Batch Culture Research Articles

Optimized batch cultivation and scale-up of Bacillus thuringiensis for high-yield production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Addition of statistically optimized concentration of electron acceptor, propionic acid (1.2 g/L) at different cultivation times (0 h, 14.86 h and 19 h) during batch cultivation of B. thuringiensis in mixed substrate (glucose and glycerol) featured production of 8 g/L of biomass and 3.57 g/L of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing 0.805 g/L of 3-hydroxyvalerate concentration. Successful scale up of batch cultivation from 7 L to a 70 L bioreactor was, thereafter, achieved using power/volume (P/V) criteria with maximum PHBV and biomass concentration of 3.57 g/L and 7.15 g/L respectively. Characterization of PHBV so produced was carried out using NMR, FTIR, DSC and TGA to elucidate its structure, thermal properties and stability to map their applications in society. These findings highlight the potential of the optimized batch cultivation and scale-up process in producing PHBV emphasizing its relevance in sustainable biopolymer production.

Production of (R)-citramalate by engineered Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, has a high tolerance to organic acids and alcohols, and thus grows well under toxic concentrations of various compounds in the culture medium, potentially allowing for highly efficient compound production. (R)-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (R)-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike Escherichia coli, S. cerevisiae has organelles, and its intracellular metabolites are compartmentalized, preventing full use of intracellular acetyl-CoA. Therefore, in this study, to increase the amount of cytosolic acetyl-CoA for highly efficient production of (R)-citramalate, we inhibited the transport of cytosolic acetyl-CoA and pyruvate to the mitochondria. We also constructed a heterologous pathway to supply cytosolic acetyl-CoA. Additionally, we attempted to export (R)-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (R)-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (R)-citramalate in batch culture flasks. This is the first report of (R)-citramalate production by recombinant S. cerevisiae, and the (R)-citramalate production by recombinant yeast achieved in this study was the highest reported to date.

Functional plasticity of HCO3– uptake and CO2 fixation in Cupriavidus necator H16

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3– uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.

Implementation of a Macroporous Polyhydroxyethylmethacrylate Cryogel-Based Mini-Bioreactor System to Improve Monoclonal Antibody Production.

Monoclonal antibodies (mAbs) are pioneers in the diagnosis and treatment of many diseases, such as cancer, asthma, poisoning, viral infections, etc. As the market value of mAbs increases in the biopharma industry, the demand for high quantities is met by upscaled production using bioreactor systems. Thus, disposable, porous matrices called cryogels have gained the primary focus for adherent support in the proliferation of hybridoma cells. In this study, a gelatin-immobilized polyhydroxyethylmethacrylate-based cryogel material (disc-shaped, 9 mL bed volume) was synthesized, and a mini-bioreactor set up developed for culturing hybridoma cells to produce mAbs continuously. The hybridoma clone, 1B4A2D5, secreting anti-human serum albumin monoclonal antibodies, was immobilized in the cryogel matrix (2 discs, 18 mL bed volume). The hybridoma cells were attached to the matrix within 12 h after inoculation, and the cells were in the lag phase for seven days, where they were secreted mAb into the circulation medium. During the initial exponential phase, the glucose consumption, lactic acid production, and mAb production were 3.36 mM/day, 3.67 mM/day, and 55.61 µg/mL/day, respectively. The medium was refreshed whenever the glucose in the media went below 50% of the initial glucose concentration. The cryogenic reactor was run continuously for 25 days, and the mAb concentration reached a maximum on the 17th day at 310.59 µg/mL. The cumulative amount of mAbs produced in 25 days of running was 246 µg/mL, 7.7 times higher than the mAbs produced from T-flask batch cultivation. These results demonstrate that the developed polyhydroxyethylmethacrylate-based cryogel reactor can be used efficiently for continuous mAb production.

Improving the Accuracy of Bulk Fitness Assays by Correcting Barcode Processing Biases.

