genes and redirection of metabolic flux for enhanced itaconate biosynthesis in engineered Escherichia coli

BackgroundItaconate or itaconic acid (IA), a sustainable and valuable platform chemical, has garnered significant attention due to its potential as an alternative petroleum-derived compound. In the past, Aspergillus terreus offered a high titer of IA, but it still faced the challenges of low productivity. To overcome the problem, a fast-growing Escherichia coli BW25113 was involved consolidating the itaconate production pathway, eliminating byproduct-forming pathways, and enhancing intermediate levels to facilitate large-scale production. MethodE. coli was engineered through the deletion of isocitrate decarboxylase (Icd) and the overexpression of pyruvate carboxylase (CgPyc), cis-aconitate decarboxylase (AtCadA) and aconitate hydratase (ENAcnA) to facilitate the IA biosynthesis pathway. A B0015 terminator was encoded behind AtCadA to improve transcription efficiency as well as poxB gene was deleted to accelerate carbon influx towards the tricarboxylic acid cycle instead of forming the byproduct acetate. A semi-continuous batch fermentation approach was employed for large-scale IA biosynthesis to address ATP insufficiency in long-termed fermentation. Significant findingThe engineered strain AB15C/Δicd improved IA titer by 20 % compared to the control strain. By deleting the acetate forming pathway under acidic condition, AB15C/ΔicdΔpoxB increased itaconate production. Overexpression of ENAcnA in AB15CEN*/ΔicdΔpoxB strain resulted in 13.8 g/L of IA. Finally, a semi-continuous batch fermentation was implemented, leading to an IA accumulation of 35.5 g/L after 4 batches.

Biogas slurry recirculation regulates food waste fermentation: Effects and mechanisms

Regularly adding biogas slurry into fermentation reactors is an effective way to enhance hydrogen or methane production. However, how this method affects the production of valuable organic acids and alcohols is still being determined. This study investigated the effects of different addition ratios on semi-continuous fermentation reactors using food waste as a substrate. The results showed that an addition ratio of 0.2 increased lactic acid production by 30% with a yield of 0.38 ± 0.01 g/g VS, while a ratio of 0.4 resulted in mixed acid fermentation dominated by n-butyric acid (0.07 ± 0.01 g/g VS) and n-caproic acid (0.06 ± 0.00 g/g VS). The introduction of Bifidobacteriaceae by biogas slurry played a crucial role in increasing lactic acid production. In contrast, exclusive medium-chain fatty acid producers enhanced the synthesis of caproic acid and heptanoic acid via the reverse β-oxidation pathway. Mechanism analyses suggested that microbial community structure and activity, substrate hydrolysis, and cell membrane transport system and structure changed to varying degrees after adding biogas slurry.

Precise fermentation coupling with simultaneous separation strategy enables highly efficient and economical sophorolipids production

Sophorolipids (SLs) represent highly promising biosurfactants. However, its widespread production and application encounter obstacles due to the significant costs involved. Here, an intelligent and precise regulation strategy was elucidated for the fermentation process coupled with in-situ separation production mode, to achieve cost-effective SLs production. Firstly, a mechanism-assisted data-driven model was constructed for “on-demand feeding of cells”. Moreover, a strategy of step-wise oxygen supply regulation based on the demand for cell metabolic capacity was developed, which accomplished “on-demand oxygen supply of cells”, to optimize the control of energy consumption. Finally, a systematic approach was implemented by integrating a semi-continuous fermentation mode with in-situ separation technology for SLs production. This strategy enhanced SLs productivity and yield, reaching 2.30 g/L/h and 0.57 g/g, respectively. These values represented a 40.2% and 18.7% increase compared to fed-batch fermentation. Moreover, the concentration of crude SLs after separation reached 793.12 g/L, facilitating downstream separation and purification processes.

Promoting Circular Economy in the Palm Oil Industry through Biogas Codigestion of Palm Oil Mill Effluent and Empty Fruit Bunch Pressed Wastewater

This research aimed to investigate the biogas production and circular economy perspective in the palm oil industry through codigestion of oil palm empty fruit bunch (EFB) pressing wastewater and palm oil mill effluent (POME). The EFB pressing method constitutes an alternative new technology used to extract the remaining oil, increasing palm oil product; however, it produces highly polluted wastewater. Batch experiments were carried out at 35 °C to investigate the optimal ratios of EFB wastewater, inoculums, and POME. The optimal condition was 45% POME + 50% seed + 5% EFB wastewater. This condition was then used in semicontinuous fermentation where the optimal hydraulic retention time (HRT) totaled 25 days. The accumulated biogas was 18,679 mL/L while the accumulated methane totaled 6778 mL/L. The methane content was 62%, and the COD removal efficiency was 67%. The sludge produced from the HRT 25-days digester complied with the organic compost standard which could be further used to nourish the soil. An economic analysis of the EFB pressing project revealed a higher internal rate ratio with shorter payback compared with the conventional process. These results provide information on the circular economic approach to promote sustainable palm oil processing.

