Successive Batch Cultures Research Articles

Continuous biohydrogen production by a degenerated strain of Clostridium acetobutylicum ATCC 824

Degenerated strains of Clostridium acetobutylicum lack the ability to produce solvents and to sporulate, allowing the continuous production of hydrogen and organic acids. A degenerated strain of Clostridium acetobutylicum was obtained through successive batch cultures. Its kinetic characterization showed a similar specific growth rate than the wild type (0.25 h−1), a higher butyric acid production of 6.8 g·L−1 and no solvents production. A steady state was reached in a continuous culture at a dilution rate of 0.1 h−1, with a constant hydrogen production of 507 mL·h−1, corresponding to a volumetric rate of 6.10 L·L−1 d−1, and a yield of 2.39 mol of H2 per mole of glucose which represents 60% of the theoretical maximum yield. These results suggest that the degeneration is an interesting alternative for hydrogen production with this strain, obtaining a high hydrogen production in a continuous culture with cells in a permanent acidogenic state.

Genomic, transcriptomic and physiological analyses of silver-resistant Saccharomyces cerevisiae obtained by evolutionary engineering.

Silver is a non-essential metal used in medical applications as an antimicrobial agent, but it is also toxic for biological systems. To investigate the molecular basis of silver resistance in yeast, we employed evolutionary engineering using successive batch cultures at gradually increased silver stress levels up to 0.25-mM AgNO3 in 29 populations and obtained highly silver-resistant and genetically stable Saccharomyces cerevisiae strains. Cross-resistance analysis results indicated that the silver-resistant mutants also gained resistance against copper and oxidative stress. Growth physiological analysis results revealed that the highly silver-resistant evolved strain 2E was not significantly inhibited by silver stress, unlike the reference strain. Genomic and transcriptomic analysis results revealed that there were mutations and/or significant changes in the expression levels of the genes involved in cell wall integrity, cellular respiration, oxidative metabolism, copper homeostasis, endocytosis and vesicular transport activities. Particularly the missense mutation in the RLM1 gene encoding a transcription factor involved in the maintenance of cell wall integrity and with 707 potential gene targets might have a key role in the high silver resistance of 2E, along with its improved cell wall integrity, as confirmed by the lyticase sensitivity assay results. In conclusion, the comparative physiological, transcriptomic and genomic analysis results of the silver-resistant S. cerevisiae strain revealed potential key factors that will help understand the complex molecular mechanisms of silver resistance in yeast.

Adaptation of neutrophilic Paracoccus denitrificans to denitrification at highly alkaline pH.

Bacterial denitrification is widely documented at neutral pH in order to improve the removal of nitrate in wastewater treatment processes. However, certain industrial contexts generate alkaline waste and effluent containing nitrate that must be denitrified. To obtain more information on denitrification at alkaline pH, this study evaluated the possibility of adapting a neutrophilic denitrifying strain, Paracoccus denitrificans, to alkaline pH. Firstly, P. denitrificans' denitrifying activity was evaluated without acclimation in batch bioreactors at pH 7.0, 8.0, 9.0 and 10.0. Then, two acclimation methods using successive batch bioreactors and a continuous bioreactor allowed P. denitrificans to be gradually exposed to alkaline pH: from 8.5 to 11.2 in 26 and 72days respectively. Results showed that P. denitrificans could grow and catalyse nitrate reduction (i) at pH 9.0 without acclimation, (ii) at pH 10.5 in successive batch cultures with progressively increasing pH and (iii) at pH 10.8 in continuously fed culture with a hydraulic retention time (HRT) of 8days. It was shown that denitrification affected the pH despite the presence of carbonate buffering of the P. denitrificans growth medium. With acetate as an electron donor, the pH of a carbonate buffered medium tends towards pH 10 during the process of denitrification. Graphical abstract.

Limnospira indica PCC8005 growth in photobioreactor: model and simulation of the ISS and ground experiments