Measuring the fitnesses of genetic variants is a fundamental objective in evolutionary biology. A standard approach for measuring microbial fitnesses in bulk involves labeling a library of genetic variants with unique sequence barcodes, competing the labeled strains in batch culture, and using deep sequencing to track changes in the barcode abundances over time. However, idiosyncratic properties of barcodes can induce nonuniform amplification or uneven sequencing coverage that causes some barcodes to be over- or under-represented in samples. This systematic bias can result in erroneous read count trajectories and misestimates of fitness. Here, we develop a computational method, named REBAR (Removing the Effects of Bias through Analysis of Residuals), for inferring the effects of barcode processing bias by leveraging the structure of systematic deviations in the data. We illustrate this approach by applying it to two independent data sets, and demonstrate that this method estimates and corrects for bias more accurately than standard proxies, such as GC-based corrections. REBAR mitigates bias and improves fitness estimates in high-throughput assays without introducing additional complexity to the experimental protocols, with potential applications in a range of experimental evolution and mutation screening contexts.

The Anti-Methanogenic Activity of Lovastatin in Batch Cultures Using Rumen Inoculum from Sheep, Goats, and Cows

Enteric methanogenesis in ruminants is identified as one of the primary anthropogenic sources of total atmospheric methane. Recent evidence suggests that rumen methanogenesis is significantly suppressed by lovastatin. Nevertheless, it has not been reported whether the methane reduction by lovastatin depends on ruminant livestock type, nor has fiber degradability been examined. The current research aimed to analyze the in vitro effect of lovastatin on the major fermentation end-products, gas production (GP) kinetics, and fiber degradation of a forage-based diet using rumen inoculum from sheep, goats, and cows. The experiment was conducted as a 3 × 3 factorial arrangement of treatments (dose of lovastatin: 0, 80, and 160 mg/L and three inoculum sources) in a completely randomized design. The results suggested that lovastatin did not affect the GP kinetics parameters. The anti-methanogenic properties of lovastatin were variable depending on dose and inoculum source. Lovastatin demonstrated a superior methane-lowering effect in sheep rumen inoculum compared with goat and cow inocula. The total volatile fatty acid (VFA) production was unaffected by lovastatin, but changes in acetate and valerate proportions were registered. Remarkably, lovastatin decreased the NH3-N concentration with goat and sheep inocula and the in vitro neutral fiber detergent (NDF) degradation for all inoculum sources.

Bioaugmentation protocols involving Methanobrevibacter thaueri and Pecoramyces ruminantium for investigating lignocellulose degradation and methane production from alfalfa stalks

Two protocols involving batch cultures were used to investigate the bioaugmentation of methane production by Pecoramyces ruminantium, and Methanobrevibacter thaueri. Protocol I examined the effect of altering the proportion of the microbial constituents in inoculum on alfalfa stalk fermentations and showed a 25 % improvement in dry matter loss in cultures where the inoculum contained just 30 % of co-culture and 70 % of fungal monoculture. Protocol II involved consecutive cultures and alternating inoculations. This protocol resulted in 17–22 mL/g DM methane production with co-cultures a 30 % increase in methane relative to the fungal monoculture. Both protocols indicate that the co-culture rapidly dominated and was more resilient than the monoculture. Synergistic interaction between fungus and methanogen, promoted more efficient lignocellulose degradation and higher methane yield. This study highlighted the potential of microbial co-cultures for enhancing methane production from lignocellulosic biomass, offering a promising bioaugmentation strategy for improving biogas yields and waste valorization.

Physiological and transcriptomic response to methyl-coenzyme M reductase limitation in Methanosarcina acetivorans.

Methane is a potent greenhouse gas that has contributed to ca. 25% of global warming in the post-industrial era. Atmospheric methane is primarily of biogenic origin, mostly produced by microorganisms called methanogens. Methyl-coenzyme M reductase (MCR) catalyzes methane formatio in methanogens. Even though MCR comprises ca. 10% of the cellular proteome, it is hypothesized to be growth-limiting during methanogenesis. In this study, we show that Methanosarcina acetivorans cells grown in substrate-replicate batch cultures produce more MCR than its cellular demand for optimal growth. The tools outlined in this study can be used to refine metabolic models of methanogenesis and assay lesions in MCR in a higher-throughput manner than isolation and biochemical characterization of pure protein.