Biochemical hydrogen potential assay for predicting the patterns of the kinetics of semi-continuous dark fermentation

The performance and kinetics response of thermophilic semi-continuous dark fermentation (DF) of simulated complex carbohydrate-rich waste was investigated at various hydraulic retention times (HRT) (2, 2.5, and 3 d) and compared with data obtained from biochemical hydrogen potential assay (BHP). A culture of Thermoanaerobacterium thermosaccharolyticum was used as the inoculum and dominated both in BHP and semi-continuous reactor. Both the modified Gompertz and first-order models described the DF kinetics well (R2 = 0.97–1.00). HRT of 2.5 d was found to be optimal in terms of maximum hydrogen production rate and hydrogen potential, which were 3.97 and 1.26 times higher, respectively, than in BHP. The hydrolysis constant was highest at HRT of 3 d and was closest to the value obtained in the BHP. Overall, BHP has been shown to be a useful tool for predicting H2 potential and the hydrolysis constant, while the maximum H2 production rate is greatly underestimated.

Efficient production of hyaluronic acid by Streptococcus zooepidemicus using two-stage semi-continuous fermentation

Hyaluronic acid is a kind of mucopolysaccharide that has wide applications in cosmetics, health food, and orthopedics. Using Streptococcus zooepidemicus ATCC 39920 as parent, a beneficial mutant SZ07 was obtained by UV mutagenesis, giving 1.42 g/L hyaluronic acid in shake flasks. To enhance the efficiency of hyaluronic acid production, a semi-continuous fermentation process consisted of two-stage 3–L bioreactors was developed, in which 1.01 g/L/h productivity and 14.60 g/L hyaluronic acid were obtained. To further enhance the titer of hyaluronic acid, recombinant hyaluronidase SzHYal was added into 2nd stage bioreactor at 6 h to reduce the viscosity of broth. The highest hyaluronic acid titer of 29.38 g/L was achieved with a productivity of 1.13 g/L/h at 300 U/L SzHYal after 24 h. This newly developed semi-continuous fermentation process provides a promising strategy for the industrial production of hyaluronic acid and related polysaccharides.

Modification and calibration of anaerobic digestion model 1 to simulate volatile fatty acids production during fermentation of municipal sludge

The present study modified the anaerobic digestion model No.1 (ADM1) to simulate biological volatile fatty acids (VFA) production from municipal sludge. A variable and constant stoichiometric approach was used to modify the ADM1. The model was implemented in the AQUASIM 2.0 modeling platform and calibrated with experimental data from the bench-scale semi-continuous flow acid fermenters to assess the model's performance in simulating the VFA generation under different operating conditions. After parameter estimation, the modified ADM1 simulated experimental results of acetic, propionic, and butyric acids with reasonable accuracy and goodness of fit values ranging from 0.90 to 0.99. The model validation using an independent data set from the calibration data successfully simulated propionic (goodness of fit of 0.89) and butyric acids (goodness of fit of 0.88) but underestimated acetic acid production (goodness of fit of 0.73). The presented results extend the applicability of the ADM1 to simulate VFA during anaerobic fermentation.

Pilot-scale fermentation of 300 t/a total solvents from sweet sorghum stalk juice

Solvent production from non-grain crops as the substrate via microbial fermentation has been projected to replace highly polluting and unsustainable chemical production methods in fossil oil refinery. Although a promising process pilot-scale studies, the prerequisite for production at industrial scale, are rare. This study reports a complete pilot test process using sweet sorghum stalk juice as feedstock, including the acquisition of sweet sorghum stalk juice, laboratory screening of suitable strains, optimization of industrial conditions, and a pilot-scale test at Yuanxing Bioenergy Co., ltd., China. Strain CIBT S0031 (Clostridium saccharobutylicum DSM13864) performed exceptionally in sweet sorghum stalk juice fermentation compared to other solventogenic clostridia tested. Furthermore, a simple and reliable non-anaerobic inoculation method applicable to extensive industrial solvent fermentation was developed. The optimal minimal medium was adapted for pilot scale fermentation, containing only sweet sorghum stalk juice diluted with industrial water, 1.5 g/L ammonium sulfate, and 1 g/L calcium carbonate. The solvent titers of single batch fermentation (2.36 m3), fed-batch (2.28 m3), and semi-continuous fermentation (7.14 m3) reached 16.5 g/L, 19.29 g/L, and 18.39 g/L, respectively, which replicated the laboratory results at the ton-scale. In the multi-stage process for continuous fermentation (total of 48.3 m3, chained with one breeding tank and three fermenters of 16 m3), 800 kg of solvents was produced with a yield of 0.45 g /g, comparable to corn-based fermentation. This study verified the effectiveness of the tested strain and the pilot process, thus laying a solid foundation for the large-scale production of solvents from a non-grain crop.