The Arthrospira-B experiment is the first experiment in space ever allowing the online measurements of both oxygen production rate and growth rate of Limnospira indica PCC8005 in batch photobioreactors running on-board ISS. Four bioreactors were integrated in the ISS Biolab facility. Each reactor was composed of two chambers (gas and liquid) separated by a PTFE membrane and was run in batch conditions. Oxygen production was monitored by online measurement of the total pressure increase in the gas chamber. The experiments are composed of several successive batch cultures for each reactor, performed in parallel on ISS and on ground. In this work, a model for the growth of the cyanobacterium Limnospira indica PCC8005 (also known as Arthrospira or spirulina) in these space membrane photobioreactors was proposed and the simulation results obtained are compared to the experimental results gathered in space and on ground.The photobioreactor model was based on a light transfer limitation model, already used to describe and predict the growth and oxygen production in small to large scale ground photobioreactors. It was completed by a model for pH prediction in the liquid phase allowing assessment of the pH increase associated to the bicarbonate consumption for the biomass growth. A membrane gas-liquid transfer model is used to predict the gas pressure increase in the gas chamber. Substrate limitation is considered in the biological model.A quite satisfactory fit was achieved between experimental and simulation results when a suitable mixing of the liquid phase was maintained. The data showed that microgravity has no first order effect on the oxygen production rate of Limnospira indica PCC8005 in a photobioreactor operating in space in zero gravity conditions.

Adaptational changes in physiological and transcriptional responses of Bifidobacterium longum involved in acid stress resistance after successive batch cultures

Bifidobacterium inhabiting the human and animal intestinal tract is known for its health-promoting effect. Tolerance to acid stress is crucial for bifidobacteria to survive and then exert their beneficial effects in the gut. A long-term adaptation in successive batch cultures was used as evolutionary engineering strategy to improve acid stress tolerance in an industrial probiotic strain, B. longum JDM301. Its derivative, JDM301AR showed higher resistance to several stress conditions, including acid stress than the parental strain, JDM301. To better understand bifidobacterial acid stress response, the changes of fatty acid (FA) in cell membrane of these two strains were determined. A shift in the production of FA in cell membrane, characterized by increased C14:0 was found, when JDM301AR was exposed to low-pH environment. It was implied that the increased production of C14:0 is associated with the acquisition of acid-tolerant phenotype for JDM301AR. High-throughput RNA-sequencing was performed to analyze the changes of gene expression profile after acid-exposure. The transcriptional profiles of JDM301AR and JDM301 under normal condition and acid stress were compared to reveal the different acid response between them. A total of 5 genes involved in FA metabolism were upregulated and no downregulated genes were found in response to acid stress in JDM301AR. The up-regulated BLJ_0565 and BLJ_1105 may play important roles in the modification of membrane FA composition of JDM301AR after acid exposure. Overall, these results suggested that successive batch cultures induced the acid stress tolerance of B. longum involved in transcriptional and physiological responses, including modification of cell wall and cell membrane, metabolism of amino acid and neutralization of internal pH by strengthening NH3 production and transport.

Physiological and Transcriptomic Analysis of a Chronologically Long-Lived Saccharomyces cerevisiae Strain Obtained by Evolutionary Engineering.

High-throughput aging studies with yeast as a model organism involve transposon-mutagenesis and yeast knockout collection, which have been pivotal strategies for understanding the complex cellular aging process. In this study, a chronologically long-lived Saccharomyces cerevisiae mutant was successfully obtained by using another high-throughput approach, evolutionary engineering, based on systematic selection in successive batch cultures under gradually increasing levels of caloric restriction. Detailed comparative physiological and transcriptomic analyses of the chronologically long-lived mutant and the reference strain revealed enhanced levels of respiratory metabolism, upregulation of genes related to carbohydrate metabolic processes, glycogen-trehalose pathways, stress response, and repression of protein synthesis-related genes in the long-lived mutant SRM11, already in the absence of caloric restriction. Interestingly, SRM11 had also significantly higher resistance to copper stress, and higher resistance to silver, ethanol, and 2-phenylethanol stresses than the reference strain. It also had lower ethanol production levels and an enhanced ethanol catabolism. To conclude, evolutionary engineering is another powerful high-throughput method for aging research, in addition to its widespread use in industrial strain development. Additionally, the interesting results revealed by this study about the potential relationship between longevity and various cellular properties are yet to be investigated further at molecular level.

Efficient transformation of Mycosphaerella fijiensis by underwater shock waves

Black leaf streak disease, also known as black Sigatoka, causes dramatic losses in production of banana and plantains fruits. The disease is caused by the pathogenic fungus Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis; Mycosphaerellaceae). Genetic transformation of M. fijiensis would allow a better understanding of molecular basis of pathogenicity and design novel approaches to control the infection caused by this pathogen. However, transformation of this fungus has not been easy. We report here a protocol for genetic transformation of M. fijiensis employing underwater shock waves and intact conidia. The recombinant strains recovered showed genetic stability over >10 generations. The frequency of transformation obtained was between 75 and 150 times higher than the efficiency reported in the only article published on transformation of M. fijiensis using spheroplasts. This improvement allowed the use of a thousand times less cells than the amount employed before, avoiding the need for cumbersome successive batch cultures. Our protocol is simple, highly efficient, fast and reproducible and together with the available genomes of M. fijiensis and Musa acuminata, it offers new possibilities to study the diverse mechanisms of pathogenesis of the fungus.

