Batch Bioreactor Research Articles

Simultaneous nitrate and sulfate biotransformation driven by different substrates: comparison of carbon sources and metabolic pathways at different C/N ratios.

Nitrate (NO3-) and sulfate (SO42-) often coexist in organic wastewater. The effects of different substrates on NO3- and SO42- biotransformation pathways at various C/N ratios were investigated in this study. This study used an activated sludge process for simultaneous desulfurization and denitrification in an integrated sequencing batch bioreactor. The results revealed that the most complete removals of NO3- and SO42- were achieved at a C/N ratio of 5 in integrated simultaneous desulfurization and denitrification (ISDD). Reactor Rb (sodium succinate) displayed a higher SO42- removal efficiency (93.79%) with lower chemical oxygen demand (COD) consumption (85.72%) than reactor Ra (sodium acetate) on account of almost 100% removal of NO3- in both Ra and Rb. Ra produced more S2- (5.96 mg L-1) and H2S (25 mg L-1) than Rb, which regulated the biotransformation of NO3- from denitrification to dissimilatory nitrate reduction to ammonium (DNRA), whereas almost no H2S accumulated in Rb which can avoid secondary pollution. Sodium acetate-supported systems were found to favor the growth of DNRA bacteria (Desulfovibrio); although denitrifying bacteria (DNB) and sulfate-reducing bacteria (SRB) were found to co-exist in both systems, Rb has a greater keystone taxa diversity. Furthermore, the potential carbon metabolic pathways of the two carbon sources have been predicted. Both succinate and acetate could be generated in reactor Rb through the citrate cycle and the acetyl-CoA pathway. The high prevalence of four-carbon metabolism in Ra suggests that the carbon metabolism of sodium acetate is significantly improved at a C/N ratio of 5. This study has clarified the biotransformation mechanisms of NO3- and SO42- in the presence of different substrates and the potential carbon metabolism pathway, which is expected to provide new ideas for the simultaneous removal of NO3- and SO42- from different media.

Biotechnological Production of Optically Pure 2,3-Butanediol by Bacillus subtilis Based on Dissolved Oxygen Control Strategy

2,3-Butanediol (2,3-BD) is a promising platform chemical, produced from microbial cells. Oxygen availability is a crucial factor driving the formation and proportion of 2,3-BD and acetoin in 2,3-BD producing bacterial strains. In this study, the ability of B. subtills GD5 to produce 2,3-BD in optimized sucrose-based media was evaluated, by investigating the impact of carbon to nitrogen (C/N) ratio and the effectiveness of alternative low-cost nitrogen sources (corn steep liquor, soybean meal, and ammonium sulphate). Subsequently, different dissolved oxygen (DO) controlling regimes were assessed in batch bioreactor fermentations. The best fermentation outcomes were obtained with uncontrolled DO, achieving 5.88 g/L of optically pure (R,R)-2,3-BD (~100% purity), accompanied by a production yield of 0.43 g/g, and a productivity of 0.2 g/L/h. Additionally, the influence of the DO controlling regime on B. subtills key enzymes involved in the reverse activity of acetoin reductase was also monitored. A fed-batch process under the most suitable DO conditions was carried out to improve 2,3-BD production, achieving 42.31 g/L 2,3-BD with a production yield of 0.52 g/g. Thus, B. subtilis GD5 is a promising strain for the efficient production of pure chiral (R,R)-2,3-BD under uncontrolled DO conditions, using alternative low-cost nitrogen sources.

Apoptosis induction in leukemic cells by L-asparaginase preparation from Bacillus indicus: bench-scale production, purification and therapeutic application.

