Repeated Batch Culture Research Articles

Bacterial diversity of the outflows of a Polichnitos (Lesvos, Greece) hot spring, laboratory studies of a Cyanobacterium sp. strain and potential medical applications

The bacterial diversity of the outflows of Polichnitos (Lesvos, Greece) hot spring has been investigated. Cyanobacteria showing high sequence homologies with Phormidium sp. and Cyanobacterium aponinum were found. Members of the Alphaproteobacteria closely related to Rhodobium sp. Albidovulum sp., Rhodobacter sp., Microvigra sp., Nitratireductor sp. and Phaeobacter sp. Gammaproteobacteria, Betaproteobacteria and Firmicutes were represented by members of Idiomarina sp., Marinobacter sp., Shinella sp., Bacillus sp. and Clostridium sp. with sequence homologies ranging from 92% to 100%. Members of the Bacteroidetes and Planctomycetes were represented by sequences of novel phylogenetic linkages exhibiting 87–90% sequence homology with type strains. When the hot spring consortium was cultivated in bioreactor repeated batch culture under photo-autotrophic growth conditions at temperature < 30 °C, Cyanobacterium sp. dominated over Phormidium sp. Cyanobacterium sp. seems to have biotechnological potential since its extracellular broth exhibited a strong insecticidal activity against larvae of Aedes aegypti (a vector of important human diseases) and significant anti-cancer activity against the PC3 human prostate cancer cell line, while its toxicity against human endothelial cells was relatively low.

Comparison between free cells and immobilized cells of Candida shehatae in ethanol production from rice straw hydrolysate using repeated batch cultivation

Ethanol production by xylose-fermenting Candida shehatae ATCC 22984 was studied in repeated batch fermentations of rice straw hydrolysate using both free cells and immobilized cells. Terracotta beads, coconut bract and corncob were investigated as supporting materials for cell immobilization. Ethanol production was stable at least 5 cycles when using cells immobilized on coconut bract. Average ethanol concentrations obtained from each batch were 16.7 g/L in free-cell system and 17.2 g/L in immobilized-cell system. Repeated batch using free cell system resulted in a slightly higher volumetric productivity than the use of immobilized cells. Ethanol that could be produced per ton of dried rice straw was 120 L using free cell system and 117 L using immobilized cell on coconut bract in glass column system.

Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations.

Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h-1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

Immobilized oleaginous microalgae for production of lipid and phytoremediation of secondary effluent from palm oil mill in fluidized bed photobioreactor

Oleaginous microalga Nannochloropsis sp. was immobilized in alginate gel beads and cultivated under optimal conditions that their growth and lipid production were comparable to those of free cells. The immobilized cells were used in phytoremediation of secondary effluent from palm oil mill and easily recovered by simple sieving method. The immobilized cells contributed to removal of nitrogen and phosphorus >90% and CO2 mitigation >99%. They also gave the biomass and lipid production of 1.300±0.050g/L and 0.356±0.097g/L, respectively. The repeated-batch cultivation improved the biomass and lipid production by 2.66 folds and 1.41 folds, respectively. The scale up in 3L-fluidized bed photobioreactor gave the maximum biomass of 3.280±0.049g/L and lipid production of 0.362±0.010g/L. Fatty acid compositions of Nannochloropsis sp. lipids showed their suitability as biodiesel feedstocks. This system not only contributes as tertiary treatment of industrial effluent and CO2 mitigation but also low-cost production of renewable energy.

Evaluation of anaerobic digestates from different feedstocks as growth media for Tetradesmus obliquus, Botryococcus braunii, Phaeodactylum tricornutum and Arthrospira maxima

In this paper, two freshwater microalgae (Tetradesmus obliquus and Botryococcus braunii), a marine diatom (Phaeodactylum tricornutum) and a photosynthetic cyanobacterium (Arthrospira maxima) were investigated for their ability to grow on liquid digestates (LDs). Three LDs were obtained from anaerobic digestion of different organic wastes: zootechnical (ZW LD), vegetable biomass (VW LD) and the organic fraction of municipal solid wastes (MW LD). All the strains showed the same growth performance on VW LD as on the respective standard media (SM), while ZW LD was efficient only for growth of T. obliquus and B. braunii. MW LD was the poorest growth medium for all the strains. Data on nutrient removal efficiency showed that A. maxima and T. obliquus made the best use of NH4+-N with removal values ranging between 98.9–99.8%, while P. tricornutum and B. braunii showed values of 79.0 and 88.5% respectively. Applying repeated batch cultivation in photobioreactors, the biochemical composition of A. maxima and T. obliquus biomass grown on ZW LD and VW LD, showed an increase of lipid, carbohydrates and ash in both microalgae. Biomass biochemical profiles suggest possible applications in feed, chemicals and energy sectors.

Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single-Use Stirred Tank Bioreactors.

Human pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.

Enhanced methane production via repeated batch bioaugmentation pattern of enriched microbial consortia

Using batch and repeated batch cultivations, this study investigated the effects of bioaugmentation with enriched microbial consortia (named as EMC) on methane production from effluents of hydrogen-producing stage of potato slurry, as well as on the indigenous bacterial community. The results demonstrated that the improved methane production and shift of the indigenous bacterial community structure were dependent on the EMC/sludge ratio and bioaugmentation patterns. The methane yield and production rate in repeated batch bioaugmentation pattern of EMC were, respectively, average 15% and 10% higher than in one-time bioaugmentation pattern of EMC. DNA-sequencing approach showed that the enhanced methane production in the repeated batch bioaugmentation pattern of EMC mainly resulted from the enriched iron-reducing bacteria and the persistence of the introduced Syntrophomonas, which led to a rapid degradation of individual VFAs to methane. The findings contributed to understanding the correlation between the bioaugmentation of microbial consortia, community shift, and methane production.

Alleviation of proteolytic degradation of recombinant human bone morphogenetic protein-4 by repeated batch culture of Chinese hamster ovary cells

The effects of culture temperature and pH on cell growth and recombinant human bone morphogenetic protein-4 (rhBMP-4) production were investigated to maximize rhBMP-4 production from Chinese hamster ovary cells. Cells were cultivated in a bioreactor at different culture pH levels (6.7, 6.9, 7.2, and 7.5) and temperatures (37°C and 33°C). The highest rhBMP-4 concentration (4.6±0.5μg/mL) was obtained at pH 7.2 and 37°C. Regardless of culture temperature and pH, significant rhBMP-4 degradation via increased proteolytic activity was observed in the decline growth phase. Protease inhibitors such as EDTA and leupeptin were toxic and thus did not enhance rhBMP-4 production. High viability was achieved in repeated batch cultures with frequent medium exchanges. These cultures showed enhanced rhBMP-4 productivity, demonstrating the potential of repeated batch cultures as a means for improved rhBMP-4 production.

Bioaugmentation of Hydrogenispora ethanolica LX-B affects hydrogen production through altering indigenous bacterial community structure

Bioaugmentation can facilitate hydrogen production from complex organic substrates, but it still is unknown how indigenous microbial communities respond to the added bacteria. Here, using a Hydrogenispora ethanolica LX-B (named as LX-B) bioaugmentation experiments, the distribution of metabolites and the responses of indigenous bacterial communities were investigated via batch cultivation (BC) and repeated batch cultivation (RBC). In BC the LX-B/sludge ratio of 0.12 achieved substantial high hydrogen yield, which was over twice that of control. In RBC one-time bioaugmentation and repeated batch bioaugmentation of LX-B resulted in the hydrogen yield that was average 1.2-fold and 0.8-fold higher than that in control, respectively. This improved hydrogen production performance mainly benefited from a shift in composition of the indigenous bacterial community caused by LX-B bioaugmentation. The findings represented an important step in understanding the relationship between bioaugmentation, a shift in bacterial communities, and altered bioreactor performance.

Microalgal TAG production strategies: why batch beats repeated-batch.

