Continuous Fermentation Process Research Articles

Online state and unknown inputs estimation for nonlinear systems with particle filter based recursive expectation‐maximization algorithm

AbstractThe article presents an innovative approach to simultaneously estimate states and unknown inputs (UIs) in nonlinear systems using a particle filter (PF) based recursive expectation‐maximization (EM) algorithm. This method is distinct from traditional iterative EM algorithms. During the E‐step, it calculates the Q‐function recursively within the maximum likelihood framework, while the PF estimates the system states. The M‐step involves local maximization of the recursive Q‐function to online estimate the UIs. The effectiveness of the PF‐based recursive EM algorithm is demonstrated with a numerical example, and comparisons with the augmented state PF are made to highlight its advantages. Finally, the proposed algorithm is implemented in a real application for the estimation of the continuous fermentation process.

Rapidly engineering an osmotic-pressure-tolerant gut bacterium for efficient non-sterile production of bulk chemicals

The concept of next-generation industrial biotechnology (NGIB) has been presented as a green biomanufacturing approach based on the use of low-cost substrates in an open and continuous non-sterilization fermentation process. However, the realization of NGIB, partially through the utilization of extremophiles, has been impeded by the challenges in engineering these extremophiles. A previously unexplored solution for advancing NGIB is to obtain readily engineerable microorganisms with osmotic-pressure tolerance. Here, Jejubacter sp. L23 strain, a Gram-negative, rod-shaped, osmotic-pressure-tolerant bacterium, was isolated from the gut of superworm Zophobas atratus and identified as a new member of the family Enterobacteriaceae, with high 16S rDNA similarity to Escherichia coli (95.2 %). The growth of L23 strain was observed at 10 to 45 °C and pH 4.0 to 10.0 in the presence of 0 to 12 % (w/v) NaCl. We systematically investigated the genetic parts, molecular tools, and manipulation approaches applicable to the L23 strain, and successfully conducted chromosomal genetic engineering, e.g. base-editing and deletion, by using high-efficiency CRISPR systems under low-salt conditions. Furthermore, the metabolic network of L23 strain was rewired to obtain the engineered L23R7 strain, which could display stable expression of exogenous genes and produce bulk chemicals at high yields using inulin, abundant non-food biomass, and salt water without sterilization. For example, 41 g/L isobutanol was produced by utilizing inulin in a 3-L bioreactor through a non-sterile fermentation process. This potent industrial strain holds the promise of promoting the development of NGIB as a sustainable biomanufacturing solution.

Global Stabilizing Control of a Continuous Ethanol Fermentation Process Starting from Batch Mode Production

Global Stabilizing Control of a Continuous Ethanol Fermentation Process Starting from Batch Mode Production

Investigation of the antioxidant properties of Persea americana seed flour altered by the fermentation process with Lactobacillus plantarum

This study aims to investigate the effect of Lactobacillus plantarum fermentation on the antioxidant potential of Persea americana seed flour. The half-maximal inhibitory concentration (IC50) value of avocado seed flour after fermentation for 24, 48, and 72 h was compared with the unfermented avocado seed flour using the measurement of free radical scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH). Additionally, the change in pH value was measured to confirm the continuous fermentation process within the desired period. The results for the DPPH assay for unfermented, 24, 48, and 72 h fermented avocado seed flour were 61.5±0.71, 51.18±0.10, 46.00±0.21, 43.73±0.21 µg/mL, respectively, which indicated a significant increase (p < 0.05) of IC50 values of avocado seed flour with the fermentation. Furthermore, with the fermentation period, there was a significant decrease in the pH value for the 72-hour fermented sample (4.15±0.03) compared to the unfermented sample (6.81±0.04). These results supported avocado seed as an important by-product source for the further development of health-promoting products, by confirming the increased antioxidant capacity of avocado seed flour after fermentation.

Metabolic Oscillation Phenomena in Clostridia Species—A Review

Clostridia are interesting candidates for biotechnological applications due to their diverse and unique metabolic abilities. Particularly in continuous fermentation processes, productivity-decreasing metabolic oscillations have been reported in many species. The resulting process instability and reduced productivity can be a serious hurdle for the development of industrially feasible processes. This review highlights the current state of knowledge about oscillatory metabolic phenomena in Clostridia, including the mechanisms, assumed and proven, behind those oscillations and methods to mitigate the phenomena if applicable. The nature of observed metabolic oscillations in Clostridia is diverse, including a wide range of periods of oscillation and different parameters in which the oscillation is observed. Some phenomena remain to be investigated further, while others are already well understood. However, knowledge of mechanisms is a very valuable asset in overcoming the metabolic oscillation to create a stable process.

