Continuous Bioreactor Research Articles

Stable partial nitritation of mature landfill leachate in a continuous flow bioreactor: Long-term performance, microbial community evolution, and mechanisms

A continuous flow bioreactor was operated for 300 days to investigate partial nitritation (PN) of mature landfill leachate, establishing the long-term performance of the system in terms of the microbial community composition, evolution, and interactions. The stable operation phase (31–300 d) began after a 30 days of start-up period, reaching an average nitrite accumulation ratio (NAR) of 94.43% and a ratio of nitrite nitrogen to ammonia nitrogen (NO2−-N/NH4+-N) of 1.16. Some fulvic-like and humic-like compounds and proteins were effectively degraded in anaerobic and anoxic tanks, which was consistent with the corresponding abundance of methanogens and syntrophic bacteria in the anaerobic tank, and organic matter degrading bacteria in the anoxic tank. The ammonia-oxidizing bacteria (AOB) Nitrosomonas was found to be the key functional bacteria, exhibiting an increase in abundance from 0.27% to 6.38%, due to its collaborative interactions with organic matter degrading bacteria. In-situ inhibition of nitrite-oxidizing bacteria (NOB) was achieved using a combination of free ammonia (FA) and free nitrous acid (FNA), low dissolved oxygen (DO) with fewer bioavailable organics conditions were employed to maintain stable PN and a specific ratio of NO2−-N/NH4+-N, without an adverse impact on AOB. The synergistic relationships between AOB and both denitrifying bacteria and organic matter degrading bacteria, were found to contribute to the enhanced PN performance and microbial community structure stability. These findings provide a theoretical guidance for the effective application of PN-Anammox for mature landfill leachate treatment.

Continuous cultivation of Debaryomyces hansenii (LAF‐3 10 U) on dodecane in synthetic desalter effluent at varying dilution rates on dodecane

AbstractDesalter effluent (DE) is typically discharged into a petroleum wastewater treatment plant, but its high salt concentration deteriorates the biological treatment. This study used various dilution rates to investigate the treatment of a synthetic DE containing dodecane under saline conditions using a halotolerant yeast, Debaryomyces hansenii, to determine the optimum substrate concentration for use in continuous stirred‐tank reactors (CSTRs). A literature review indicated that this study was the first to examine the biological treatment of DE using D. hansenii in a CSTR system. At a low dodecane substrate concentration, DE did not inhibit D. hansenii growth, and the experimental data approached the Monod model, with μmax and Ks selected as 0.08 h−1 and 1575 mg L−1, respectively. The optimum removal of chemical oxygen demand (95.7% and 85%) was obtained at dilution rates of 0.007 and 0.026 d−1. Using D. hansenii in a CSTR system appeared to be a sustainable approach for the biological treatment of DE. Scale‐up of these laboratory findings to the industrial scale is required to confirm that petroleum DE can be treated using equalization and filtration tanks as a continuous bioreactor. Adjusting the dilution rate can provide sufficient time for biodegradation and hydrocarbon removal from high salt DE by halotolerant yeasts like D. hansenii.

Promising Application of Automated Liquid Culture System and Arbuscular Mycorrhizal Fungi for Large-Scale Micropropagation of Red Dragon Fruit

Red dragon fruit (Hylocereus polyrhizus) is an economic and promising fruit crop in arid and semi-arid regions with water shortage. An automated liquid culture system using bioreactors is a potential tool for micropropagation and large-scale production. In this study, axillary cladode multiplication of H. polyrhizus was assessed using cladode tips and cladode segments in gelled culture versus continuous immersion air-lift bioreactors (with or without a net). Axillary multiplication using cladode segments (6.4 cladodes per explant) was more effective than cladode tip explants (4.5 cladodes per explant) in gelled culture. Compared with gelled culture, continuous immersion bioreactors provided high axillary cladode multiplication (45.9 cladodes per explant) with a higher biomass and length of axillary cladodes. Inoculation of H. polyrhizus micropropagated plantlets with arbuscular mycorrhizal fungi (Gigaspora margarita and Gigaspora albida) significantly increased the vegetative growth during acclimatization. These findings will improve the large-scale propagation of dragon fruit.