BackgroundFor a commercially feasible microalgal triglyceride (TAG) production, high TAG productivities are required. The operational strategy affects TAG productivity but a systematic comparison between different strategies is lacking. For this, physiological responses of Nannochloropsis sp. to nitrogen (N) starvation and N-rich medium replenishment were studied in lab-scale batch and repeated-batch (part of the culture is periodically harvested and N-rich medium is re-supplied) cultivations under continuous light, and condensed into a mechanistic model.ResultsThe model, which successfully described both strategies, was used to identify potential improvements for both batch and repeated-batch and compare the two strategies on optimized TAG yields on light (amount of TAGs produced per mol of supplied PAR photons). TAG yields on light, for batch, from 0.12 (base case at high light) to 0.49 g molph−1 (at low light and with improved strain) and, for repeated-batch, from 0.07 (base case at high light) to 0.39 g molph−1 (at low light with improved strain and optimized repeated-batch settings). The base case yields are in line with the yields observed in current state-of-the-art outdoor TAG production.ConclusionsFor continuous light, an optimized batch process will always result in higher TAG yield on light compared to an optimized repeated-batch process. This is mainly because repeated-batch cycles start with N-starved cells. Their reduced photosynthetic capacity leads to inefficient light use during the regrowth phase which results in lower overall TAG yields compared to a batch process.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0475-4) contains supplementary material, which is available to authorized users.

High molecular weight β-poly(l-malic acid) produced by A. pullulans with Ca2+ added repeated batch culture

β-Poly(malic acid) (PMLA) has attracted increasing attentions because of its potential application in medicine and other industries. In this study, the variation of PMLA molecular weight (Mw) in the batch culture and the strategies to enhance PMLA Mw were studied. Adding exogenous Ca2+ (0.1g/L CaCl2) to the medium caused a significant increase in both PMLA concentration and Mw (11.38% and 26.3%, respectively) when Na2CO3 was used as the neutralizer. The Mw of PMLA during the process of batch culture, which associated with the specific PMLA production per unit cell mass (Yp/x) before glucose was depleted, increased from 12.522KDa to its maximum 18.693KDa and then kept decreasing until the end of the culture. Compared with the results in batch culture, Mw increased by 84.4% (up to 19.51kDa) with a productivity of 1.1gh−1L−1 when the cells were maintained in exponential growth phase during Ca2+ added repeated batch culture. The present work provides an efficient approach to obtain superior quality PMLA product with high Mw.

Long term impact of stressing agents on fermentative hydrogen production: Effect on the hydrogenase flux and population diversity

In this study, the long term effect of different microbial stressing agents on hydrogen (H2) production was examined using repeated batch cultivations. When compared to thermophilic cultures, higher H2 yields were observed in mesophilic cultures receiving repeated glucose addition. Methane production was only observed in control mesophilic cultures receiving repeated 5 glucose additions. Lower hydrogenase evolution specific activity was observed in thermophilic cultures (except alkali-treated cultures) compared to mesophilic cultures. For both mesophilic and thermophilic cultures, the hydrogenase uptake specific activity of the untreated control cultures exhibited higher levels of activity than the pretreated cultures. A flux balance analysis (FBA) showed negligible homoacetogenic flux in mesophilic cultures pretreated with linoleic acid (LA) and loading shock (LS) after successive batch cultivations. The homoacetogenic flux accounted for approximately 98% loss in the H2 yield in untreated mesophilic control cultures. Both homoacetogens (Eubacterium sp.) and aceticlastic methanogens (Methanosaeta sp. and Methanosarcina sp.) were abundant in the control cultures. In comparison, Clostridium sp. were dominant in mesophilic stress treated cultures whereas under thermophilic conditions, the dominant microorganisms were Flavobacterium sp., Bacillus sp., Thermoanaerobacter sp., Bacteroides sp., Lactobacillus sp. and Thioalkalivibrio sp.

Batch and continuous thermophilic hydrogen fermentation of sucrose using anaerobic sludge from palm oil mill effluent via immobilisation technique

