Semi-continuous Fermentation Research Articles

Shaping microbial consortia in coupling glycerol fermentation and carboxylate chain elongation for Co-production of 1,3-propanediol and caproate: Pathways and mechanisms

Glycerol is presently being generated in surplus with the rapid growth of the biodiesel industry and seeks ways to be upcycled, rather than to be treated with costs. Glycerol for the co-production of 1,3-propanediol (1,3-PDO) and caproate has a great prospect. Yet, its technical difficulty lies in the enhancement of caproate productivity, which requires the presence of ethanol as a co-substrate and necessitates the co-existence of functional microbes for glycerol fermentation and chain elongation. This study successfully achieved 6.38 mM C 1,3-PDO d−1 and 2.95 mM C caproate d−1 in a 2-L mixed-cultured semi-continuous fermenter with a glycerol-ethanol-acetate stoichiometric ratio of 4:3:1. Such conversions were mainly facilitated by a microbial community of Eubacterium limosum, Clostridium kluyveri and Massilibacterium senegalense. With such a synergistic microbiome, the co-production of 1,3-PDO and caproate was achieved from glycerol without ethanol addition. Based on metagenomics, E. limosum is capable of converting glycerol to 1,3-PDO, ethanol and H2, and also redirecting the electron potential of H2 into acetate via the Wood–Ljungdahl pathway, which is then used for chain elongation. C. kluyveri worked synergistically with E. limosum by consuming ethanol and acetate for caproate production. M. senegalense encodes for ethanol oxidation to acetate and butyrate, facilitating the generation of these intermediates for C. kluyveri elongation to caproate. During the transition between fermentation and elongation, an unexpected observation of poly-β-hydroxybutyrate (PHB) formation and reutilization by M. senegalense may be associated with butyrate formation for further caproate generation. The knowledge gleaned from the substrate constitute, microbial consortium and their synergetic metabolism demonstrates a resource upgrade potential for crude glycerol or glycerol-containing wastewater generated from the biodiesel industry.

Modified Anaerobic Digestion Model No. 1 for modeling methane production from food waste in batch and semi-continuous anaerobic digestions

A modified Anaerobic Digestion Model No. 1 (ADM1) with optimized kinetic parameters was presented to model methane production in the anaerobic digestion of food waste. Experimental data from batch and semi-continuous fermentations were used to calibrate and verify the model. Modified ADM1 simulation was carried out using AQUASIM 2.0 software. Sensitivity analysis was used to identify and evaluate the most sensitive kinetic parameters during biogas production. The decay constant of microorganisms, the disintegration constant, the hydrolysis constant of carbohydrates, the Monod maximum specific substrate uptake rate, and the half-saturation constants affected biogas production significantly. The optimized values of these parameters were 0.001, 0.16, 3, 1 and 0.23, respectively. Optimization results were validated using batch and semi-continuous experiments. The modified ADM1 well-predicted methane production, with R2 values for the validation experiments all above 90%. These results can be used as basic data to simulate methane production in full-scale reactors.

ThermoFlex: Heat Storage in Secondary Digesters for Flexible Power Generation of Biogas Plants

AbstractFor flexible power generation of biogas plants and effective heat utilization, efficient heat storage technologies will play a decisive role. For this purpose, a process is being developed in the ThermoFlex project, based on controlled operation of a secondary digester in thermophilic temperature ranges after a mesophilic main digester. This allows effective, flexible heat storage in the secondary digester for digester heating and external heat utilization at an acceptable temperature level without additional storage. The process biology of the secondary digester tolerates controlled temperature variations. An overview of the application potential and design of the ThermoFlex process is given. Batch and semicontinuous fermentation tests at various temperatures are described.