An Extended Super-Twisting Algorithm for simultaneous estimation of reaction rates and input disturbance in bioprocess

The present contribution deals with the simultaneous estimation of the reaction rates and input disturbances in a bioprocess. Online estimation of just the reaction rates is possible by sliding mode observer techniques or high-gain observers, in finite-time or with exponential convergence, respectively. However, these techniques are not robust against unpredictable input bioreactor disturbances and are not readily adaptable to treat external disturbances. Motivated by these facts, we propose a novel extended super-twisting algorithm for a class of nonlinear systems, such that the estimation of uncertain parameters, unknown internal dynamics, and external disturbances converge in a finite time or a neighborhood near its nominal values. The effectiveness of the developed algorithm is tested through simulations in an anaerobic digestion process using a batch and a continuous bioreactor. Simulation results show good robustness performance of the extended super-twisting algorithm.

Continuous bioreactors enable high-level bioremediation of diesel-contaminated seawater at low and mesophilic temperatures using Antarctic bacterial consortia: Pollutant analysis and microbial community composition

In 2020, more than 21,000 tons of diesel oil were released accidently into the environment with most of it contaminating water bodies. There is an urgent need for sustainable technologies to clean up rivers and oceans to protect wildlife and human health. One solution is harnessing the power of bacterial consortia; however isolated microbes from different environments have shown low diesel bioremediation rates in seawater thus far. An outstanding question is whether Antarctic microorganisms that thrive in environments polluted with hydrocarbons exhibit better diesel degrading activities when propagated at higher temperatures than those encountered in their natural ecosystems.Here, we isolated bacterial consortia, LR-30 (30 °C) and LR-10 (10 °C), from the Antarctic rhizosphere soil of Deschampsia antarctica (Livingston Island), that used diesel oil as the only carbon substrate. We found that LR-30 and LR-10 batch bioreactors metabolized nearly the entire diesel content when the initial concentration was 10 (g/L) in seawater. Increasing the initial diesel concentration to 50 gDiesel/L, LR-30 and LR-10 bioconverted 33.4 and 31.2 gDiesel/L in 7 days, respectively. The 16S rRNA gene sequencing profiles revealed that the dominant bacterial genera of the inoculated LR-30 community were Achromobacter (50.6%), Pseudomonas (25%) and Rhodanobacter (14.9%), whereas for LR-10 were Pseudomonas (58%), Candidimonas (10.3%) and Renibacterium (7.8%). We also established continuous bioreactors for diesel biodegradation where LR-30 bioremediated diesel at an unprecedent rate of (34.4 g/L per day), while LR-10 achieved (24.5 g/L per day) at 10 °C for one month. The abundance of each bacterial genera present significantly fluctuated at some point during the diesel bioremediation process, yet Achromobacter and Pseudomonas were the most abundant member at the end of the batch and continuous bioreactors for LR-30 and LR-10, respectively.

Biological treatment of cadmium (Cd2+)-containing wastewater with sulfate as the electron acceptor and its microbial community

The pollution of cadmium (Cd) to environment is becoming increasingly serious. In this study, the ability of the continuous flow bioreactor with sulfate as the electron acceptor to remove pollutants from Cd-containing wastewater was investigated. The results show that Cd2+ could be effectively removed and the concentration of Cd2+ in the anaerobic effluent is less than 0.01 ​mg/L. Cd can affect the decomposition of urea by anaerobic sludge. In addition, ammonia nitrogen could be also effectively removed with the removal rate was approximately 72.4%. The activated sludge mainly removed Cd through the precipitation of cadmium sulfide outside the bacteria after sulfide was generated by bacterial sulfate metabolism, facilitated continuous biological removal of Cd from wastewater. Microbial community structure analysis revealed Trichococcus and Hyphomicrobium mainly occurred in anaerobic and anoxic reaction, and Sulfurovum was unique to the sulfur oxidation reaction, with a content of 10.20% and the ability to oxidize sulfide. The content of Nitrosomonas in aerobic reaction zone reached 36.75%, but no nitrifying bacteria were detected. The technology in this study, which used sulfate as the electron acceptor for the continuous biological removal of Cd2+, has future engineering application value for the treatment of wastewater containing low concentrations of Cd.

