Batch Culture Conditions Research Articles

Unveiling the Cultivation of Nostoc sp. under Controlled Laboratory Conditions.

Cyanobacteria, photoautotrophic Gram-negative bacteria, play a crucial role in aquatic and terrestrial environments, contributing significantly to fundamental ecological processes and displaying potential for various biotechnological applications. It is, therefore, critical to identify viable strains for aquaculture and establish accurate culture parameters to ensure an extensive biomass supply for biotechnology purposes. This study aims to establish optimal laboratory batch culture conditions for Nostoc 136, sourced from Alga2O, Coimbra, Portugal. Preliminary investigations were conducted to identify the optimal culture parameters and to perform biomass analysis, including protein and pigment content. The highest growth was achieved with an initial inoculum concentration of 1 g.L-1, using modified BG11 supplemented with nitrogen, resulting in a Specific Growth Rate (SGR) of 0.232 ± 0.017 μ.day-1. When exposed to white, red, and blue LED light, the most favourable growth occurred under a combination of white and red LED light exhibiting an SGR of 0.142 ± 0.020 μ.day-1. The protein content was determined to be 10.80 ± 2.09%. Regarding the pigments, phycocyanin reached a concentration of 200.29 ± 30.07 µg.mL-1, phycoerythrin 148.29 ± 26.74 µg.mL-1, and allophycocyanin 10.69 ± 6.07 µg.mL-1. This study underscores the influence of light and nutrient supplementation on the growth of the Nostoc biomass.

Hernandulcin Production in Elicited Hairy Roots of Phyla scaberrima: Toward Sustainable Production of a Non-Caloric Sweetener with Nutraceutical Properties.

Herein we report on the generation of hairy root lines of P. scaberrima able to produce hernandulcin (HE), a non-caloric sweetener with nutraceutical properties. From ten different lines analyzed, three synthesized up to 100 mg ⋅ L-1 HE under the batch culture conditions standardized in this investigation. Adding elicitors (salicylic acid, chitin, Glucanex, polyethylene glycol) and biosynthetic precursors (farnesol and (+)-epi-alpha-bisabolol) significantly altered HE accumulation. Chitin and Glucanex enhanced HE production from 130 to 160 mg ⋅ L-1 , whereas farnesol and (+)-epi-alpha-bisabolol from 165 to 200 mg ⋅ L-1 without dependence on biomass accumulation. Improved batch cultures containing liquid Murashige & Skoog medium (MS; pH 7), added with 4 % sucrose, 0.5 mg ⋅ L-1 naphthaleneacetic acid, 100 mg ⋅ L-1 Glucanex, 150 mg ⋅ L-1 chitin, 250 mg ⋅ L-1 farnesol, and 150 mg ⋅ L-1 (+)-epi-alpha-bisabolol at 25 °C (12 h light/12 h darkness), triggered HE accumulation to 250 mg ⋅ L-1 in 25 days. The efficiency of each recombinant line is discussed.

Disruption of trehalose periplasmic recycling dysregulates cAMP-CRP signaling in Escherichia coli during stationary phase.

Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such reserves. How Escherichia coli manage carbon storage systems under starvation stress, as well as transpose changes in intracellular metabolite levels into regulatory signals, is not well understood. Endogenous trehalose metabolism may be at the center of these processes, coupling carbon storage with carbon starvation responses. The coupled transport to the periplasm and subsequent hydrolysis of trehalose back to glucose for transport to the cytoplasm may function as a crucial metabolic signaling pathway. Although trehalose has been characterized as a stress protectant in E. coli, the disaccharide also functions as both an energy storage compound and a regulator of carbohydrate metabolism in fungi, plants, and other bacteria. Our research explores the metabolic regulatory properties of trehalose in E. coli and a potential mechanism by which the intracellular carbon pool is interconnected with regulatory circuits, enabling long-term survival.