With the emergence of multiple side effects on the usage of commercial L-asparaginase formulations, keen interest is provoked to investigate new sources of L-asparaginases that possess antileukemic properties with minimal side effects. The present study reports the cost-effective bench-scale production, homogeneity purification and apoptosis induction potential of a new L-asparaginase preparation from Bacillus indicus against human leukemia cells. The enzyme is highly specific toward the natural substrate L-asparagine. The study initiated with the enzyme production using cost-effective substrates in which a 3.28-fold enhancement of enzyme activity was achieved in comparison with an unoptimized medium using the central composite experimental design approach. The scale-up of the process in a 3.7-L batch bioreactor resulted in 16.42 ± 0.17IU/mL of L-asparaginase activity in 24h. The crude extracellular enzyme was purified to homogeneity using anion exchange chromatography followed by gel filtration chromatography. A single band of approximately 35kDa molecular weight was obtained on SDS-PAGE, while native PAGE analysis confirmed it to be a tetramer of four identical subunits. The circular dichroism spectroscopic study revealed the α + β mixed type of secondary structure with 38.7% α-helices and 27.4% β pleated sheets. The antitumor toxicity exhibited on the MOLT-4 leukemia cells by the new L-asparaginase was revealed using the MTT assay and acridine orange/propidium iodide dual staining for live/dead cells. The flow cytometry analysis established the potential of the purified L-asparaginase to induce the apoptotic cell death mechanism in MOLT-4 leukemia cells. Conclusively, the L-asparaginase of Bacillus indicus is a highly promising candidate that can be introduced as a new enzyme therapeutic against various leukemia disorders.

Enhanced production of biosurfactant by Bacillus subtilis RSL2 in semicontinuous bioreactor utilizing molasses as a sole substrate

The growth-associated metabolites are produced during the exponential phase; however, this phase terminates due to substrate depletion or product inhibition. In the present study, a semicontinuous mode with a fill-and-draw strategy was applied to extend the exponential phase of the biosurfactant production to overcome the product inhibition and in turn, enhance the yield. Bioreactor studies were performed in batch mode, followed by the semicontinuous operation. A potential biosurfactant producer Bacillus subtilis RSL2 was used in this study at the previously optimized conditions of pH 6.6, temperature 41 °C and 5% (w/v) of molasses. A better mass transfer was achieved in the bioreactor as compared to the shake flask study. In the batch bioreactor study, 90% of sugar was utilized with simultaneous 13.7 g L−1 of biosurfactant production. The sugar utilization was further improved to > 98% in the case of semicontinuous operation employing a fill-and-draw strategy. The exponential phase got extended up to 18 days and a total of 13 L of media was fed in the semicontinuous operation of 21 days as compared to 1.5 L of working volume in the batch reactor. The biosurfactant yield was enhanced by 1.5 folds and was found to be 0.97 g g−1. The produced biosurfactant was identified as a lipopeptide. The interfacial properties of the biosurfactant along with colloidal and thermal stability have been investigated. The critical micelle concentration of the produced biosurfactant was 70 mg L−1. The present study highlighted the efficient utilization of molasses for the production of biosurfactant, an alternative metabolite, in a semicontinuous mode of bioreactor.

Concept of Batch and Fed-Batch Cultures of Yarrowia lipolytica as a Valuable Source of Sterols with Simultaneous Valorization of Molasses and Post-Frying Rapeseed Oil

Food byproduct streams can potentially be transformed into value-added products such as microbial lipids in bioprocesses based on the non-conventional Yarrowia yeast. The effect of culture conditions of Y. lipolytica KKP 379 wild strain in waste media on the efficiency of lipid accumulation, fatty acid composition, presence of selected sterols, yield and elemental composition of biomass has been studied. Batch and fed-batch bioreactor cultures were carried out in media with molasses hydrolysate (MH) and post-frying rapeseed oil. It was determined that biomass grown in MH contained more minerals than in medium with rapeseed post-frying oil. Considering the PDSC study, the Tmax of oxidation induction ranged from 10.04–26.36 min for the analyzed samples. The biomass from fed-batch cultures with MH had the highest total sterol content (68.40 mg/goil), dominated by ergosterol at 60.16 mg/g. Feeding with post-frying rapeseed oil with new doses of mineral medium promoted maintaining the cellular lipid content at a high level (30.75–31.73%) for 50 h, with maximum yield at 37.50%. The results of the experiment showed that the cellular lipid accumulation efficiency of Y. lipolytica yeast and the content of sterols in the cell membrane can be manipulated by selecting waste substrates and culture mode.

Evaluation of the efficiency of the mixing system of the laboratory SBR-type reactor

Requirements for the continuous improvement of the quality of wastewater discharged to a receiving water body have led to the development of many devices used in biological treatment. Current research in water and wastewater solutions often focuses on finding energy-efficient solutions that have the least negative impact on the environment while reducing operating costs. Treatment systems commonly use the properties of microorganism structures in the form of activated sludge flocs to remove organic and nutrient compounds contained in wastewater. An important part of this solution is the use of a mixing and aeration system in the appropriate configuration. In a wastewater treatment plant with a biological part operating with SBR technology, many types of mixing equipment are used. This article presents the use of an innovative mixing system in sequential batch bioreactors in which activated sludge is the process medium.