Effect of Soil Type and In Vitro Proliferation Conditions on Acclimation and Growth of Willow Shoots Micropropagated in Continuous Immersion Bioreactors.

Salix viminalis L. is a species with high capacity for micropropagation and acclimation and could therefore be used to evaluate emergent techniques in the field of plant propagation. The aims of this study were to propagate willow in liquid medium with a continuous immersion system, to explore the application of photoautotrophic conditions and to investigate the adaptation of willow plantlets to different soils that could be used as alternatives to commercial peat. For proliferation, we used 3% sucrose or sugar-free medium, and as substrates, we used commercial peat, a soil from an oak forest with high organic matter content and a crop soil with low organic matter content. The effect of sugar supplementation during proliferation and the soil characteristics during acclimation and growth were evaluated on the basis of aerial and root growth and the hydrolytic and dehydrogenase enzymatic activities of the soils. The results indicate that under photoautotrophic conditions, the supplementation of sucrose during micropropagation did not affect the subsequent growth of the plantlets. All plants acclimated without loss, but the type of soil influenced the height and vigor. Plants produced the highest shoots in peat, whereas the most root development occurred in crop soil. Soil enzyme activities were more influenced by the type of soil than by the presence of plants.

Synergistic inhibition of Pseudomonas fluorescens growth and proteases activities via sodium chlorite-based oxyhalogen

Pseudomonas fluorescens is considered among the main spoilage microorganisms due to its ability to produce proteases. Food deterioration caused by spoilage microorganisms has a major impact on food quality and the environment. The inactivation of Pseudomonas fluorescens growth and protease production was intensively investigated with the use of Salmide®, A Sodium Chlorite-Based Oxy­halogen Disinfectant. A unique M9 media was also developed to assure sufficient protease productions with different mutants of Pseudomonas fluorescens as a microbioreactor. Mutations were induced by classical whole-cell mutagenesis using N-methyl-N′- nitro-N-nitrosoguanidine (NTG). A dramatic decrease occurred in protease activity when different Salmide concentrations (5, 10, and 15 ppm) were added to the growth culture followed by a complete inhibition concentration (20, 25, 50, and 100 ppm) of Salmide. However, no significant inhibition occurred once it is secreted out of cells. Some mutants were resistant and remains highly stable with high protease production under stressful conditions of Sodium Chlorite-Based Oxy­halogen. The production of the protease showed a linear correlation with the increase in incubation time using a continuous culture bioreactor system and recorded maximum protease activity after 40 h. Our findings would offer alternative antimicrobial procedures for food and industrial sectors.

Kendali Optimal Model Pertumbuhan Mikroalga dalam Chemostat

Microalgae is one of Indonesia's natural potentials that can be utilized in various needs such as producing energy products (bioethanol, biodiesel), biopolymer producing plastic and as a soil mixture for construction engineering, even as waste treatment by absorbing the remaining organic compounds and nutrients or absorb some of the hazardous compounds contained in the waste. One of the microalgae culture media is chemostat. Chemostat is a type of continuous bioreactor that functions for the cultivation of microalgae and other microorganisms that are usually used in laboratory and industrial scales. In this study, control efforts on the microalgae growth model in the chemostat were carried out. This is so that the growth of microalgae reaches the equilibrium value in the minimum possible time. By applying Pontryagin's Minimum Principle and dilution parameters as control variables, it is obtained that the type of control carried out is bang-bang control. Then the control problem is solved numerically using the ICLOCS2 toolbox. The simulation results showed that microalgae were able to reach equilibrium conditions faster by 40.9% under the control. So, it can be said that the system can be controlled optimally.

Improvement of Biohydrogen Production from Date Wastes by Thermotoga maritima Using a Continuous Anaerobic Membrane Bioreactor

Improvement of Biohydrogen Production from Date Wastes by Thermotoga maritima Using a Continuous Anaerobic Membrane Bioreactor

Controlling microbial co-culture based on substrate pulsing can lead to stability through differential fitness advantages.