Aroma profile of pilot plant-scale produced fruit vinegar using a thermotolerant Acetobacter pasteurianus strain isolated from Moroccan cactus

In this study, new thermotolerant Acetobacter pasteurianus CV01 strain recently isolated from local product of Morocco has been investigated for its ability to perform efficient acetous fermentation at a large-scale. Firstly, the thermotolerance basis bioconversion of CV01 strain was compared to other mesophilic and thermotolerant acetic acid bacteria. Subsequently, CV01 strain was assessed for its ability to produce and tolerate high amount of acetic acid at optimal and thermal stress conditions in lab-scale bioreactor. It was found that the studied strain exhibited thermotolerant properties compared to reference strains and could withstand the increase in temperature during acetous fermentation in fermenter. Furthermore, gas chromatography-mass spectrometry (GC-MS) was used on the samples prepared with solid phase microextraction (SPME) to determine the volatile compounds of the pilot plant produced apple vinegar based on developed start-up and semi-continuous fermentation protocol. The operation strategy in the 500-L pilot plant scale acetator allowed achieving 7.3% (w/v) of final acetic acid concentration recording high yield and acetification rate. The aroma profile of experimentally produced vinegar was found different from that of the commercial reference one. According to the literature, the results obtained show that major volatile compounds found in pilot-plant produced apple vinegar are related to good aromatic note descriptors which could have a positive impact on the organoleptic quality of industrial vinegar. Consequently, it can be concluded that CV01 Acetobacter strain is well suited for large-scale production of high quality fruit vinegar.

The effect of encapsulated nitrate and monensin on ruminal fermentation using a semi-continuous culture system.

Because enteric methane (CH4) production from ruminants represents a source of greenhouse gas emissions and an energy loss for the host animal alternatives to minimize emissions is a current research priority. Seven 37-d trials tested the effect of encapsulated nitrate (EN) and sodium monensin (MON) in diets commonly fed to dairy (DAIRY; 50:50 forage to concentrate; four trials) and beef cattle (BEEF; 15:85 forage to concentrate; three trials) on rumen fermentation and CH4 production using a semi-continuous fermentation system. A 3 × 2 factorial arrangement was used and additives (0, 1.25, and 2.5% of EN; 0 and 4 mg/L of MON) were tested alone and combined (EN + MON) totaling six treatments. Rumen fluid was pooled from five nonadapted lactating cows fed 50:50 forage to concentrate diet 3 h after morning feeding, and 1 L of processed inoculum was transferred to 2.2-L vessels. Treatment diets were added to nylon bags which remained in the anaerobic fermentation of mixed rumen microorganisms for 48 h. Nitrate decreased CH4 production in DAIRY (24.7 vs. 32.1 mM/d; P < 0.01) and BEEF trials (33.5 vs. 43.5 mM/d; P < 0.01). Methane production was decreased by MON in DAIRY (26.3 vs. 32.1; P < 0.01) and BEEF (26.6 vs. 43.5 mM/d; P < 0.01). The combination of EN + MON further decreased CH4 in DAIRY (21.3 vs. 32.1 mM/d; P = 0.03) and BEEF (19.3 vs. 43.5 mM/d; P = 0.01). Nitrate did not affect major VFA production in DAIRY and BEEF trials, but significantly decreased digestion of protein (96.8 vs. 97.6%; P < 0.01) and starch (79.0 vs. 80.4%; P < 0.01) in DAIRY and NDF (29.3 vs. 32.5%; P < 0.01) and starch (88.5 vs. 90.3%; P < 0.01) in BEEF. Monensin significantly affected VFA pattern with an increase in propionate (P < 0.01) and a decrease on acetate (P < 0.01) production with consequent decrease on acetate-to-propionate ratio in DAIRY (1.6 vs. 2.0; P < 0.01) and BEEF (1.6 vs. 1.9; P < 0.01). Monensin decreased NDF digestion in BEEF only (29.3 vs. 32.5 %; P < 0.01). Significant concentrations of nitrate and nitrite were detected only for EN and EN + MON (P < 0.01). Nitrate and MON effectively decreased CH4 production when fed separately and the combination of additives additively decreased CH4 production.

Volatile fatty acids production from household food waste

The present study aims at determining key factors and current constrains of household food waste (HFW) fermentation process and its large-scale implementation within the frame of biorefinery concept. The production of Volatile Fatty Acids (VFA) from HFW by a mixed microbial culture fermentation (MMC) was studied in both, batch and semi-continuous scale reactors.Results from batch scale trials without pH control pointed out that highest conversion yield obtained, 111 ± 20 mg of chemical oxygen demand, as VFA, per gram of volatile solids fed to the system, was reached after 6-7days of fermentation. Moreover, during semi-continuous fermentation at uncontrolled pH and hydraulic retention time (HRT) of 6 d, was observed a VFA production of 3.3 ± 0.8 g·L-1 on average, being increased up to a maximum of 30.1 g·L-1 when pH was controlled at pH 5.5.In light of the results obtained, HFW should be considered a promising feedstock for the production of chemical intermediates like carboxylic acids and platform chemicals in general. Nevertheless, best optimal operational conditions have not been still properly deciphered and further research must be done to foster economic viability of VFA production in future urban biorefineries.