Optimization, Scale-Up, and Economic Analysis of the Ethanol Production Process Using Sargassum horneri

Recently, the extensive spread of some algae along coastlines has surged into unmanageable thick decomposition layers. This study aimed to demonstrate the use of Sargassum horneri as a biomass resource for ethanol production through the continuous hydrolysis, enzymatic saccharification, and fermentation process. Sugars from S. horneri were obtained using a combination of thermal acid hydrolysis and enzymatic saccharification. The optimal conditions for thermal acid hydrolysis involved a 10% (w/v) S. horneri slurry treated with 100 mM H2SO4 at 121 °C for 60 min; enzymatic saccharification using 16 U/mL Cellic CTec2 further boosted the monosaccharide concentration to 23.53 g/L. Fermentation experiments were conducted with mannitol-adapted Saccharomyces cerevisiae BY4741 using S. horneri hydrolysate. Enhanced ethanol production was observed in the hydrolysate, particularly with mannitol-adapted S. cerevisiae BY4741, which yielded 10.06 g/L ethanol. Non-adapted S. cerevisiae produced 8.12 g/L ethanol, as it primarily utilized glucose and not mannitol. Regarding ethanol fermentation using 5 L- and 500 L-scale fermenters, the ethanol concentrations reached 10.56 g/L and 7.88 g/L with yields of 0.51 and 0.45, respectively, at 48 h. This study confirmed the economic viability of ethanol production using waste seaweed with optimized pretreatment conditions and the adaptive evolution of S. cerevisiae to mannitol.

Application of High Gain and Sliding Mode Observers for Estimating Key Variables in Bioprocesses

The monitoring and control of bioreactors is essential for any bioprocess. Therefore, measuring some essential biochemical key variables is crucial, such as the reaction rates, whose structure is usually barely known. Although many high gain and sliding mode observers-based estimators were designed to estimate these variables, these strategies are not robust against external disturbances. This work extends the high gain and sliding mode techniques to simultaneously estimate the reaction rates and an external disturbance under a persistent excitation condition that is easy to verify. Furthermore, the proposed algorithms are exponentially and finite-time stable, respectively. Finally, both observers are tested in a continuous fermentation process simulation.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79g/L and 6.8u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025h-1 , DCW increased to 88.39g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80mL/h, a recombinant uricase activity was 4.13, 7.2, and 0u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04u/mL/h (0.0017mg/mL/h) to 0.18u/mL/h (0.0078mg/mL/h).

Continuous production of succinic acid from glycerol: A complete experimental and computational study

In this work, a bioprocess for the fermentation of A. succinogenes for the production of succinic acid from glycerol was developed, employing a continuous bioreactor with recycle. Moreover, a new bioprocess model was constructed, based on an existing double substrate limitation model, which was validated with experimental results for a range of operating parameters. The model was used to successfully predict the dynamics of the continuous fermentation process and was subsequently employed in optimisation studies to compute the optimal conditions, dilution rate, reflux rate and feed glycerol concentration, that maximise the productivity of bio-succinic acid. In addition, a Pareto front for optimal volumetric productivity and glycerol conversion combinations was computed. Maximum volumetric productivity of 0.518 g/L/h, was achieved at the optimal computed conditions, which were experimentally validated. This is the highest bio-succinic acid productivity reported so far, for such a continuous bioprocess.

Continuous ethanol production via ultrasound-enhanced yeast sedimentation

Continuous glucose fermentation produces bioethanol at higher volumetric rates than conventional batch or fed-batch systems. The retention of yeast cells via ultrasonic sedimentation in a lab-scale fermenter allowed for shearless, continuous cell upconcentration, and consequently process intensification. The cell separation efficiency of the ultrasonic system was predicted with a Response Surface Model (RSM) developed for yeast cells based on the linear, mixed, and quadratic effects of the operating variables and flow rates (3 levels, 5 variables). The experiments for the RSM calibration were designed via a central composite design. The efficiency model was validated and showed dependency to the Biomass concentration, Power input, and Harvest rate (R2calibration = 0.92, R2prediction = 0.83). A lab-scale fermenter fed with yeast growth medium was operated at varying dilution rates (0.1 – 0.6 h−1) based on the RSM to maximize the cell retention efficiency (23%−90%). Yeast cell concentration in the fermenter reached up to 31.5 ± 0.7 g/L while it remained at around 3 g/L in the harvest stream for all the dilution rates tested. The ethanol concentration ranged between 17 and 23 g/L and reached high volumetric productivity (8.8 g/L/h). A control run without ultrasonic sedimentation led to the washout of biomass at a dilution rate of 0.6 h−1. Ultrasonic yeast sedimentation is a promising technology for cell retention and enhanced productivity in continuous fermentation processes.