Microbial consortia are an exciting alternative for increasing the performances of bioprocesses for the production of complex metabolic products. However, the functional properties of microbial communities remain challenging to control, considering the complex interaction mechanisms occurring between co-cultured microbial species. Indeed, microbial communities are highly dynamic and can adapt to changing environmental conditions through complex mechanisms, such as phenotypic diversification. We focused on stabilizing a co-culture of Saccharomyces cerevisiae and Escherichia coli in continuous cultures. Our preliminary data pointed out that transient diauxic shifts could lead to stable co-culture by providing periodic fitness advantages to the yeast. Based on a computational toolbox called MONCKS (for MONod-type Co-culture Kinetic Simulation), we were able to predict the dynamics of diauxic shift for both species based on a cybernetic approach. This toolbox was further used to predict the frequency of diauxic shift to be applied to reach co-culture stability. These simulations were successfully reproduced experimentally in continuous bioreactors with glucose pulsing. Finally, based on a bet-hedging reporter, we observed that the yeast population exhibited an increased phenotypic diversification process in co-culture compared with mono-culture, suggesting that this mechanism could be the basis of the metabolic fitness of the yeast.

Effect of Operational Variables on the Yield of Chemoenzymatic Oxidation of 2,5-Furandicarboxaldehyde to 2,5-Furandicarboxylic Acid in Fed-Batch and Continuous Packed-Bed Millibioreactor

This work explores for the first time the use of a fed-batch and continuous packed-bed millibioreactor for the chemoenzymatic oxidation of 2,5-furandicarboxaldehyde (DFF) to 2,5-furandicarboxylic acid (FDCA). Different operational variables were studied: temperature, substrate concentration, and flow rate using different reactors (batch, fed-batch, and a continuous packed-bed bioreactor). The best yield (100%) was achieved using the fed-batch reactor at an H2O2 flow rate of 3 µL/min with a substrate concentration (DFF) of 100 mM. Regarding the specific productivity, the highest values (>0.05 mg product/min g biocatalyst) were reached with the operation in the fed-batch bioreactor and the continuous packed-bed bioreactor. The yield of the biocatalyst decreased by 98% after the first reaction cycle during the operational stability tests, due to a substantial inactivation of the biocatalyst by H2O2 and peracid. In this study, it is possible to select the operational variables in fed-batch and continuous reactors for chemoenzymatic oxidation that can increase the yield and specific productivity; however, the stability of the biocatalyst should be improved in future research.

Different contribution of exoelectrogens in methanogenesis via direct interspecies electron transfer (DIET) by the different substrate in continuous anaerobic bioreactor

Direct interspecies electron transfer (DIET) is a syntrophic mechanism for electron transfer between exo- and endoelectrogens. Previous studies have demonstrated that methanogenesis performance was significantly improved via the DIET mechanism through conductive materials (CMs) under batch conditions with a single substrate, while that under continuous condition is still under investigation. To investigate how the DIET via CM on methanogenesis performance was changed in response to the different substrates (acetate versus glucose)-fed in continuous anaerobic bioreactors, continuous bioreactors were operated by cross-feeding with acetate and glucose. Acetate-fed conditions showed 0.40 day shorten lagtime, 1.88- and 1.22-folds higher methane production rate, and ultimate methane production than glucose-fed conditions, respectively. Burkholderiaceae- and Anaerolineaceae-related exo-electrogenic populations were enriched with low abundance of Geobacter species in batch reactors. Furthermore, influent substrates affected the distribution of the enriched populations. Taken together, the results suggested that different syntrophic associations contributed methane production by DIET in continuous bioreactors.