Effects of pH adjustment on the hydrolysis of Al-enhanced primary sedimentation sludge for volatile fatty acid production

Aluminum-based chemically enhanced primary sedimentation has seen increasing use in wastewater treatment plants. However, the derived sludge (Al-sludge) has low biodegradability due to the inhibition of Al-coagulants on organic hydrolysis, which increases the difficulty of sludge treatment and limits the potential for resource recovery. In this study, the effects of pH adjustment on the pretreatment and fermentation of Al-sludge was investigated, with a focus on sludge hydrolysis and production of volatile fatty acids (VFAs). The effects of abiotic and biotic hydrolysis on sludge disintegration, organics solubilization, and hydrolytic enzyme activity were also evaluated. The results show that adjustment of the pH to between 8.0 and 10.0 was more effective than adjustment to between 2.0 and 6.0 for sludge hydrolysis and subsequent VFA production. The enzymatic activities of protease and α-glucosidase showed a positive correlation with the pH values, leading to the highest VFA yield (275 mg-COD/g-VS) for the initial pH 10.0 condition, with 46% improvement over the control reactor (without pH adjustment). Semi-continuous fermentation was found to be more effective than batch fermentation for the biotic hydrolysis rate, kH. Little phosphorus (<0.1 mg/L) was released from the Al-sludge into the fermented sludge liquor, so P removal was not necessary before the VFAs were used. The wastewater treatment process with Al-based chemically enhanced primary sedimentation and pH adjustment for enhanced sludge fermentation can recover 24% of organic carbon from domestic wastewater.

Development of a semi-continuous two-stage simultaneous saccharification and fermentation process for enhanced 2,3-butanediol production by Klebsiella oxytoca.

There is growing interest in the production of 2,3-butanediol (2,3-BD) from renewable resources by microbial fermentation because of its wide applications to specialty and commodity chemical industries. Klebsiella oxytoca usually produces 2,3-BD as a major end product during the fermentation of carbohydrates. This is the first study to provide a high-efficiency simultaneous saccharification and 2,3-BD fermentation process. Also, this study proves the stability of a metabolically engineered 2,3-BD overproducing K. oxytoca strain for industrial application.

To study the effect of glucose feeding rate for sustainable voltage generation from microbial fuel cell containing wastewaters

The sustainable voltage generation was extensively experimented through semi batch using isolated pure microorganism. Though higher voltage generation of 911 mV can be possible through optimisation of process parameters by batch fermentation, however, sustainable power generation was not found in batch process. Therefore, the potential influence of several parameters for voltage generation was addressed by semi continuous fermentation process. In this present work sustainable voltage generation (550 mV) was achieved through fed batch mode when initial glucose was kept 8 g/L in the production medium. However, 18 g/L of glucose (25 ml) was consecutively fed at interval of 72 and 108 h during semi continuous fermentation.

Semicontinuous sophorolipid fermentation using a novel bioreactor with dual ventilation pipes and dual sieve-plates coupled with a novel separation system.

SummarySophorolipids (SLs) are biosurfactants with widespread applications. The yield and purity of SLs are two important factors to be considered during their commercial large‐scale production. Notably, SL accumulation causes an increase in viscosity, decrease in dissolved oxygen and product inhibition in the fermentation medium. This inhibits the further production and purification of SLs. This describes the development of a novel integrated system for SL production using Candida albicans O‐13‐1. Semicontinuous fermentation was performed using a novel bioreactor with dual ventilation pipes and dual sieve‐plates (DVDSB). SLs were separated and recovered using a newly designed two‐stage separation system. After SL recovery, the fermentation broth containing residual glucose and oleic acid was recycled back into the bioreactor. This novel approach considerably alleviated the problem of product inhibition and accelerated the rate of substrate utilization. Production of SLs achieved was 477 g l−1, while their productivity was 1.59 g l−1 h−1. Purity of SLs improved by 23.3%, from 60% to 74%, using DVDSB with the separation system. The conversion rate of carbon source increased from 0.5 g g−1 (in the batch fermentation) to 0.6 g g−1. These results indicated that the integrated system could improve the efficiency of production and purity of SLs.