This study aimed to investigate the feasibility to use immobilised cells on granular activated carbon (GAC) operated in fifteen cycles of repeated batch mode for enhancement of biohydrogen production under thermophilic conditions. The effects of the initial pH (5.0–6.5), sucrose concentration (13–43mM) and repeated batch cultivation on biohydrogen production from sucrose were investigated using anaerobic sludge from a palm oil mill effluent treatment plant. The cumulative hydrogen production results were fitted into a modified Gompertz equation in order to find the optimum operational conditions. The optimal hydrogen yield (2.8molH2/molhexose) was obtained at an initial pH of 5.5 and sucrose concentration of 13mM after fifteen cycles of repeated batch. Enriched granular activated carbon (GAC)-immobilised cells from the repeated batch cultivation were used as the immobilised seed culture for anaerobic fermentation of sucrose into hydrogen in continuous operation using a fluidised-bed column reactor (FBCR). The maximum hydrogen production rate (HPR) was found to be 2.7mmolH2/L/h and the hydrogen yield peaked at 2.8molH2/molhexose_consumed after a hydraulic retention time of 12h. The main soluble metabolites were identified as acetic acid, butyric acid and ethanol. The hydrogen content ranged from 48 to 50% of the total biogas.

Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200pF/cm (where 1pF/cm is equivalent to 1×106viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37°C to 29°C was studied during 13 days at cell densities above 100pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31°C was applied at cell density 100pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

Production of technical-grade sodium citrate from glycerol-containing biodiesel waste by Yarrowia lipolytica

The production of technical-grade sodium citrate from the glycerol-containing biodiesel waste by Yarrowia lipolytica was studied. Batch experiments showed that citrate was actively produced within 144h, then citrate formation decreased presumably due to inhibition of enzymes involved in this process. In contrast, when the method of repeated batch cultivation was used, the formation of citrate continued for more than 500h. In this case, the final concentration of citrate in the culture liquid reached 79–82g/L. Trisodium citrate was isolated from the culture liquid filtrate by the addition of a small amount of NaOH, so that the pH of the filtrate increased to 7–8. This simple and economic isolation procedure gave the yield of crude preparation containing trisodium citrate 5.5-hydrate up to 82–86%.

Mycelial Pellet Formation in Fungal Lipid Production by Cunninghamella bainieri 2A1 Using Repeated Batch Culture

Repeated batch culture of Cunninghamella bainieri 2A1 was performed to investigate mycelial pellet formation in fungal lipid production process. Experimental studies were performed based on the size and structural morphology of pellets formed with consideration of harvesting times and harvested volumes of repeated batch culture. Electron microscope images showed that fungal cells formed mycelial pellets with varied size from 575 µm × 586 µm in 60 % harvesting volume with a hairy loose morphology to 1.01 mm × 1.18 mm in 90 % harvesting volume with a tightly packed dense morphology.

Batch culture and repeated-batch culture of Cunninghamella bainieri 2A1 for lipid production as a comparative study

This research was performed based on a comparative study on fungal lipid production by a locally isolated strain Cunninghamella bainieri 2A1 in batch culture and repeated-batch culture using a nitrogen-limited medium. Lipid production in the batch culture was conducted to study the effect of different agitation rates on the simultaneous consumption of ammonium tartrate and glucose sources. Lipid production in the repeated-batch culture was studied by considering the effect of harvesting time and harvesting volume of the culture broth on the lipid accumulation. The batch cultivation was carried out in a 500ml Erlenmeyer flask containing 200ml of the fresh nitrogen-limited medium. Microbial culture was incubated at 30°C under different agitation rates of 120, 180 and 250rpm for 120h. The repeated-batch culture was performed at three harvesting times of 12, 24 and 48h using four harvesting cultures of 60%, 70%, 80% and 90%. Experimental results revealed that nitrogen source (ammonium tartrate) was fully utilized by C. bainieri 2A1 within 24h in all agitation rates tested. It was also observed that a high amount of glucose in culture medium was consumed by C. bainieri 2A1 at 250rpm agitation speed during the batch fermentation. Similar results showed that the highest lipid concentration of 2.96g/L was obtained at an agitation rate of 250rpm at 120h cultivation time with the maximum lipid productivity of 7.0×10−2mg/ml/h. On the other hand, experimental results showed that the highest lipid concentration produced in the repeated-batch culture was 3.30g/L at the first cycle of 48h harvesting time using 70% harvesting volume, while 0.23g/L gamma-linolenic acid (GLA) was produced at the last cycle of 48h harvesting time using 80% harvesting volume.