Modelling the effect of fluctuating herbicide concentrations on algae growth

Herbicide concentrations fluctuate widely in watercourses after crop applications and rain events. The level of concentrations in pulses can exceed the water chronic quality criteria. In the present study, we proposed modelling the effects of successive pulse exposure on algae. The deterministic model proposed is based on two parameters: (i) the typical growth rate of the algae, obtained by monitoring growth rates of several successive batch cultures in growth media, characterizing both the growth of the control and during the recovery periods; (ii) the growth rate of the algae exposed to pulses, determined from a dose–response curve obtained with a standard toxicity test. We focused on the herbicide isoproturon and on the freshwater alga Scenedesmus vacuolatus, and we validated the model prediction based on effect measured during five sequential pulse exposures in laboratory. The comparison between the laboratory and the modelled effects illustrated that the results yielded were consistent, making the model suitable for effect prediction of the herbicide photosystem II inhibitor isoproturon on the alga S. vacuolatus. More generally, modelling showed that both pulse duration and level of concentration play a crucial role. The application of the model to a real case demonstrated that both the highest peaks and the low peaks with a long duration affect principally the cell density inhibition of the alga S. vacuolatus. It is therefore essential to detect these characteristic pulses when monitoring of herbicide concentrations are conducted in rivers.

Decolouration of azo dyes by Phanerochaete chrysosporium immobilised into alginate beads

Because of high discharged volumes and effluent composition, wastewater from the textile industry can be considered as the most polluting amongst all industrial sectors, thus greatly requiring appropriate treatment technologies. Although some abiotic methods for the reduction of several dyes exist, these require highly expensive catalysts and reagents. Biotechnological approaches were proven to be potentially effective in the treatment of this pollution source in an eco-efficient manner. The white-rot fungi are, so far, the most efficient microorganisms in degrading synthetic dyes. This white-rot fungi's property is due to the production of extracellular lignin-modifying enzymes, which are able to degrade a wide range of xenobiotic compounds because of their low substrate specificity. In this paper, we studied the ability of the white-rot fungus Phanerochaete chrysosporium immobilised into Ca-alginate beads to decolourise different recalcitrant azo dyes such as Direct Violet 51 (DV), Reactive Black 5 (RB), Ponceau Xylidine (PX) and Bismark Brown R (BB) in successive batch cultures. To the best of our knowledge, this is the first study on the immobilisation of P. chrysosporium into Ca-alginate beads for its application in dye decolouration. P. chrysosporium was immobilised into Ca-alginate beads using a method of gel recoating to minimise cellular leaking. The immobilised fungus was transferred to 250-ml Erlenmeyer flasks containing 50 ml of growth medium and incubated on an orbital shaker at 150 rpm and 30 degrees C for 7 days. The ratio of beads/medium used was 10% (w/v). The dyes were added into the culture flasks when MnP production started (50 U l(-1)), which corresponded with the seventh cultivation day. MnP activity and dye decolouration were measured spectrophotometrically. The dyes DV, RB and PX were almost totally decolourised at the end of each batch during the course of three successive batches. However, the dye BB was more resistant to decolouration and it was not completely decolourised (86.7% in 144 h). Further, the beads were kept in sterilised calcium chloride (2 g l(-1)) for 3 weeks at 4 degrees C. After these three storage weeks, the immobilised P. chrysosporium was again efficiently reused for azo dye decolouration during two successive batches, decolouration being more effective even for BB. Also, the in vitro decolouration of the aforementioned azo dyes by crude MnP from P. chrysosporium was performed. The decolouration levels obtained were lower than those attained with the whole cultures especially for RB and BB dyes, in spite of the fact that dye concentrations used were considerable lower. The good performance of the immobilisation system was likely due to the gel re-coating method utilised to prepare the alginate beads which not only maintained the beads integrity but also avoided cellular leaking. The lower decolouration percentages obtained by the enzyme indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or other compounds that favour dye decolouration. Immobilised P. chrysosporium efficiently decolourised different types of azo dyes. In this decolouration process, the MnP secreted by the fungus played the main role whilst adsorption was found to be negligible except for the dye BB. Efforts should be made to scale up and apply fungal decolouration techniques to real industrial dye-containing wastewater. Further, detailed characterisation of the intermediates and metabolites produced during biodegradation must be done to ensure the safety of the decolourised wastewater.