High yield production of lipid and carotenoids in a newly isolated Rhodotorula mucilaginosa by adapting process optimization approach

Rhodotorula sp. is a robust microbe, which can synthesize a wide range of products such as lipids and carotenoids. In the present study, we have attempted to enhance the production of lipids and carotenoids from a newly isolated Rhodotorula mucilaginosa using a process optimization approach. Among the carbon source screened, glucose showed enhanced lipid and carotenoid production of 46.19% (w/w) and 36.2 mg/g, respectively. The low C/N has influenced lipid production by 43.5% and the high C/N ratio favored carotenoid production (41.06 µg/g). The sodium acetate with 1.5% (w/v) concentration showed a lipid content of 48.28% and carotenoid of 41.16 mg/g. Further with the optimum levels of RPM (180), pH (6), and temperature (30 °C), the maximum lipid and carotenoid were found to be 51.89%, and 43.34 µg/g, respectively. In a batch bioreactor, MSM medium with sodium acetate showed a maximum lipid content of 54%. The lipid and carotenoid were successfully characterized by laser Raman spectroscopy, a label-free method and the FAME analysis clearly showed the high content of palmitic acid (C16:0) 21.05 (% w/w), oleic acid (C18:1) 56.18 (%w/w) and linoleic acid (C18:2) 12.25 (% w/w), which is suitable for biodiesel preparation.

Polylactide (PLA) as a Cell Carrier in Mesophilic Anaerobic Digestion-A New Strategy in the Management of PLA.

The management of waste polylactide (PLA) in various solutions of thermophilic anaerobic digestion (AD) is problematic and often uneconomical. This paper proposes a different approach to the use of PLA in mesophilic AD, used more commonly on the industrial scale, which consists of assigning the function of a microbial carrier to the biopolymer. The study involved the testing of waste wafers and waste wafers and cheese in a co-substrate system, combined with digested sewage sludge. The experiment was conducted on a laboratory scale, in a batch bioreactor mode. They were used as test samples and as samples with the addition of a carrier: WF-control and WFC-control; WF + PLA and WFC + PLA. The main objective of the study was to verify the impact of PLA in the granular (PLAG) and powder (PLAP) forms on the stability and efficiency of the process. The results of the analysis of physicochemical properties of the carriers, including the critical thermal analysis by differential scanning calorimetry (DSC), as well as the amount of cellular biomass of Bacillus amyloliquefaciens obtained in a culture with the addition of the tested PLAG and PLAP, confirmed that PLA can be an effective cell carrier in mesophilic AD. The addition of PLAG produced better results for bacterial proliferation than the addition of powdered PLA. The highest level of dehydrogenase activity was maintained in the WFC + PLAG system. An increase in the volume of the methane produced for the samples digested with the PLA granules carrier was registered in the study. It went up by c.a. 26% for WF, from 356.11 m3 Mg-1 VS (WF-control) to 448.84 m3 Mg-1 VS (WF + PLAG), and for WFC, from 413.46 m3 Mg-1 VS, (WFC-control) to 519.98 m3 Mg-1 VS (WFC + PLAG).

Heterotrophic denitrification: An overlooked factor that contributes to nitrogen removal in n-DAMO mixed culture

Nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) has been recognized as a sustainable process for simultaneous removal of nitrogen and methane. The metabolisms of denitrifying anaerobic methanotrophs, including Candidatus Methanoperedens and Candidatus Methylomirabilis, have been well studied. However, potential roles of heterotrophs co-existing with these anaerobic methanotrophs are generally overlooked. In this study, we pulse-fed methane and nitrate into an anaerobic laboratory sequencing batch bioreactor and enriched a mixed culture with stable nitrate removal rate (NRR) of ∼28 mg NO3−-N L−1 d−1. Microbial community analysis indicates abundant heterotrophs, e.g., Arenimonas (5.3%–18.9%) and Fimbriimonadales ATM1 (6.4%), were enriched together with denitrifying anaerobic methanotrophs Ca. Methanoperedens (10.8%–13.2%) and Ca. Methylomirabilis (27.4%–34.3%). The results of metagenomics and batch tests suggested that the denitrifying anaerobic methanotrophs were capable of generating methane-derived intermediates (i.e., formate and acetate), which were employed by non-methanotrophic heterotrophs for denitrification and biomass growth. These findings offer new insights into the roles of heterotrophs in n-DAMO mixed culture, which may help to optimize n-DAMO process for nitrogen removal from wastewater.