Acidogenic fermentation of iron-enhanced primary sedimentation sludge under different pH conditions for production of volatile fatty acids

Iron-based chemically enhanced primary sedimentation (CEPS) is increasingly adopted for wastewater treatment in mega cities, producing a large amount of sludge (Fe-sludge) with a high content of organics for potential organic resource recovery. In this experimental study, acidogenic fermentation was applied treat FeCl3-based CEPS sludge for production of volatile fatty acids (VFAs) at different pHs. Batch fermentation tests on the Fe-sludge with an organic content of 10 g-COD/L showed that the maximum VFAs production reached 2782.2 mg-COD/L in the reactor without pH control, and it reached 688.4, 3095.3, and 2603.7 mg-COD/L in reactors with pHs kept at 5.0, 6.0 and 8.0, respectively. Analysis of the acidogenesis kinetics and enzymatic activity indicated that the alkaline pH could accelerate the rate of organic hydrolysis but inhibited the further organic conversion to VFAs. In semi-continuous sludge fermentation tests, the VFAs yield in the pH6 reactor was 20% higher than that in the control reactor without pH regulation, while the VFAs yield in the pH8 reactor was 10% lower than the control. Illumina MiSeq sequencing revealed that key functional microorganisms known for effective sludge fermentation, including Bacteroidia and Erysipelotrichi, were enriched in the pH6 reactor with an enhanced VFAs production, while Clostridia became more abundant in the pH8 reactor to stand the unfavorable pH condition. The research presented acidogenic fermentation as an effective process for CEPS sludge treatment and organic resource recovery and provided the first insight into the related microbial community dynamics.

Continuous liquid fermentation of pretreated waste activated sludge for high rate volatile fatty acids production and online nutrients recovery

Raw sludge was pretreated, and the separated sludge liquid was used as substrate in a continuous operated up-flow anaerobic sludge blanket reactor to produce volatile fatty acids (VFAs). The highest VFA productivity of continuous fermentation with sludge liquid at an organic loading rate (OLR) of 10.0 kg COD/m3/d was about 5.0-fold and 4.0-fold higher than batch and semi-continuous fermentation with pretreated sludge slurry, respectively. Moreover, the liquid fermentation with an OLR of 10.0 kg COD/m3/d consumed the least energy, which was about 10.57% and 12.12% of batch and semi-continuous sludge fermentation, respectively. When combined with online nitrogen and phosphorus recovery, VFA production further increased by 20.67% and struvite recovery efficiency reached 1.98 ± 0.28 g/g PO43−. The process showed high VFA production, low energy consumption and good nutrients recovery by continuous liquid anaerobic fermentation, significantly increasing the economic potential of VFA production from waste activated sludge.

Hydrogel Film-Immobilized Lactobacillus brevis RK03 for γ-Aminobutyric Acid Production.

Hydrogels of 2-hydroxyethyl methacrylate/polyethylene glycol diacrylate (HEMA/PEGDA) have been extensively studied for their use in biomedical and pharmaceutical applications owing to their nontoxic and highly hydrophilic characteristics. Recently, cells immobilized by HEMA/PEGDA hydrogels have also been studied for enhanced production in fermentation. Hydrogel films of HEMA/PEGDA copolymer were generated by Ultraviolet (UV)-initiated photopolymerization. The hydrogel films were used to immobilize viable Lactobacillus brevis RK03 cells for the bioconversion of monosodium glutamate (MSG) to γ-aminobutyric acid (GABA). The mechanical properties and fermentation yields of the L. brevis RK03 cells immobilized on polyacrylate hydrogel films with different monomeric formulations were investigated. Fermentation was carried out in 75 mL de Man, Rogosa and Sharpe (MRS) medium containing various concentrations of MSG. We found that HEMA (93%)/PEGDA (3%) hydrogels (sample H) maximized GABA production. The conversion rate of MSG to GABA reached a maximum value of 98.4% after 240 h. Bioconversion activity gradually declined after 420 h to 83.8% after five cycles of semi-continuous fermentation. Our results suggest that HEMA (93%)/PEGDA (3%) hydrogels have great potential for use in GABA production via semi-continuous fermentation.

