Sequential Batch Fermentation Research Articles

Sequential Dark-Photo Batch Fermentation and Kinetic Modelling for Biohydrogen Production Using Cheese Whey as a Feedstock.

The present work describes the utilisation of cheese whey to produce biohydrogen by sequential dark-photo fermentation. In first stage, cheese whey was fermented by Enterobacter aerogenes 2822 cells in a 2 L double-walled cylindrical bioreactor to produce hydrogen/organic acids giving maximum biohydrogen yield and cumulative hydrogen of 2.43 ± 0.12molmol-1 lactose and 3270 ± 143.5mL at cheese whey concentration of 105mM lactose L-1. The soluble metabolites of dark fermentation when utilised as carbon source for photo fermentation by Rhodobacter sphaeroides O.U.001, the yield, and cumulative hydrogen was increased to 4.22 ± 0.20molmol-1 VFA and 3800 ± 170mL, respectively. Meanwhile, an overall COD removal of about 38.08% was also achieved. The overall biohydrogen yield was increased from 2.43 (dark fermentation) to 6.65 ± 0.25molmol-1 lactose. Similarly, the modelling for biohydrogen production in bioreactor was done using modified Gompertz equation and Leudeking-Piret model, which gave adequate simulated fitting with the experimental values. The carbon material balance showed that acetic acid, lactic acid, and CO2 along with microbial biomass were the main by-products of dark fermentation and comprised more than 75% of carbon consumed.

Immobilization of Spathaspora passalidarum NRRL Y-27907 in Calcium Alginate Aiming the Production of Second-Generation Ethanol

The immobilization of S. passalidarum in calcium alginate beads for second-generation ethanol production (2G ethanol) was evaluated in a medium that simulated a hemicellulosic hydrolysate of sugarcane bagasse pretreated with diluted sulfuric acid in terms of sugar composition. Three sets of sequential batch fermentations (SBF) were carried out with free cells or immobilized cells in high (HSC) and moderate (MSC) initial sugar concentration (120 and 70 g/L, respectively). SBF were characterized by five consecutive batches performed in shaker, at 30 °C and 110 rpm. Better results were observed for the SBF with immobilized cells in MSC medium when compared to HSC (Y’P/S of 0.27 ± 0.02 and 0.19 ± 0.03 g/g, respectively), in the second batch cycle. The value for YP/S in MSC was similar to the obtained with free cells (0.30 ± 0.02 to 0.33 ± 0.02 g/g). However, QP was lower for MSC with immobilized cells, reaching 0.81 ± 0.04 g/L.h in the second batch, while for free cells the QP varied from 1.06 ± 0.02 to 1.16 ± 0.22 g/L.h. A technique for determining the concentration of immobilized cells in the alginate beads was applied, which made it possible to determine the specific rates for the SBF performed. According to the results obtained, it was possible to demonstrate that S. passalidarum can be immobilized in calcium alginate and reused through SBF, with performance similar to free cells, which can be a good strategy for fermentation of hemicellulosic hydrolysates.

Effect of Air Sparging on Ethanol Production from Xylose and Glucose in Continuous Chemostat Fermentation Process Utilizing High Cell Density of <i>Candida intermedia</i> 4-6-4T2

Reducing fermentation periods and increasing ethanol productivity are cost effective for ethanol production from lignocellulosic biomass. Increasing the density of cells for fermentation typically increases ethanol productivity, but also increases the concentration of dissolved carbon dioxide (dCO2) in the fermented broth. Such accumulated dCO2 sometimes reduces ethanol production. The Continuous Chemostat Fermentation (CCF) process utilizing high density of Candida intermedia 4-6-4T2 with and without air sparging was evaluated for the effect on ethanol production and rapid fermentation using 24-h cycles. Synthetic fermentation solution without nitrogen sources containing 20 g/L xylose and 30 g/L glucose plus 5 g/L acetic acid as fermentation inhibitor was supplemented into a culture vessel at 15 mL/h, and fermented broth was recovered from the same flask at 15 mL/h. Various conditions were tested to reduce the accumulated dCO2 in the fermented broth, but air sparging at 0.056 vvm was the most effective for ethanol production in the CCF process. For the 24-h startup-batch and 6-cycle CCF process (144 h), the ethanol yield was 0.4 g/g and the cell density of the used C. intermedia 4-6-4T2 for one cycle was one-third compared to that of sequential batch fermentation.