Metabolic Engineering and Adaptive Evolution for Efficient Production of l-Lactic Acid in Saccharomyces cerevisiae.

l-Lactic acid (LA) is a three-carbon hydroxycarboxylic acid with extensive applications in food, cosmetic, agricultural, pharmaceutical, and bioplastic industries. However, microbial LA production is limited by its intrinsic inefficiency of cellular metabolism. Here, pathway engineering was used to rewire the biosynthetic pathway for LA production in Saccharomyces cerevisiae by screening heterologous l-lactate dehydrogenase, reducing ethanol accumulation, and introducing a bacterial acetyl coenzyme A (acetyl-CoA) synthesis pathway. To improve its intrinsic efficiency of LA export, transporter engineering was conducted by screening the monocarboxylate transporters and then strengthening the capacity of LA export, leading to LA production up to 51.4 g/L. To further enhance its intrinsic efficiency of acid tolerance, adaptive evolution was adopted by cultivating yeast cells with a gradual increase in LA levels during 12 serial subcultures, resulting in a 17.5% increase in LA production to 60.4 g/L. Finally, the engineered strain S.c-NO.2-100 was able to produce 121.5 g/L LA, with a yield of up to 0.81 g/g in a 5-L batch bioreactor. The strategy described here provides a guide for developing efficient cell factories for the production of the other industrially useful organic acids. IMPORTANCE Saccharomyces cerevisiae is one of the most widely engineered cell factories for the production of organic acids. However, microbial production of l-lactic acid is limited by its intrinsic inefficiency of cellular metabolism in S. cerevisiae. Here, the transmission efficiency of the biosynthetic pathway was improved by pathway optimization to increase l-lactic acid production. Then, the synthetic ability for l-lactic acid was further enhanced by adaptive evolution to improve acid tolerance of S. cerevisiae. Based on these strategies, the final engineered S. cerevisiae strain achieved high efficiency of l-lactic acid production. These findings provide new insight into improving the intrinsic efficiency of cellular metabolism and will help to construct superior industrial yeast strains for high-level production of other organic acids.

Optical Evaluation of Effects of Energy Substrates on PHB Accumulation for Bioplastic Production

To date, hundreds of millions tons of plastics has been produced worldwide. Their production and disposal are associated with high pollution and carbon release into the atmosphere. A more environmentally friendly alternative is bioplastics, and the most popular is polyhydroxybutyrate (PHB) polymer. Large amounts of PHB can be obtained from activated sludge where used cooking oil or other industrial waste can be used as potential substrates. In this work, efficient bioplastic production strategies are studied, and the considered substrate is a mixture of oil and peptone. Pseudomonas fluorescens bacteria are used to accumulate PHB, and the cultivation of microorganisms is carried out in batch and continuous-flow bioreactors. Microscopic observations and laboratory essays are performed to confirm presence of PHB and other key parameters. The obtained results allow us to determine the optimal feeding strategy.

Revealing the Phenotypic and Genomic Background for PHA Production from Rapeseed-Biodiesel Crude Glycerol Using Photobacterium ganghwense C2.2.

Polyhydroxyalkanoates (PHA) are promising biodegradable and biocompatible bioplastics, and extensive knowledge of the employed bacterial strain's metabolic capabilities is necessary in choosing economically feasible production conditions. This study aimed to create an in-depth view of the utilization of Photobacterium ganghwense C2.2 for PHA production by linking a wide array of characterization methods: metabolic pathway annotation from the strain's complete genome, high-throughput phenotypic tests, and biomass analyses through plate-based assays and flask and bioreactor cultivations. We confirmed, in PHA production conditions, urea catabolization, fatty acid degradation and synthesis, and high pH variation and osmotic stress tolerance. With urea as a nitrogen source, pure and rapeseed-biodiesel crude glycerol were analyzed comparatively as carbon sources for fermentation at 20 °C. Flask cultivations yielded 2.2 g/L and 2 g/L PHA at 120 h, respectively, with molecular weights of 428,629 g/mol and 81,515 g/mol. Bioreactor batch cultivation doubled biomass accumulation (10 g/L and 13.2 g/L) in 48 h, with a PHA productivity of 0.133 g/(L·h) and 0.05 g/(L·h). Thus, phenotypic and genomic analyses determined the successful use of Photobacterium ganghwense C2.2 for PHA production using urea and crude glycerol and 20 g/L NaCl, without pH adjustment, providing the basis for a viable fermentation process.