Semi-Continuous Fermentation of Onion Vinegar and Its Functional Properties.

For the fermentation of vinegar using onion, acetic acid bacteria and yeast strains with high fermentation ability were screened. Among them, Saccharomyces cerevisiae 1026 was selected as a starter for ethanol production and Acetobacter orientalis MAK88 was selected as a vinegar producer. When the two-stage fermentation of onion vinegar was performed at 28 °C, the titratable acidity reached 4.80% at 24 h of fermentation. When semi-continuous fermentation proceeded to charge-discharge consisting of three cycles, the acetic acid content reached 4.35% at 48 h of fermentation. At this stage, the fermentation efficiency, acetic acid productivity, and specific product formation rate were 76.71%, 17.73 g/(L·d), and 20.58 g/(g·h), respectively. The process in this study significantly reduced the fermentation time and simplified the vinegar production process. The content of total flavonoids and total polyphenols in onion vinegar were 104.36 and 455.41 μg/mL, respectively. The antioxidant activities of onion vinegar in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS+) radical scavenging activity, and reducing power were 75.33%, 98.88%, and 1.28, respectively. The nitrite scavenging abilities of onion vinegar were 95.38 at pH 1.2. The onion vinegar produced in this study showed higher organoleptic acceptability than commercial onion vinegar.

Application of metabolic controls for the maximization of lipid production in semicontinuous fermentation

Acetic acid can be generated through syngas fermentation, lignocellulosic biomass degradation, and organic waste anaerobic digestion. Microbial conversion of acetate into triacylglycerols for biofuel production has many advantages, including low-cost or even negative-cost feedstock and environmental benefits. The main issue stems from the dilute nature of acetate produced in such systems, which is costly to be processed on an industrial scale. To tackle this problem, we established an efficient bioprocess for converting dilute acetate into lipids, using the oleaginous yeast Yarrowia lipolytica in a semicontinuous system. The implemented design used low-strength acetic acid in both salt and acid forms as carbon substrate and a cross-filtration module for cell recycling. Feed controls for acetic acid and nitrogen based on metabolic models and online measurement of the respiratory quotient were used. The optimized process was able to sustain high-density cell culture using acetic acid of only 3% and achieved a lipid titer, yield, and productivity of 115 g/L, 0.16 g/g, and 0.8 g⋅L-1⋅h-1, respectively. No carbon substrate was detected in the effluent stream, indicating complete utilization of acetate. These results represent a more than twofold increase in lipid production metrics compared with the current best-performing results using concentrated acetic acid as carbon feed.

Comparison of the anaerobic digestion at the mesophilic and thermophilic temperature regime of organic wastes from the agribusiness

An overall kinetic power law model has been successfully applied to study the anaerobic digestion of agricultural wastes. In this comparative kinetic study feed composition, organic load rate, residence time and process temperature have been systematically varied in an automated semi-continuous fermentation system to obtain the dependency of the rate of degradation as biogas production on the organic load rate and temperature.The results show that the overall reaction order depend only on the Chemical Oxygen Demand (COD) at values between 3.6 and 3.7. The Arrhenius approach shows a shift in the rate determining step between the mesophilic and thermophilic temperature regimes. The activation energy at the temperature insensitive mesophilic regime is very small at 8.9 (kJ/mole), while the activation energy at the temperature sensitive thermophilic regime lies around 117 (kJ/mole).