Production of Siamenoside I and Mogroside IV from Siraitia grosvenorii Using Immobilized β-Glucosidase

Siraitia grosvenorii is a type of fruit used in traditional Chinese medicine. Previous studies have shown that the conversion of saponins was often carried out by chemical hydrolysis, which can be problematic because of the environmental hazards it may cause and the low yield it produces. Therefore, the purpose of this study is to establish a continuous bioreactor with immobilized enzymes to produce siamenoside I and mogroside IV. The results show that the immobilization process of β-glucosidase exhibited the best relative activity with a glutaraldehyde (GA) concentration of 1.5%, carrier activation time of 1 h and binding enzyme time of 12 h. After the immobilization through GA linkage, the highest relative activity of β-glucosidase was recorded through the reaction with the substrate at 60 °C and pH 5. Subsequently, the glass microspheres with immobilized β-glucosidase were filled into the reactor to maintain the optimal active environment, and the aqueous solution of Siraitia grosvenorii extract was introduced by controlling the flow rate. The highest concentration of siamenoside I and mogroside IV were obtained at a flow rate of 0.3 and 0.2 mL/min, respectively. By developing this immobilized enzyme system, siamenoside I and mogroside IV can be prepared in large quantities for industrial applications.

Simultaneous removal of selenite and heavy metals from wastewater and their recovery as nanoparticles using an inverse fluidized bed bioreactor

Occurrence of selenium oxyanions and different heavy metals as co-pollutants in various waste streams is common from industries such as mining, coal burning, thermal power plants, petrochemical refinery, etc. Among the available technologies to treat such wastewater, biological removal using anaerobic microbes is one of the most advantageous. This study examined simultaneous removal and recovery of selenite and heavy metals using an inverse fluidized bed bioreactor (IFBR). The effect of commonly occurring heavy metals namely Zn, Cu and Cd on selenite removal was evaluated at 24 h hydraulic retention time (HRT). The maximum selenite removal of 93.9% was obtained in the absence of any heavy metals. The presence of heavy metals negatively impacted the selenite removal (45.8–85.3%), and the inhibitory effect due to Cu was the most significant among the three metals at 50 mg/L of influent concentration (47% lower). The heavy metal removal efficiency values were more than 70%, except in the case of Cd at 50 mg/L. The recovery values of selenium and heavy metals using the continuous bioreactor were in the range 45.1–73.9% and 59.2–69.8%, respectively, under different experimental conditions. Elemental selenium nanoparticles were formed inside the bioreactor due to selenite bioreduction and the nanoparticle size ranged from 50 to 300 nm. Furthermore, formation of small sized (<10 nm) metal selenide nanoparticles of the respective metals were detected during the bioreactor operation. Overall, this study clearly demonstrated the potential of the IFBR system for the removal of selenite and heavy metals from wastewater as well as their recovery as elemental selenium and metal selenide nanoparticles, respectively.

Synthesis, Characterization, and Performance Evaluation of Hybrid Waste Sludge Biochar for COD and Color Removal from Agro-Industrial Effluent

Agro-waste management processes are evolving through the development of novel experimental approaches to understand the mechanisms in reducing their pollution levels efficiently and economically from industrial effluents. Agro-industrial effluent (AIE) from biorefineries that contain high concentrations of COD and color are discharged into the ecosystem. Thus, the AIE from these biorefineries requires treatment prior to discharge. Therefore, the effectiveness of a continuous flow bioreactor system (CFBS) in the treatment of AIE using hybrid waste sludge biochar (HWSB) was investigated. The use of a bioreactor with hydraulic retention time (HRT) of 1–3 days and AIE concentrations of 10–50% was used in experiments based on a statistical design. AIE concentration and HRT were optimized using response surface methodology (RSM) as the process variables. The performance of CFBS was analyzed in terms of COD and color removal. Findings indicated 76.52% and 66.97% reduction in COD and color, respectively. During biokinetic studies, the modified Stover models were found to be perfectly suited for the observed measurements with R2 values 0.9741 attained for COD. Maximum contaminants elimination was attained at 30% AIE and 2-day HRT. Thus, this study proves that the HWSB made from biomass waste can potentially help preserve nonrenewable resources and promote zero-waste attainment and principles of circular economy.

Study on the Changes in Immobilized Petroleum-Degrading Bacteria Beads in a Continuous Bioreactor Related to Physicochemical Performance, Degradation Ability, and Microbial Community.