Insights into the effects of operating parameters on sulfate reduction performance and microbial pathways in the anaerobic sequencing batch reactor

Sulfate-reducing bacteria (SRB)-based anaerobic process has aroused wide concern in the treatment of sulfate-containing wastewater. Chemical oxygen demand-to-sulfate ratio (COD/SO42−) and HRT are two key factors that affect not only the anaerobic treatment performance but also the activity of SRB. In this study, an anaerobic sequencing batch reactor was constructed, and the effects of different operating parameters (COD/SO42−, HRT) on the relationship of sulfate (SO42−) reduction performance, microbial communities, and metabolic pathways were comprehensively investigated. The results indicated that the SO42− removal rates could achieve above 95% under different operating parameters. Bioinformatics analysis revealed that microbial community changed with reactor operation. At the genus level, the enrichment of Propionicclava and Peptoclostridium contributed to the establishment of a homotrophic relationship with Desulfobulbus, the dominant SRB in the reactor, which indicated that they took vital part in maintaining the structural and functional stability of the bacterial community under different operating parameters. In particular, an increasing trend of the relative abundance of functional genes encoding dissimilatory sulfate reduction was detected with the increase of COD/SO42−, which indicated high SO42− reduction potentials. This knowledge will help to reveal the mechanism of the effect of operating parameters on the anaerobic sulfate removal process, thus providing effective guidance for the targeted regulation of anaerobic sequencing batch bioreactors treating SO42--containing wastewater.

Process Development of Polyhydroxyalkanoates Production by Halophiles Valorising Food Waste.

Polyhydroxyalkanoates (PHA) is an emerging biodegradable plastic family that can replace a broad spectrum of conventional thermoplastics and is promising in the sustainable process development and valorization of organic waste. This study established a novel production system of PHA from food waste through halophilic microbial fermentation with spent medium recycling. The essential processing parameters for batch cultivation of Haloferax mediterranei were optimized for food waste substrate (a 40 g/L loading and 2.5 vvm of aeration), which achieved a yield of 0.3 g PHA/g COD consumed. A batch bioreactor system was developed, which produced 7.0 ± 0.7 g/L cell dry mass and 4.5 ± 0.2 g/L PHA with a 20% dissolved oxygen (DO) level. A DO above 50% saturation resulted in faster cell growth and similar cell mass production but 25% less PHA production. The spent saline medium, treated with H2O2 and rotary evaporation, was successfully reused for four consecutive batches and provided consistent PHA concentrations and product qualities.

Wastewater treatment potential, light penetration profile and biomass settling performance of a photo‐sequencing batch reactor

AbstractBACKGROUNDThe performance of microalgal and bacterial systems devoted to wastewater treatment might be limited by the poor settling of microalgal biomass and the inhibition of nitrifying bacteria under high irradiance conditions. This work implemented a photo‐sequencing batch bioreactor (PSBR) treating medium‐ and low‐strength wastewaters. The impact of a short settling time on the formation of microalgal–bacterial aggregates (MABAs) was evaluated. Moreover, online photosynthetically active radiation (PAR) measurements at the bottom of the PSBR were used to identify potential nitrifying activity inhibition.RESULTSA chemical oxygen demand removal efficiency of up to 88% was obtained for the medium‐ and low‐strength wastewaters. Photoinhibition of the nitrifying activity was observed at PAR values from 73 to 480 μmol m−2 s−1, and it was confirmed under non‐limiting dissolved oxygen conditions. The PSBR operating conditions, including a short settling time (30 min) and treatment of medium‐strength wastewater, favored the rapid formation of MABAs in only 19 days, with a biomass settling velocity (vs) of 0.74 m h−1. The microalgal genera Acutudesmus, Desmodesmus, and Chlorella were predominant in the MABAs. Experimental estimation of the light extinction coefficient (λ = 0.056 m2 g−1 total suspended solids) allowed the simulation of light penetration profiles at different reactor liquid depths and biomass concentrations.CONCLUSIONA short settling time favors the rapid formation of MABAs in PSBR‐type reactors treating medium‐strength wastewater. Online monitoring and simulation of light penetration is a valuable tool to determine photoinhibition of the nitrifying activity in microalgal and bacterial systems. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Lipid production by Rhodosporidium toruloides from biodiesel-derived glycerol in shake flasks and bioreactor: Impact of initial C/N molar ratio and added onion-peel extract