&lt;b&gt;&lt;i&gt;Zymomonas mobilis&lt;/i&gt; immobilized on loofa sponge: levan and ethanol production in semi-continuous fermentation

Zymomonas mobilis is a promising microorganism in the biotechnological production of ethanol and levan due to its low biomass and high tolerance to ethanol concentrations. Ethanol and levan production by Z. mobilis CDBB-603 immobilized on loofa sponge using semi-continuous fermentation is evaluated. The first experiment, conducted with initial volume 50 mL and incubated for 24 hours, produced maximum ethanol and levan production, respectively 13.56 g L -1 and 23.94 g L -1 , with 250 g L -1 sucrose and without agitation, at 30°C. The second experiment was based on the best condition obtained in the first, using fermenter with production scale of 200 mL and by semi-continuous process. The second experiment also assessed immobilized biomass reuse during 10 days (240 hours) and produced higher ethanol (34.64 g L -1 ) and levan (26.40 g L -1 ) production rates than in the first experiment. Experiment 2 also verified that the microorganism remained viable and produced ethanol and levan until the last (10 th ) recycle day.

Maximizing the production of butyric acid from food waste as a precursor for ABE-fermentation

The current study reports on the maximization of butyric acid production from food waste using a mixed microbial fermentation. In semi-continuous fermentations the effect of three different pH values (5.5, 7.0 and 9.0), three different temperatures (37°C, 55°C and 70°C) and two levels of hydraulic retention time (HRT, 2days and 6days) on the formation of butyric acid as well as total volatile fatty acid production (tVFA) were investigated.Overall, pH5.5 provided the lowest butyric acid concentrations regardless of the temperature and the HRT. At mesophilic temperature (37°C) alkaline conditions (pH9.0) lead to a strong incline of tVFA as well as butyric acid concentration probably due to a decreased solubilization of the substrate. However, most efficient in terms of butyric acid production was the fermentation conducted at 55°C and pH7 where a butyric acid concentrations of 10.55g/L (HRT 2days) and 13.00g/L (HRT 6days) were achieved. Additional experiments at 70°C showed declining butyric acid production. Increase of the HRT from 2days to 6days provided an increment of butyric acid concentration throughout almost all experimental settings. However, regarding volumetric productivity the increase in concentration does not compensate for the bigger reactor volume required to establish a higher HRT. At pH7 and 55°C the resulting volumetric production rates were 5.27g/L∗d at a HRT 2days and only 2.17g/L∗d at a HRT of 6days.

Effects of organic loading rate on biogas production from macroalgae: Performance and microbial community structure

Macroalgae biomass has been considered as a promising feedstock for biogas production. In order to improve the efficiency of anaerobic digestion (AD) of macroalgae, semi-continuous fermentation was conducted to examine the effects of organic loading rate (OLR) on biogas production from Macrocystis pyrifer. Results showed that, under OLRs of 1.37, 2.74, 4.12 and 6.85kgVSsubstrate/(m3·d), the average unit biogas yields were 438.9, 477.3, 480.1 and 188.7mL/(gVSsubstrated), respectively. It indicated that biogas production was promoted by the increased OLR in an appropriate range while inhibited by the OLR beyond the appropriate range. The investigation on physical-chemical parameters revealed that unfavorable VFAs concentration, pH and salinity might be the main causes for system failure due to the overrange OLR, while the total phenols failed to reach the inhibitory concentration. Microbial community analysis demonstrated that several bacterial and archaeal phyla altered with increase in OLR apparently.

Consolidated Bioprocessing for Butyric Acid Production from Rice Straw with Undefined Mixed Culture.

Lignocellulosic biomass is a renewable source with great potential for biofuels and bioproducts. However, the cost of cellulolytic enzymes limits the utilization of the low-cost bioresource. This study aimed to develop a consolidated bioprocessing without the need of supplementary cellulase for butyric acid production from lignocellulosic biomass. A stirred-tank reactor with a working volume of 21 L was constructed and operated in batch and semi-continuous fermentation modes with a cellulolytic butyrate-producing microbial community. The semi-continuous fermentation with intermittent discharging of the culture broth and replenishment with fresh medium achieved the highest butyric acid productivity of 2.69 g/(L· d). In semi-continuous operation mode, the butyric acid and total carboxylic acid concentrations of 16.2 and 28.9 g/L, respectively, were achieved. Over the 21-day fermentation period, their cumulative yields reached 1189 and 2048 g, respectively, corresponding to 41 and 74% of the maximum theoretical yields based on the amount of NaOH pretreated rice straw fed in. This study demonstrated that an undefined mixed culture-based consolidated bioprocessing for butyric acid production can completely eliminate the cost of supplementary cellulolytic enzymes.