Effect of Complex Nutrients and Repeated-Batch Cultivation of Halobacterium salinarum on enhancing Bacteriorhodopsin Production

The extreme halophilic bacteria Halobacterium salinarum is known to produce bacteriorhodopsin (BR) protein in its purple membrane (PM) as a light-driven pump for the synthesis ATP. Its growth does not utilize simple carbon sources such as glucose, but relies on the complex carbon/nitrogen sources. The production yield of BR in the culture of H. salinarum also strongly depends on the complex nitrogen sources employed. From the various complex carbon/nitrogen sources employed in this work, tryptone rather than commonly used peptone was found to be the best complex nutrient for the growth of H. salinarum and its BR production. Bubble column photobioreactor operated in a repeated-batch mode was also employed to enhance the growth of H. salinarum by intermittent removing growth inhibitory metabolites. By employing 0.5% tryptone as carbon/nitrogen source in the culture medium, 201.8 mg/L of BR was obtained after 210 h repeated-batch cultivation which is about 50% higher than that obtained in shaker flask cultivation.

Improvement in bacterial cellulose production using Gluconacetobacter xylinus ATCC 10245 and characterization of the cellulose pellicles produced

Aim This paper deals with the improvement in bacterial cellulose (BC) production by Gluconacetobacter xylinus ATCC 10245 cultivated under shaking condition. Materials and methods The study was performed using two different approaches. First, through the addition of some thickening agents to the fermentation medium; second, by applying a two-stage fermentation process in which cells of G. xylinus were grown under two successive static and shaking conditions. During the first fermentation stage, the experimental microorganism was cultivated under static condition to produce thick, leather-like, white BC pellicles on which the bacterial cells were firmly adhered. However, during the second fermentation stage, these pellicles were reused to inoculate fresh, more economic medium, and incubated under shaking condition. Results The results showed that the addition of sodium alginate at a concentration of 0.4 g/l resulted in a BC production of 8.25 g/l compared with only 1.7 g/l obtained using the control medium without thickeners under similar shaking condition. The second method resulted in a substantial increase in the produced BC pellicle up to about 16.9 g/l. Further increase in the weight of the BC gels was obtained by adopting a repeated batch cultivation method during the second fermentation stage. After seven successive repeated batches, which extended for 56 days, the total BC mass reached 81.25 g/l. The properties of some BC pellicles were studied using thermogravimetric analyses and then compared with those of cotton linter. Conclusion The results showed that these two methods are very promising for BC production on a large scale.

Comparative studies of free and immobilized phytase, produced by Penicillium purpurogenu GE1, using grafted alginate/carrageenan beads

Aim The aim of the study was to immobilize the phytase enzyme produced by Penicillium purpurogenu GE1 on grafted alginate/carrageenan beads and study the properties of the immobilized enzyme in comparison with free ones. Materials and methods The immobilization conditions were first optimized and then the optimum conditions of temperature and pH for the maximum activity of the immobilized and free enzyme were studied and compared. The stabilities of both immobilized and free phytase at moderate and low temperatures of 50 and 4°C, as well as their stability at the acidic pH of 4, were also studied. Finally, the activity of the immobilized enzyme was monitored over 20 successive repeated batches. Results The maximum loading capacity was obtained after 20 h at the enzyme/acetate buffer dilution ratio of 1 : 2. The optimum temperature and pH of the immobilized enzyme, as compared with free state, were found to have shifted from 37 to 45°C and from pH 5.5 to 4, respectively. Moreover, the results also proved that when phytase in both immobilized and free states was subjected to an acidic pH of 4 for 45 min, or to a moderately high temperature of 50°C for 60 min, the activity of the former remained stable, whereas that of the latter showed substantial losses. In contrast, at the refrigerator shelf temperature of 4°C, dry and wet immobilized forms retained 100% activity for 12 weeks, whereas that of the free enzyme was completely lost within a shorter period of 4 weeks. Furthermore, the activity of the phytase enzyme immobilized on gel beads was maintained at the 100% level for more than 12 repeated batch utilizations of the beads. Conclusion The results revealed that the physiological parameters of the immobilized enzyme were greatly improved compared with the free state. Further, the activity of the phytase enzyme immobilized on gel beads was maintained at the 100% level for more than 12 repeated batch cultivations of the beads.