Rhodosporidium toruloides NRRL Y-27012 grown in glycerol, produced yeast biomass and secondary metabolites as lipids (L) and intra-cellular polysaccharides (IPS) in trials under nitrogen limitation (initial C/N molar ratios ≈ 50, ≈ 100, ≈ 160 and ≈ 240). The initial glycerol (Glol0) concentration for all experiments was ≈ 90 g/L. The yeast demonstrated significant dry cell weight (DCW) production irrespective of the C/N ratio employed, ranging between 19 and 29 g/L. The optimum C/N ratio medium was that of 100 moles/moles, where a DCWmax = 24.5 g/L containing c. 50% w/w of lipids was recorded. Endopolysaccharides presented indeed impressive values (≥ 50% w/w) even at the very early growth steps, while IPS/DCW values decreased as the fermentation proceeded, with simultaneous increase of L/DCW values. Experiments were also conducted in media with a hydroglycerolic extract of onion peels (at 40 mL/L), while a blank experiment (no extract) was also realized. The trial with the added extract was performed under both aseptic (previous sterilization) and non-aseptic (previous thermal treatment at T = 100 °C; 5 min) conditions. The aseptic experiment with the added extract resulted in a relative decrease in lipid production in relation to the control (L=8.6 g/L in the control vs L = 6.2 g/L with the extract). Under non-aseptic conditions, lipid production was slightly lower (L = 5.2 g/L). Finally, the optimum trial in shake flasks was scaled-up in a batch bioreactor, and higher glycerol assimilation rate, and biomass and lipid production occurred compared to the flask trial (final DCW and lipid values ≈27 g/L and 13.5 g/L respectively). Cellular lipids, rich in palmitic and oleic acid, were mainly composed of neutral fractions. Phospholipids were more saturated than the neutral lipids.

Improved Gamma-Decalactone Synthesis by Yarrowia lipolytica Yeast Using Taguchi Robust Design Method

The production of gamma-decalactone (GDL) by Yarrowia lipolytica is mainly based on the biotransformation of ricinoleic acid, derived from castor oil triglycerides. The main difficulty in this process is the multitude of factors that determine the growth rate of microorganisms, and thus affect the efficiency of lactone synthesis. In order to improve the technological aspects of GDL biosynthesis in batch culture, the influence of three factors was determined: substrate concentration, medium mixing intensity, and its pH, using the Taguchi solid design method (based on orthogonal array design). On the basis of four bioreactor batch cultures, the most favorable culture conditions in terms of GDL synthesis were selected using the statistical Taguchi method. The statistical method of experimental planning has shown that the optimal parameters of lactone biosynthesis are a constant pH at the level of 7, a variable mixing speed in the range of 200–500 rpm, and a substrate concentration at the level of 75 g/L. Using these parameters, about 2.93 ± 0.33 g/L of aroma was obtained. The intensity of mixing turned out to be the most important factor influencing the increase in GDL concentration in the medium.

Extractive Fermentation as A Novel Strategy for High Cell Mass Production of Hetero-Fermentative Probiotic Strain Limosilactobacillus reuteri

This study reports on a novel technique to enhance the high cell mass and viable cell counts of the heterofermentative probiotic strain, Limosilactobacillus reuteri. This is the first report on the cultivation of L. reuteri, which was incorporated with weak base anion-exchange resins to remove the accumulating lactic acid in the fermentation broth. Two anion-exchange resins—Amberlite IRA 67 and IRA 96—were found to have a high adsorption capacity with lactic acid. Batch fermentation and fed-batch cultivation were further analyzed using IRA 67 resins, as this application resulted in a higher maximum number of viable cells. The in situ application of anion-exchange resins was found to create shear stress, and thus, it does not promote growth of L. reuteri; therefore, an external and integrated resin column system was proposed. The viable cell count from batch fermentation, when incorporated with the integrated resin column, was improved by 71 times (3.89 × 1011 ± 0.07 CFU mL−1) compared with control batch fermentation (5.35 × 109 ± 0.32 CFU mL−1), without the addition of resins. The growth improvement was achieved due to the high adsorption rate of lactic acid, which was recorded by the integrated IRA 67 resin system, and coupled with the stirred tank bioreactor batch fermentation process.