Immobilization of Lactobacillus bulgaricus with gellan gum and its application in continuous fermentation of D-lactic acid from corn straw

Biorefinery of chemicals from straw is an effective approach to alleviate the environmental pollution caused by straw burning. In this paper, we prepared gellan gum immobilized Lactobacillus bulgaricus T15 gel beads (LA-GAGR-T15 gel beads), characterized their properties, and established a continuous cell recycle fermentation process for D-lactate (D-LA) production using the LA-GAGR-T15 gel beads. The fracture stress of LA-GAGR-T15 gel beads was (91.68±0.11) kPa, which was 125.12% higher than that of the calcium alginate immobilized T15 gel beads (calcium alginate-T15 gel beads). This indicated that the strength of LA-GAGR-T15 gel beads was stronger, and the strain was less likely to leak out. The average D-LA production was (72.90±2.79) g/L after fermentation for ten recycles (720 h) using LA-GAGR-T15 gel beads as the starting strain and glucose as the substrate, which was 33.85% higher than that of calcium alginate-T15 gel beads and 37.70% higher than that of free T15. Subsequently, glucose was replaced by enzymatically hydrolyzed corn straw and fermented for ten recycles (240 h) using LA-GAGR-T15 gel beads. The yield of D-LA reached (1.74±0.79) g/(L·h), which was much higher than that of using free bacteria. The wear rate of gel beads was less than 5% after ten recycles, which indicated that LA-GAGR is a good carrier for cell immobilization and can be widely used in industrial fermentation. This study provides basic data for the industrial production of D-LA using cell-recycled fermentation, and provides a new way for the biorefinery of D-LA from corn straw.

Case Study of Continuous Itaconic Acid Fermentation by Aspergillus Terreus in a Bench-Scale Bioreactor

Itaconic acid (IA) is a highly important bioproduct. The traditional biotechnological production of organic acids generates large quantities of biomass and water streams as waste. Bipolar membrane electrodialysis is a suitable technique for directly recovering IA from clarified fermentation broth whereby itaconic acid is separated without chemical loading. As a result, the remaining diluate stream can be reintroduced into the fermentation process. This membrane operation can be integrated into a continuous fermentation process in order to carry out the process using a high glucose concentration in the effluent, opening up new possibilities for the implementation of continuous itaconic acid fermentation. In this study, a possible highly successful implementation of this technique is presented. The applied gentle stirring technique as well as the low dilution rate of 0.007 s-1 is necessary to be complemented by highly efficient oxygenation, thereby promoting itaconic acid generation. With an inlet supplying pure oxygen gas, the acid accumulated and an average of 30.1 g·L-1 titer of IA was achieved in the effluent. The volumetric oxygen uptake rate was monitored during the fermentation which fluctuated from 40-88% so further investigation of this may be worthwhile in the future.

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains.

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S.cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S.cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S.cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5°C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S.cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.

Immobilization of Clostridium perfringens type D in calcium alginate beads: toxin production mimics free cell culture.

Cell-immobilization is used to maintain microbial culture to produce metabolites in repeated-batch or continuous fermentations, thereby reducing the time and resources spent on delivering mass production of microbe. The technique also enables shortening of the detoxification phase and the amount of formaldehyde required due to low incidence of viable bacteria in the extract. A solution of sodium alginate containing Clostridium perfringens cells was dropped into stirring CaCl solution via a sterile syringe needle. Optimizations resulted in reasonably uniform beads containing C. perfringens. Beads were externally stabilized by poly L-lysine, followed by immersion in a solution of Na-alginate to coat them with a new layer of alginate forming an alginate-PLL-alginate cortex. This study proved successful in immobilizing C. perfringens cells inside uniform alginate microspheres. Cell loading and cell propagation inside the beads were measured. The cell loaded beads were cultivable in liquid media producing 550 minimum lethal doses per milliliter (MLD/ml) in a 72 h. The research paved the way for further investigations to optimize and establish an efficient bacterial encapsulation method. Thus, it seems possible to produce toxins from beads engulfing C. perfringens on larger scales via repeated-batch or continuous fermentation processes.