Continuous bioreactors for petroleum degradation and the effect factors of these bioreactors have rarely been mentioned in studies. In addition, indigenous bacteria living in seawater could influence the performance of continuous bioreactors with respect to petroleum degradation in practice. In this paper, a bioreactor fitted with immobilized petroleum–degrading bacteria beads was designed for further research. The results indicated that the diesel degradation rate of the bioreactor could remain above 50% over 27 days, while degradation performance decreased with bioremediation time. Intriguingly, the diameters of immobilized petroleum–degrading bacteria beads were reduced by 32.49% after 45 days remediation compared with the initial size of the immobilized petroleum–degrading bacteria beads. Change in immobilized petroleum–degrading bacteria beads was considered to correlate remarkably with reduced degradation efficiency. Therefore, this paper will be helpful for further study and improvement of bioreactors in the practical context of oil-spill accident recovery.

Kinetics and hydrodynamics of Candida antartica lipase-catalyzed synthesis of glycerol dioleate (GDO) in a continuous flow packed-bed millireactor

Diacylglycerols (DAG) have been widely used in many industries due to their remarkable capabilities as emulsifiers and stabilisers. However, developing a sustainable and an effective synthesis method for DAG remains a challenge. Continuous flow bio-reactor is recognized to be more productive, controllable, and reliable instrument for developing green and intensified processes. In this work, a continuous flow packed bed millireactor was employed for the synthesis of glycerol dioelate (GDO) catalyzed by immobilized lipase namely Candida antartica. Experiments were carried out to evaluate the kinetic parameters as well as to assess the internal and external mass transfer limitations. Using one-factor-at-a-time variables method, maximum oleic acid conversion and GDO selectivity were achieved at 85% and 74% respectively, at 0.15 g of lipase, 77 min of residence time with 1.6:1 molar ratio of oleic acid/glycerol. Hydrodynamic studies showed that the esterification reaction is kinetically controlled and unaffected by external and internal mass transfer. Employing Lilly–Hornby model for kinetic evaluation, Km values increased with increasing flow rates, whereas, Vmax appeared to be flow rate independent. Reusability tests revealed that the activity of immobilized lipase remained the same after 9 successive reaction cycles. At 11 days of operation, the stability of the lipase in the continuous packed bed millireactor decreased only 5–7%, indicating satisfying operational results and recyclability. This work may promote the enzymatic engineering synthesis of DAG, facilitating the creation of a cleaner and safer process. It has the potential to broaden the application of enzymes in continuous flow micro or millireactors.

Static and Dynamic Analysis of a Continuous Bioreactor Model for the Production of Biofuel from Refinery Wastewater Using Rhodococcus opacus

This paper studied the bifurcation phenomena that can occur in a continuous bioreactor for lipid-rich biomass production using Rhodococcus opacus that feeds on refinery wastewater. An unstructured model of the bioreactor was developed based on experimentally validated studies reported in the literature. The analysis of the unsteady-state model was carried out both analytically and through numerical simulations. It was shown that the inhibition effect of the biomass growth rate can lead to the occurrence of static bistability in the model. Periodic behavior was also found for some range of model parameters. The effect of model parameters on the productivity of the bioreactor was also investigated. The analysis carried out in this paper allowed for the detection of unsafe operating regions in the bioreactor which, would help in the attenuation of these operational problems in the early stage of process design.

An accelerated approach of biogas production through a two-stage BioH2/CH4 continuous anaerobic digestion system from Napier grass

Napier grass found to be greatest potential for gaseous bioenergy production. The biohydrogen and biomethane productions from untreated Napier grass in single and two-stage continuous bioreactors was evaluated using anaerobic digestion technology. The bioreactors were fed Napier grass with organic loading rate (OLR) of 0.5, 1.0 and 2.0 kg VS/m3-d, respectively. The hydrogen, methane, and energy yields were evaluated. The methane yield of single-stage system was 282.08 CH4/g vS with OLR of 0.5 kg VS/m3-d. For two-stage system, the biohydrogen and biomethane yields were 90.06 mL H2/g vS and 367.00 mL CH4/g vS with OLRs of 1.0 and 0.5 kg VS/m3-d, respectively. The energy yields of single and two-stage systems were 13.14 and 10.10 kJ/g vS respectively. The peak OLR of Napier grass was 0.5 kg VS/m3-d for the two-stage system whereas the total energy recovery was 30 % higher than the single-stage system.