Adaptive laboratory evolution of Pseudomonas putida and Corynebacterium glutamicum to enhance anthranilate tolerance.

Microbial bioproduction of the aromatic acid anthranilate (ortho-aminobenzoate) has the potential to replace its current, environmentally demanding production process. The host organism employed for such a process needs to fulfil certain demands to achieve industrially relevant product levels. As anthranilate is toxic for microorganisms, the use of particularly robust production hosts can overcome issues from product inhibition. The microorganisms Corynebacterium glutamicum and Pseudomonas putida are known for high tolerance towards a variety of chemicals and could serve as promising platform strains. In this study, the resistance of both wild-type strains towards anthranilate was assessed. To further enhance their native tolerance, adaptive laboratory evolution (ALE) was applied. Sequential batch fermentation processes were developed, adapted to the cultivation demands for C. glutamicum and P. putida, to enable long-term cultivation in the presence of anthranilate. Isolation and analysis of single mutants revealed phenotypes with improved growth behaviour in the presence of anthranilate for both strains. The characterization and improvement of both potential hosts provide an important basis for further process optimization and will aid the establishment of an industrially competitive method for microbial synthesis of anthranilate.

Glucose fermentation with biochar amended consortium: Sequential fermentations

The aim of this work was to study sequential batch fermentation of glucose with a biological consortium amended with nine different biochars or with an activated carbon. The glucose fermentation was enhanced by carbon amendment, with activated carbon being more effective than biochars as cell carriers and electron conductors between functional species. The volatile fatty acid distributions were shifted in the consumption of the produced H2 and CO2. The types of biochars were irrelevant to glucose glycolysis and the subsequent H2 and CO2 consumption reactions. Biofilm growth affects the detailed mechanisms occurred in fermentation broth to the yielded volatile fatty acid distributions.

Preparation and characterization of a substitute for Ruditapes philippinarum conglutination mud as a natural bioflocculant

In this study a preparation strategy was attempted to produce a substitute (BBF) for the Ruditapes philippinarum conglutination mud (RPM), which was newly discovered to be a promising natural bioflocculant resource. A stable yield of 73.77 ± 1.79 g L−1 BBF was established by a sequential batch fermentation under optimized conditions via single factor experiments. BBF attained similar flocculation performance as RPM, showing a maximum flocculation rate of 87.92 ± 0.65%. BBF had significant decolorization efficiency on methylene blue, crystal violet and malachite green by 98.78 ± 0.46%, 89.37 ± 0.35% and 99.11 ± 0.17%, respectively. BBF could harvest microalgae Chlorella salina by 84.38 ± 0.57%. High throughput sequencing revealed that Vibrio and Bacillus might be the extracellular polysaccharides producers. The successful preparation will enable a potential industrial production of BBF thus avoid scattered collection of RPM.

Practical considerations for a simple ethanol concentration from a fermentation broth via a single adsorptive process using molecular-sieving carbon

Abstract A simple ethanol concentration process composed of a simple adsorptive apparatus using a single adsorbent, i.e., molecular-sieving carbon (MSC), is proposed for small-scale bioethanol production. The process designed to comprise pre-concentration of ethanol through gaseous-phase adsorption onto MSC and selective dehydration driven by molecular-sieving during desorption was tested under various conditions. Among the candidate adsorbents, MSC 5A showed the highest maximum ethanol adsorption capacity (0.163 g g−1). It was confirmed that the adsorption temperature and the initial ethanol concentration in the broth are crucial factors for the adsorption stage. For the desorption stage, the water recovery temperature significantly affected the final ethanol concentration and the ethanol recovery. As a practical option for the application of the proposed system, sequential batch fermentation and ethanol recovery was successfully demonstrated.

Propionic acid production from soy molasses by Propionibacterium acidipropionici: Fermentation kinetics and economic analysis

Propionic acid (PA) is a specialty chemical; its calcium salt is widely used as food preservative. Soy molasses (SM), a low-value byproduct from soybean refinery, contains sucrose and raffinose-family oligosaccharides (RFO), which are difficult to digest for most animals and industrial microorganisms. The feasibility of using SM for PA production by P. acidipropionici, which has genes encoding enzymes necessary for RFO hydrolysis, was studied. With corn steep liquor as the nitrogen source, stable long-term PA production from SM was demonstrated in sequential batch fermentations, achieving PA productivity of >0.8 g/L h and yield of 0.42 g/g sugar at pH 6.5. Economic analysis showed that calcium propionate as the main component (63.5%) in the product could be produced at US $1.55/kg for a 3000-MT plant with a capital investment of US $10.82 million. At $3.0/kg for the product, the process offers attractive 40% return of investment and is promising for commercial application.