Novel Saccharomyces uvarum x Saccharomyces kudriavzevii synthetic hybrid with enhanced 2-phenylethanol production

BackgroundOver the last two decades, hybridization has been a powerful tool used to construct superior yeast for brewing and winemaking. Novel hybrids were primarily constructed using at least one Saccharomyces cerevisiae parent. However, little is known about hybrids used for other purposes, such as targeted flavor production, for example, 2-phenylethanol (2-PE). 2-PE, an aromatic compound widely utilised in the food, cosmetic, and pharmaceutical industries, presents challenges in biotechnological production due to its toxic nature. Consequently, to enhance productivity and tolerance to 2-PE, various strategies such as mutagenesis and genetic engineering are extensively explored to improved yeast strains. While biotechnological efforts have predominantly focused on S. cerevisiae for 2-PE production, other Saccharomyces species and their hybrids remain insufficiently described.ResultsTo address this gap, in this study, we analysed a new interspecies yeast hybrid, II/6, derived from S. uvarum and S. kudriavzevii parents, in terms of 2-PE bioconversion and resistance to its high concentration, comparing it with the parental strains. Two known media for 2-PE biotransformation and three different temperatures were used during this study to determine optimal conditions. In 72 h batch cultures, the II/6 hybrid achieved a maximum of 2.36 ± 0.03 g/L 2-PE, which was 2–20 times higher than the productivity of the parental strains. Our interest lay not only in determining whether the hybrid improved in productivity but also in assessing whether its susceptibility to high 2-PE titers was also mitigated. The results showed that the hybrid exhibited significantly greater resistance to the toxic product than the original strains.ConclusionsThe conducted experiments have confirmed that hybridization is a promising method for modifying yeast strains. As a result, both 2-PE production yield and tolerance to its inhibitory effects can be increased. Furthermore, this strategy allows for the acquisition of non-GMO strains, alleviating concerns related to additional legislative requirements or consumer acceptance issues for producers. The findings obtained have the potential to contribute to the development of practical solutions in the future.

Optimizing the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators.

Crop pests and pathogens annually cause over $220 billion in global crop damage, with insects consuming 5%-20%of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes, and agricultural practices. Double-stranded RNA(dsRNA) is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialization of dsRNA-based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimized the design of plasmid DNA constructs to produce dsRNA biocontrols in Escherichia coli, by employing a wide range of alternative synthetic transcriptional terminators before measurement of dsRNA yield. We demonstrate that a 7.8-fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstratethat batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small-scale batch culture and large-scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight.

From yeast screening for suitability as single cell protein to fed-batch cultures.

Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors. SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively. C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 gxgs-1) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5%CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50%CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43%CDW). Total nucleic acid content of the yeasts was similar (6.8%CDW and 6.3%CDW, for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80. This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae.

Delineating acetaminophen biodegradation kinetics and metabolomics using bacterial community.

Acetaminophen [N-(4-hydroxyphenyl) acetamide, APAP]is an extensively and frequently consumed over-the-counter analgesic and antiphlogistic medication. It is being regarded as an emerging pollutant due to its continuous increment in the environment instigating inimical impacts on humans and the ecosystem. Considering its wide prevalence in the environment, there is an immense need of appropriate methods for the removal of APAP. The present study indulged screening and isolation of APAP degrading bacterial strains from pharmaceuticals-contaminated sites, followed by their molecular characterization via 16SrRNA sequencing. The phylogenetic analyses assigned the isolates to the genera Pseudomonas, Bacillus, Paracoccus, Agrobacterium, Brucella, Escherichia, and Enterobacter based on genetic relatedness. The efficacy of these strains in batch cultures tested through High-performance Liquid Chromatography (HPLC) revealed Paracoccus sp. and Enterobacter sp. as the most promising bacterial isolates degrading up to 88.96 and 85.92%, respectively of 300mg L-1 of APAP within 8days of incubation. Michaelis-Menten kinetics model parameters also elucidated the high degradation potential of these isolates. The major metabolites identified through FTIR and GC-MS analyses were 4-aminophenol, hydroquinone, and 3-hydroxy-2,4-hexadienedioic. Therefore, the outcomes of this comprehensive investigation will be of paramount significance in formulating strategies for the bioremediation of acetaminophen-contaminated sites through a natural augmentation process via native bacterial strains.

Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering

L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.

Total RNA analysis of the active microbiome on moving bed biofilm reactor carriers under incrementally increasing micropollutant concentrations.

Micropollutants are increasingly prevalent in the aquatic environment. A major part of these originates from wastewater treatment plants since traditional treatment technologies do not remove micropollutants sufficiently. Moving bed biofilm reactors (MBBRs), however, have been shown to aid in micropollutant removal when applied to conventional wastewater treatment as a polishing step. Here, we used Total RNA sequencing to investigate both the active microbial community and functional dynamics of MBBR biofilms when these were exposed to increasing micropollutant concentrations over time. Concurrently, we conducted batch culture experiments using biofilm carriers from the MBBRs to assess micropollutant degradation potential. Our study showed that biofilm eukaryotes, in particular protozoa, were negatively influenced by micropollutant exposure, in contrast to prokaryotes that increased in relative abundance. Further, we found several functional genes that were differentially expressed between the MBBR with added micropollutants and the control. These include genes involved in aromatic and xenobiotic compound degradation. Moreover, the biofilm carrier batch experiment showed vastly different alterations in benzotriazole and diclofenac degradation following the increased micropollutant concentrations in the MBBR. Ultimately, this study provides essential insights into the microbial community and functional dynamics of MBBRs and how an increased load of micropollutants influences these dynamics.

The 'Erlenmeter': a low-cost, open-source turbidimeter for no-sampling phenotyping of microorganism growth.

This work presents a low-cost, open-source turbidimeter, the 'Erlenmeter', designed to monitor the growth of microorganisms in batch cultures. It is easy to build, based exclusively on inexpensive off-the-shelf electronic components and 3D-printed parts. The Erlenmeter allows measuring the optical density of cultures on standard Erlenmeyer flasks without the need to open the flasks to collect aliquots, ensuring speed, minimal use of consumables, and elimination of the risk of contamination. These features make it particularly well-suited not just for routine research assays but also for experimental teaching. Here we illustrate the use of the Erlenmeter turbidimeter to record the growth of the microalga Phaeodactylum tricornutum, of the bacterium Escherichia coli, and of the yeast Saccharomyces cerevisiae, model organisms that are widely used in research and teaching. The Erlenmeter allows a detailed characterization of the growth curves of all organisms, confirming its usefulness for studying microbial populations dynamics both for research purposes and in classroom settings.

Competitive advantages of T-even phage lysis inhibition in response to secondary infection.

T-even bacteriophages are known to employ lysis inhibition (LIN), where the lysis of an infected host is delayed in response to secondary adsorptions. Upon the eventual burst of the host, significantly more phage progenies are released. Here, we analysed the competitive advantage of LIN using a mathematical model. In batch culture, LIN provides a bigger phage yield at the end of the growth where all the hosts are infected due to an exceeding number of phage particles and, in addition, gives a competitive advantage against LIN mutants with rapid lysis by letting them adsorb to already infected hosts in the LIN state. By simulating plaque formation in a spatially structured environment, we show that, while LIN phages will produce a smaller zone of clearance, the area over which the phages spread is actually comparable to those without LIN. The analysis suggests that LIN induced by secondary adsorption is favourable in terms of competition, both in spatially homogeneous and inhomogeneous environments.

Rapid Start-up of Continuous Autotrophic Nitrogen Removal Reactor by Hybrid-inoculating PN and PN/A Granular Sludges