Formation mechanism and metabolic pathways of photogranule in batch and continuous-flow mode under high salinity stress

Photogranules have considerable advantages for treating high saline wastewater economically and efficiently. However, photogranulation mechanism needs to be further investigated. In this study, a continuous-flow photogranular bioreactor (R1) and sequencing batch photogranular bioreactor (R2) were setup for exploring the formation mechanism of photogranules. Results showed that R1 achieved satisfying pollutant removal efficiencies with TOC of 85.36 ± 2.84 %, NH4+ of 93.30 ± 2.07 % and PO43- of 77.68 ± 5.81 %. Microscopy and 16S/18S rRNA sequencing indicated that the dominate bacteria showed discrepancy with Phormidium sp. in R1 and Leptolyngbya sp. in R2, and a specific network structure was formed on photogranules in R1. Additionally, more abundant polysaccharides in soluble microbial products (SMP) were observed in R1, which facilitated cyanobacteria gliding motility, thereby accelerating the photogranulation. Meanwhile, three dimensional excitation-emission matrix (3D-EEM) analysis implied that the pivotal SMP components driving photogranulation were different, with aromatic protein-like substances accumulating in R1 and humic acid-like substances and soluble microbial by-product-like substances in R2. In contrast, R1 exhibited lower abundance of extracellular polymeric substances (EPS) but higher proteins to polysaccharides ratio (PN/PS) than those in R2. High PN/PS showed advantages for accelerating photogranule formation with a compact structure. Furthermore, more enriched aromatic protein-like and humic acid-like EPS were identified in R2 compared with R1, suggesting they were more significant contributors to granulation in R2. Finally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) results revealed that metabolism and genetic information processing were the most abundant metabolic pathways, which were responsible for excellent pollutants removal and stable granulation. Therefore, this study demonstrates the impacts of SMP and EPS on photogranulation and reveals the biological mechanism that drives granular structure and function.

Cultivation of previously uncultured microorganisms with a continuous-flow down-flow hanging sponge (DHS) bioreactor, using a syntrophic archaeon culture obtained from deep marine sediment as a case study.

In microbiology, cultivation is a central approach for uncovering novel physiology, ecology, and evolution of microorganisms, but conventional methods have left many microorganisms found in nature uncultured. To overcome the limitations of traditional methods and culture indigenous microorganisms, we applied a two-stage approach: enrichment/activation of indigenous organisms by using a continuous-flow down-flow hanging sponge bioreactor and subsequent selective batch cultivation. Here, we provide a protocol for this bioreactor-mediated technique using activation of deep marine sediment microorganisms and downstream isolation of a syntrophic co-culture containing an archaeon closely related to the eukaryote ancestor (Candidatus Promethearchaeum syntrophicum strain MK-D1) as an example. Both stages can easily be tailored to target other environments and organisms by modifying the inoculum, feed solution/gases, attachment material and/or cultivation media. We anaerobically incubate polyurethane sponges inoculated with deep-sea methane seep sediment in a reactor at 10 °C and feed anaerobic artificial seawater medium and methane. Once phylogenetically diverse and metabolically active microorganisms are adapted to synthetic conditions in the reactor, we transition to growing community samples in glass tubes with the above medium, simple substrates and selective compounds (e.g., antibiotics). To accommodate for the slow growth anticipated for target organisms, primary cultures can be incubated for ≥6-12 months and analyzed for community composition even when no cell turbidity is observed. One casamino acid- and antibiotic-amended culture prepared in this way led to the enrichment of uncultured archaea. Through successive transfer in vitro combined with molecular growth monitoring, we successfully obtained the target archaeon with its partner methanogen as a pure syntrophic co-culture.