Dynamic cell responses in Thermoanaerobacterium sp. under hyperosmotic stress

As a nongenetic engineering technique, adaptive evolution is an effective and easy-to-operate approach to strain improvement. In this work, a commercial Thermoanaerobacterium aotearoense SCUT27/Δldh-G58 was successfully isolated via sequential batch fermentation with step-increased carbon concentrations. Mutants were isolated under selective high osmotic pressures for 58 passages. The evolved isolate rapidly catabolized sugars at high concentrations and subsequently produced ethanol with good yield. A 1.6-fold improvement of ethanol production was achieved in a medium containing 120 g/L of carbon substrate using the evolved strain, compared to the start strain. The analysis of transcriptome and intracellular solute pools suggested that the adaptive evolution altered the synthesis of some compatible solutes and activated the DNA repair system in the two Thermoanaerobacterium sp. evolved strains. Overall, the results indicated the potential of adaptive evolution as a simple and effective tool for the modification and optimization of industrial microorganisms.

Sucrose purification and repeated ethanol production from sugars remaining in sweet sorghum juice subjected to a membrane separation process.

The juice from sweet sorghum cultivar SIL-05 (harvested at physiological maturity) was extracted, and the component sucrose and reducing sugars (such as glucose and fructose) were subjected to a membrane separation process to purify the sucrose for subsequent sugar refining and to obtain a feedstock for repeated bioethanol production. Nanofiltration (NF) of an ultrafiltration (UF) permeate using an NTR-7450 membrane (Nitto Denko Corporation, Osaka, Japan) concentrated the juice and produced a sucrose-rich fraction (143.2gL-1 sucrose, 8.5gL-1 glucose, and 4.5gL-1 fructose). In addition, the above NF permeate was concentrated using an ESNA3 NF membrane to provide concentrated permeated sugars (227.9gL-1) and capture various amino acids in the juice, enabling subsequent ethanol fermentation without the addition of an exogenous nitrogen source. Sequential batch fermentation using the ESNA3 membrane concentrate provided an ethanol titer and theoretical ethanol yield of 102.5-109.5gL-1 and 84.4-89.6%, respectively, throughout the five-cycle batch fermentation by Saccharomyces cerevisiae BY4741. Our results demonstrate that a membrane process using UF and two types of NF membranes has the potential to allow sucrose purification and repeated bioethanol production.

Bioethanol production by recycled Scheffersomyces stipitis in sequential batch fermentations with high cell density using xylose and glucose mixture

Here, it is shown three-step investigative procedures aiming to improve pentose-rich fermentations performance, involving a simple system for elevated mass production by Scheffersomyces stipitis (I), cellular recycle batch fermentations (CRBFs) at high cell density using two temperature strategies (fixed at 30°C; decreasing from 30 to 26°C) (II), and a short-term adaptation action seeking to acclimatize the microorganism in xylose rich-media (III). Cellular propagation provided 0.52gdrycellweightgRS−1, resulting in an expressive value of 45.9gdrycellweightL−1. The yeast robustness in CRBF was proven by effective ethanol production, reaching high xylose consumption (81%) and EtOH productivity (1.53gL−1h−1). Regarding the short-term adaptation, S. stipitis strengthened its robustness, as shown by a 6-fold increase in xylose reductase (XR) activity. The short fermentation time (20h for each batch) and the fermentation kinetics for ethanol production from xylose are quite promising.