Enhanced production of laccase in Coriolopsis rigida grown on barley bran in flask or expanded-bed bioreactor

In this work, the effect on laccase activity of adding xylidine, veratryl alcohol and copper sulphate to cultures of Coriolopsis rigida under submerged cultivation conditions have been investigated. The highest activities (approximately 6 × 105 nkat/l) were obtained when the inducers copper sulphate (2 mM) and xylidine (10 mM) were added simultaneously. In addition, operating in the optimal conditions, it was possible to maintain the sustained production of laccase (around 3 × 105 nkat/l) for successive repeated batch cultures in an expanded-bed laboratory scale bioreactor. On the other hand, in vitro phenol degradation by laccase obtained in the bioreactor was studied with/without an effective mediator 1-hydroxybenzotriazol (HBT). The presence of a radical mediator plays an important role inducing the degradation of phenol, and without mediator the polymerization of phenol was detected.

Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.

Degradation of polychlorinated biphenyl by cells of Rhodococcus opacus strain TSP203 immobilized in alginate and in solution

Rhodococcus opacus strain TSP203, a Gram-positive biphenyl utilizer, can degrade trichloro-, tetrachloro-, and pentachlorobiphenyl congeners in PCB concentrations of 100 μg ml −1 Kaneclor 300 (equivalent to Aroclor 1242). The effect of Ca-alginate entrapment on the PCB-degrading competence of strain TSP203 was studied. Initially, the ability of immobilized cells of strain TSP203 and cells of TSP203 in solution to degrade congeners in 20 μg ml −1 Kaneclor 300, Kaneclor 400 (equivalent to Aroclor 1248), and Kaneclor 500 (equivalent to Aroclor 1254) was compared. In Kaneclor 300 and Kaneclor 400, PCB degradation by alginate immobilized and cells in solution were almost equivalent while in Kaneclor 500, degradation by cells in solution was slightly higher. The effect of increasing concentrations of Kaneclor 300 on the PCB-degrading ability of alginate-immobilized cells was evaluated. At 50, 100, and 150 μg ml −1 Kaneclor 300, an improvement in degradation by immobilized cells compared to cells in solution was observed. For degradation of 100 μg ml −1 Kaneclor 300, the optimum pH was 8.4 and optimum temperature 30°C. The biomass concentration was found to be critical for degradation. At low biomass concentration, trichloro-, tetrachloro-, and pentachlorobiphenyls were poorly degraded. In semicontinuous experiments, the immobilized cells could be used for three successive batch cultures with decreasing degrees of efficiency while cells in solution lysed after the first use. Although Ca-alginate afforded protection against toxicity of PCBs, we were unable to obtain a stable regeneration of the biocatalyst probably due to the absence of phosphate in the regeneration medium.

Palm petiolar felt-sheath as a new and convenient material for the immobilization of microalgal cells

This communication reports the use of the petiolar felt-sheath of palm as a novel biomatrix for the immobilization of microalgal cells. Immobilized cells, as compared with free cells, were observed to have significantly higher biomass and polysaccharide production after 27 days of culture growth. Immobilized cells were successfully maintained through 12 successive batch cultures over 96 days. Extracellular polysaccharide production during this period ranged from 382 to 440 mg L−1. The new immobilization material is cheap, stable and easily available, and the procedure developed for entrapment of the microalga is simple, reliable and practical.

Production of endo-1,4-β-glucanase and xylanase with nylon-web immobilized and free Trichoderma reesei

On all media studied, the endo-1,4-β-glucanase and xylanase activities obtained with the nylon-web immobilized Trichoderma reesei were higher than the activities with the free mycelium. The nitrogen source had a marked effect on endoglucanase and xylanase production on lactose, cellulose, and l-sorbose-based medium with immobilized T. reesei. The highest enzyme activities with this medium, 880 nkat ml −1 for endoglucanase and 8,020 nkat ml −1 for xylanase, were obtained in the presence of proteose peptone and yeast extract. Urea alone was an insufficient nitrogen source for enzyme production. A lactose-containing industrial by-product-based medium offered a good alternative for endoglucanase and xylanase production. In this case, the maximum endoglucanase activity was 940 nkat ml −1, xylanase activity was 4,410 nkat ml −1, and filter paper activity was 1.9 FPU ml −1. On synthetic cellulose-based medium, the activities were 840 nkat ml −1, 4.790 nkat ml −1, and 3.0 FPU ml −1, respectively. The nylon-web immobilized biocatalyst could be used for two successive batch cultures with only a little decrease in enzyme production observed.