The rapid cultivation of partial nitritation/ANAMMOX （PN/A） granular sludge in a continuous-flow mode is one of the key technologies for efficient biological nitrogen removal in domestic wastewater treatment. Compared with that in PN/A granular sludge, PN granular sludge demonstrates a shorter incubation period and suitability for batch culture. It is also a good carrier for enriching ANAMMOX （AMX） bacteria. In this study, we established a continuous-flow autotrophic nitrogen removal process in three continuously stirred tank reactors （CSTR） （R1-R3） by hybrid-inoculating PN/A and PN granular sludge at the mass ratios of 3∶1, 1∶1, and 1∶3, respectively. By implementing high ammonium nitrogen loading and short hydraulic retention time, continuous autotrophic nitrogen removal processes were successfully started up in the three CSTRs. The results showed that compared with that of R1 and R2, R3 had a longer start-up time but a similar steady-state nitrogen removal performance. The total nitrogen removal load of R3 could be more than 2.6 kg·（m3·d）-1. Intriguingly, the inoculated PN granular sludge served as a precursor for PN/A granular sludge cultivation. This approach facilitated the enrichment of anaerobic ammonia-oxidizing bacteria （AMX） by introducing abundant ammonium-oxidizing bacteria （AOB） and nitrite nitrogen substrates into the CSTR. According to the results of high-throughput sequencing, the microbial abundance and diversity of the mature granules in R1-R3 were significantly higher than those of the inoculation sludge. AOB （genus Nitrosomonas）, AMX （genera Candidatus Kuenenia and Candidatus Brocadia）, and symbiotic heterotrophs, such as Chloroflexi, Bacteroidetes, and Chlorobi, drove the autotrophic nitrogen removal process and maintained the stability of the granular structure. In summary, a novel start-up strategy of hybrid-inoculating granular sludge was provided for a continuous-flow autotrophic nitrogen removal in engineering application.

Dynamic Energy Budget model for E. coli growth in carbon and nitrogen limitation conditions

The simulations and predictions obtained from mathematical models of bioprocesses conducted by microorganisms are not overvalued. Mechanistic models are bringing a better process understanding and the possibility of simulating unmeasurable variables. The Dynamic Energy Budget (DEB) model is an energy balance that can be formulated for any living organism and can be classified as a structured model. In this study, the DEB model was used to describe E. coli growth in a batch reactor in carbon and nitrogen substrate limitation conditions. The DEB model provides a possibility to follow the changes in the microbes’ cells including their elemental composition and content of some important cell ingredients in different growth phases in substrate limitation conditions which makes it more informative compared to Monod’s model. The model can be used as an optimal choice between Monod-like models and flux-based approaches.Key points• The DEB model can be used to catch changes in elemental composition of E. coli• Bacteria batch culture growth phases can be explained by the DEB model• The DEB model is more informative compared to Monod’s based models

Production and molecular weight variation of poly-γ-glutamic acid using a recombinant Bacillus subtilis with various pgs-component ratios.

Poly-γ-glutamic acid (PGA) has been of interest as a sustainable biopolymer in industrial applications. PGA biosynthesis in Bacillus subtilis is catalyzed by a transmembrane protein complex comprising PgsB, PgsC, and PgsA. To determine the Pgs component responsible for PGA overproduction, we constructed recombinants in which the promoter of the host-derived pgs gene was replaced with another host-derived gene promoter. These recombinants were then transformed using high-copy-number plasmids with various pgs-gene combinations to enhance Pgs component in different ratios. Subsequently, PGA production was investigated in batch cultures with l-glutamate supplemented medium. The recombinant strain enhanced with pgsB alone significantly overproduced PGA (maximum production 35.8 gL-1) than either the pgsC- or pgsA-enhanced strain. The molecular weight of the PGA produced with pgsB-enhanced strain was also greater than the pgsC- or pgsA-enhanced strain (approximately 10-fold). Hence, PgsB enhancement alone contributes to PGA overproduction with increased molecular weight.

Effects of microplastics on reproductive characteristics and mechanisms of the marine rotifer Brachionus plicatilis

Microplastic pollution, especially secondary microplastics (MPs), poses a significant threat to marine ecosystems. Despite its prevalence, the impact of natural-aged MPs on marine organisms, hindered by collection challenges, remains poorly understood. This study focused on 1–3 μm natural-aged MPs collected from Japan's coastal sea, investigating their effects on the rotifer Brachionus plicatilis sensu stricto and its reproductive mechanisms. Rotifers exposed to varying MP concentrations (0, 20, and 200 particles/mL) over 14-day batch cultures exhibited reduced population growth and fertilization rates. Down-regulation of reproductive genes and up-regulation of oxidative stress-related genes were observed, indicating MP-induced disruptions. Enhanced activities of superoxide dismutase and acetylcholinesterase and elevated malondialdehyde levels further emphasized oxidative stress. These findings underscore the detrimental impact of MPs on rotifer reproductivity, shedding light on the underlying mechanisms.