Production and storage of biohydrogen during sequential batch fermentation of Spirogyra hydrolyzate by Clostridium butyricum

The biological hydrogen production from Spirogyra sp. biomass was studied in a SBR (sequential batch reactor) equipped with a biogas collecting and storage system. Two acid hydrolysis pre-treatments (1N and 2N H2SO4) were applied to the Spirogyra biomass and the subsequent fermentation by Clostridium butyricum DSM 10702 was compared. The 1N and 2N hydrolyzates contained 37.2 and 40.8 g/L of total sugars, respectively, and small amounts of furfural and HMF (hydroxymethylfurfural). These compounds did not inhibit the hydrogen production from crude Spirogyra hydrolyzates. The fermentation was scaled up to a batch operated bioreactor coupled with a collecting system that enabled the subsequent characterization and storage of the biogas produced. The cumulative hydrogen production was similar for both 1N and 2N hydrolyzate, but the hydrogen production rates were 438 and 288 mL/L.h, respectively, suggesting that the 1N hydrolyzate was more suitable for sequential batch fermentation. The SBR with 1N hydrolyzate was operated continuously for 13.5 h in three consecutive batches and the overall hydrogen production rate and yield reached 324 mL/L.h and 2.59 mol/mol, respectively. This corresponds to a potential daily production of 10.4 L H2/L Spirogyra hydrolyzate, demonstrating the excellent capability of C. butyricum to produce hydrogen from microalgal biomass.

Repeated ethanol production from sweet sorghum juice concentrated by membrane separation

Sequential batch fermentation from sweet sorghum juice concentrated by membrane separation (ultrafiltration permeation and nanofiltration concentration) to increase sugar contents, was investigated. Ethanol production at 5th batch fermentation by Saccharomyces cerevisiae BY4741 attained 113.7±3.1gL−1 (89.1±2.2% of the theoretical ethanol yield) from 270.0±22.6gL−1 sugars, corresponding to 98.7% of ethanol titer attained at the 1st batch fermentation. This titer was comparable to ethanol production of 115.8±0.6gL−1 (87.1±2.7% of the theoretical ethanol yield) obtained at 5th batch fermentation with 3gL−1 yeast extract and 6gL−1 polypeptone. Increase of cell density in the concentrated sweet sorghum juice was observed during sequential batch fermentation, as indicated by increased OD600. Utilization of sweet sorghum juice as the sole source, membrane separation, and S. cerevisiae was a cost-effective process for high ethanol production.

Improved propionic acid production from glycerol: Combining cyclic batch- and sequential batch fermentations with optimal nutrient composition

Propionic acid was produced from glycerol using Propionibacterium acidipropionici. In this study, the impact of the concentrations of carbon and nitrogen sources, and of different modes of high cell density fermentations on process kinetics and -efficiency was investigated. Three-way ANOVA analysis and batch cultivations at varying C/N ratios at pH 6.5 revealed that propionic acid production rate is significantly influenced by yeast extract concentration. Glycerol to yeast extract ratio (ww−1) of 3:1 was required for complete glycerol consumption, while maintaining the volumetric productivity. Using this optimum C/N ratio for propionic acid production in cyclic batch fermentation gave propionate yield up to 93mol% and productivity of 0.53gL−1h−1. Moreover, sequential batch fermentation with cell recycling resulted in production rates exceeding 1gL−1h−1 at initial glycerol up to 120gL−1, and a maximum of 1.63gL−1h−1 from 90gL−1 glycerol.

Adaptive evolution of Saccharomyces cerevisiae with enhanced ethanol tolerance for Chinese rice wine fermentation.

High tolerance towards ethanol is a desirable property for the Saccharomyces cerevisiae strains used in the alcoholic beverage industry. To improve the ethanol tolerance of an industrial Chinese rice wine yeast, a sequential batch fermentation strategy was used to adaptively evolve a chemically mutagenized Chinese rice wine G85 strain. The high level of ethanol produced under Chinese rice wine-like fermentation conditions was used as the selective pressure. After adaptive evolution of approximately 200 generations, mutant G85X-8 was isolated and shown to have markedly increased ethanol tolerance. The evolved strain also showed higher osmotic and temperature tolerances than the parental strain. Laboratory Chinese rice wine fermentation showed that the evolved G85X-8 strain was able to catabolize sugars more completely than the parental G85 strain. A higher level of yeast cell activity was found in the fermentation mash produced by the evolved strain, but the aroma profiles were similar between the evolved and parental strains. The improved ethanol tolerance in the evolved strain might be ascribed to the altered fatty acids composition of the cell membrane and higher intracellular trehalose concentrations. These results suggest that adaptive evolution is an efficient approach for the non-recombinant modification of industrial yeast strains.