Mass Transfer, Gas Holdup, and Kinetic Models of Batch and Continuous Fermentation in a Novel Rectangular Dynamic Membrane Airlift Bioreactor

Compared with conventional cylinder airlift bioreactors (CCABs) that produce coarse bubbles, a novel rectangular dynamic membrane airlift bioreactor (RDMAB) developed in our lab produces fine bubbles to enhance the volumetric oxygen mass transfer coefficient (kLa) and gas holdup, as well as improve the bioprocess in a bioreactor. In this study, we compared mass transfer, gas holdup, and batch and continuous fermentation for RNA production in CCAB and RDMAB. In addition, unstructured kinetic models for microbial growth, substrate utilization, and RNA formation were established. In batch fermentation, biomass, RNA yield, and substrate utilization in the RDMAB were higher than those in the CCAB, which indicates that dynamic membrane aeration produced a high kLa by fine bubbles; a higher kLa is more beneficial to aerobic fermentation. The starting time of continuous fermentation in the RDMAB was 20 h earlier than that in the CCAB, which greatly improved the biological process. During continuous fermentation, maintaining the same dissolved oxygen level and a constant dilution rate, the biomass accumulation and RNA concentration in the RDMAB were 9.71% and 11.15% higher than those in the CCAB, respectively. Finally, the dilution rate of RDMAB was 16.7% higher than that of CCAB during continuous fermentation while maintaining the same air aeration. In summary, RDMAB is more suitable for continuous fermentation processes. Developing new aeration and structural geometry in airlift bioreactors to enhance kLa and gas holdup is becoming increasingly important to improve bioprocesses in a bioreactor.

The Influence of Sugar Composition and pH Regulation in Batch and Continuous Acetone–Butanol–Ethanol Fermentation

Acetone–butanol–ethanol (ABE) fermentation is influenced by external conditions. This work aimed to study the influence of pH regulation on monosaccharide composition in batch and continuous fermentation processes to determine butanol production and productivity. Batch fermentations with ammonium acetate or calcium carbonate combined with minimum pH control (pH ≥ 4.8 or 5.1) were assessed with pure xylose and glucose/xylose mixtures (ratios of 1:1 and 3:1). Continuous two-stage fermentation was developed using plastic rings to retain the biomass. Although batch fermentations with pure xylose performed better without active minimum pH control with both buffers, minimum pH control was necessary to metabolize xylose in the presence of glucose. Xylose uptake was favored by the use of calcium carbonate and pH ≥ 5.1 at a ratio of 1:1, while ammonium acetate and a pH ≥ 4.8 was the best option for a 3:1 ratio. The best butanol production and productivity values with sugar mixtures in batch reactors were 8.8 g L−1 and 0.61 g L−1 h−1 with an ammonium acetate pH ≥ 4.8 (ratio 3:1). The glucose/xylose ratio combined with pH regulation thus modulated xylose metabolism and solvent production in batch modes. Immobilized cells combined with operating at D = 0.333 h−1 and pH regulation increased butanol productivity almost fourfold up to 2.4 ± 0.2 g L−1 h−1.

Metabolic engineering of Clostridium ljungdahlii for the production of hexanol and butanol from CO2 and H2

BackgroundThe replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging.ResultsUsing a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol.ConclusionsWe thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

Monitoring of total acidity content in continuous vinegar fermentation using a self-made modification of a micro-electro-mechanical system near-infrared spectrometer

This study evaluated the potential of a micro-electro-mechanical system (MEMS) near-infrared spectrometer for the on-line monitoring of total acids in the continuous fermentation process of rice vinegar and white vinegar. A self-made modification of the digital light processing NIRscan nano spectrometer was used for this purpose. As best pretreatment method, the multiplicative scatter correction was used to pretreat the raw spectra, which were subsequently subjected to a partial least square regression calibration. The results showed that total acids could be determined by this experimental setup with root-mean-square errors of predictions of 0.12% (w/v) and 0.27% (w/v) for rice vinegar and white vinegar, respectively. In conclusion, this low-cost MEMS near-infrared instrument can be applied to monitor total acids in the industrial continuous fermentation process of rice and white vinegar.

Robust nonlinear model predictive control of cascade of fermenters with recycle for efficient bioethanol production

In this paper, dynamical behavior and control of the continuous ethanol fermentation process are studied. The process productivity is improved by using two fermenters connected in series with recycle of cells. First, dynamical behavior and productivity of the cascade system are studied by using numerical bifurcation analysis. Then, multi-stage nonlinear model predictive control (MS-NMPC), classical and offset-free NMPCs algorithms are designed to eliminate the undesired oscillations. To ensure acceptable efficiency of the cascade process, a minimal ethanol concentration is defined and included as additional constraint in the controller design. The unmeasured state variables are estimated by using the asymptotic observer. It is shown that for smaller sampling times the predictive controllers are very effective, even in presence of parameter uncertainties. It is also observed that offset-free NMPC algorithm is more sensitive to measurement noise, but MS-NMPC and classical NMPC are more sensitive to estimation errors for longer sampling times.

Mathematical Modeling of a Biotechnological Continuous Fermentation Process for Lactic Acid Production: A Review

Mathematical Modeling of a Biotechnological Continuous Fermentation Process for Lactic Acid Production: A Review