Effects of yeast immobilization on bioethanol production

The current study evaluated a newer method, which includes a dehydration step, of immobilizing Saccharomyces cerevisiae L-77 and S. cerevisiae L-73 onto hydroxylapatite and chamotte ceramic supports. The efficiency of cell immobilization on chamotte was significantly higher than hydroxylapatite. Immobilized yeast preparations were investigated for their ethanol-producing capabilities. The glucose concentration in a fermentation medium was 100mg/mL. Immobilized preparations produced the same amount of ethanol (48 ± 0.5mg/mL) as free cells after 36 H of fermentation. During the early stages of fermentation, immobilized yeast cells produced ethanol at a higher rate than free cells. Yeast preparations immobilized on both supports (hydroxylapatite and chamotte) were successfully used in six sequential batch fermentations without any loss of activity. The chamotte support was more stable in the fermentation medium during these six cycles of ethanol production. In addition to the high level of ethanol produced by cells immobilized on chamotte, the stability of this support and its low cost make it a promising material for biotechnologies associated with ethanol production.

Generation of Novel Wine Yeast Strains by Adaptive Evolution

As a non-recombinant means of strain improvement, adaptive evolution is a technique with great potential. In this first report of the use of adaptive evolution in the improvement of a commercial wine yeast strain, a sequential batch fermentation system was used to adaptively evolve the wine strain L-2056 and its haploid derivative, C9. Mutants were isolated under the selective pressures of a winelike fermentation after approximately 350 generations (from L-2056) and 250 generations (from C9) and were demonstrated to have altered production of metabolites, including ethanol, glycerol, and succinic and acetic acid. Additionally, the evolved isolate of the commercial wine yeast was able to more rapidly catabolize all available sugars under these conditions. These results endorse the potential of adaptive evolution as a tool for the non-recombinant modification and optimization of industrial yeast strains.

Estudo da reciclagem de células na produção biológica de etanol

Nas destilarias de álcool o conteúdo de etanol, na suspensão de levedura reciclada, gira em torno de 2% a 3%. A reciclagem de células é realizada tanto em fermentações em batelada, como nas contínuas. A fermentação alcoólica e a recuperação de células de leveduras foram avaliadas em laboratório. A levedura recuperada, por centrifugação, nos experimentos laboratoriais, foi tratada e novamente inoculada em novo meio fermentativo para avaliar o efeito tóxico do etanol e de outros metabólitos nas fermentações subseqüentes. A cepa Saccharomyces cerevisiae IZ-1904 utilizada foi inoculada em meio de melaço diluído contendo 180g L-1 e 310g L-1 de açúcares redutores totais (ART), com três repetições. O tratamento da levedura recuperada constou de dupla centrifugação e ressuspensão em água e vinho. Foram realizadas oito fermentações, a primeira com fermento multiplicado e as demais com a reciclagem de células das fermentações anteriores. O elevado teor de açúcar foi necessário para obtenção da concentração alcoólica desejada no vinho e, assim, conferir inibições e toxicidades detectáveis. Nas fermentações com concentração de etanol menor que 6,4% (v v-1) o efeito tóxico ou inibidor não foi detectado. A inibição pelo etanol e por outros componentes foi detectada sobre as leveduras e os resultados mostraram que a redução dos componentes reciclados com a levedura pode melhorar o processo fermentativo.

Development and solution of a cell mass population balance model applied to the SCF process

Self-cycling fermentation (SCF) is a process in which sequential batch fermentations are performed using a computerized feedback control scheme. During the fermentation, half of the reactor volume is periodically removed and replaced by fresh medium. The periodicity of the system is determined by continuously monitoring a growth associated parameter such as dissolved oxygen concentration or carbon dioxide evolution. The system cycles when an extremum in the monitored parameter is detected, corresponding to a decrease in metabolic activity. This results in very stable and repeatable growth cycles and synchronized cell populations. In this work, a segregated, structured microbial population balance model is formulated and used to numerically simulate the SCF process. The model uses cell mass as the index of physiological state. In order to solve the cell mass population balance model for SCF, a numerical scheme using the Galerkin finite element method (GFEM) and the predictor–corrector Euler method was developed. The model outputs were compared and validated with previously obtained experimental data. The cell mass model as described by Eakman et al. (1966) was able to capture the major growth profiles of the system. However, the model was not able to describe the cell number profile or cell synchrony. By introducing a feedback mechanism between the critical division mass m c and the limiting substrate into the classical cell population model, cell synchrony and qualitative agreement with experimental data are obtained.