Improvement of ethanol and 2,3-butanediol production in Saccharomyces cerevisiae by ATP wasting

Background“ATP wasting” has been observed in 13C metabolic flux analyses of Saccharomyces cerevisiae, a yeast strain commonly used to produce ethanol. Some strains of S. cerevisiae, such as the sake strain Kyokai 7, consume approximately two-fold as much ATP as laboratory strains. Increased ATP consumption may be linked to the production of ethanol, which helps regenerate ATP.ResultsThis study was conducted to enhance ethanol and 2,3-butanediol (2,3-BDO) production in the S. cerevisiae strains, ethanol-producing strain BY318 and 2,3-BDO-producing strain YHI030, by expressing the fructose-1,6-bisphosphatase (FBPase) and ATP synthase (ATPase) genes to induce ATP dissipation. The introduction of a futile cycle for ATP consumption in the pathway was achieved by expressing various FBPase and ATPase genes from Escherichia coli and S. cerevisiae in the yeast strains. The production of ethanol and 2,3-BDO was evaluated using high-performance liquid chromatography and gas chromatography, and fermentation tests were performed on synthetic media under aerobic conditions in batch culture. The results showed that in the BY318-opt_ecoFBPase (expressing opt_ecoFBPase) and BY318-ATPase (expressing ATPase) strains, specific glucose consumption was increased by 30% and 42%, respectively, and the ethanol production rate was increased by 24% and 45%, respectively. In contrast, the YHI030-opt_ecoFBPase (expressing opt_ecoFBPase) and YHI030-ATPase (expressing ATPase) strains showed increased 2,3-BDO yields of 26% and 18%, respectively, and the specific production rate of 2,3-BDO was increased by 36%. Metabolomic analysis confirmed the introduction of the futile cycle.ConclusionATP wasting may be an effective strategy for improving the fermentative biosynthetic capacity of S. cerevisiae, and increased ATP consumption may be a useful tool in some alcohol-producing strains.

Semi-continuous production of polyhydroxybutyrate (PHB) in the Chlorophyta Desmodesmus communis

Polyhydroxyalkanoates (PHAs) are promising alternatives that accumulate as energy and carbon storage material in various microorganisms, including bacteria and microalgae, being biodegradable and suitable for a wide variety of applications. Among these compounds, the most prevalent and well-characterized biopolymer is polyhydroxybutyrate (PHB), which belongs to the short-chain PHAs.The present study was designed to evaluate algae-based PHB production in two Chlorophyta (Desmodesmus communis and Chlorella vulgaris) under a two-phase nutritional mode of cultivation, namely a phototrophic growth phase (PGP) and a mixotrophic stress phase (MSP) with N,P-depleted media and organic carbon supply (i.e., glucose or sodium acetate, NaOAc). The highest PHB productivity (0.11 g PHB/g biomass/d; 0.015 g PHB/L/d), corresponding to 32.1 % w/w of intracellular PHB, was observed for D. communis after 3 days of cultivation under mixotrophic conditions in batch cultures (e.g., low light, phosphorus-free medium, 1 g/L of NaOAc). A scaled-up cultivation (10 L) was set up to evaluate for the first time PHB yields and biomass composition in a semi-continuous system. A PHB content of 34 % w/w was achieved on day 8, corresponding to a maximum PHB productivity of 0.10 g PHB/g biomass/d (or 0.011 g PHB/L/d), which increased up to 54 % w/w on day 15. The biomass was composed of about 30 % w/w proteins, 6 % w/w polysaccharides, and 11 % w/w lipids, which can be valorised from a biorefinery perspective. The scaled-up D. communis cultivation in 10 L PBRs confirmed the potential utilization of this algal species for PHB production with productivity up to 2-times higher than those reported for several cyanobacterial species and similar to the maximum value obtained with batch cultures in previous works performed with Scenedesmaceae.

Assessment of batch culture conditions for cyanobacterial propagation for a bioreactor in space

One important point in human space exploration is the reliable air, water and food production for the space crew, less dependent from cargo supply. Bioregenerative life support systems aim to overcome this challenge. The life support program MELiSSA of the European Space Agency uses the cyanobacterium Limnospira indica for air revitalization and food production. In the Space flight experiments ArtEMISS-B and -C, L. indica is tested on the International Space Station. In this study we elucidate which conditions are most favorable for cell propagation from inoculum to a full culture in space to enable a high final biomass concentration, with high pigment composition for an efficient bioprocess. We found that lower light intensities (36–75 µmol photons m-2 s-1) show higher maximum biomass densities and higher pigment contents than cultures grown above 100 µmol photons m-2 s-1. 36 μmol photons m-2 s-1 resulted in maximum biomass concentrations of 3.36 ± 0.15 g L-1 (23 °C), while cultures grown at 140 µmol photons m-2 s-1 only achieved concentrations of 0.82 ± 0.10 g L-1 (25°C) (−75.8%). Colder temperatures (21°C–25°C) showed a negative effect on the pigment content. At 36 µmol photons m-2 s-1, a temperature of 30°C gave a phycocyanin concentration of 0.122 ± 0.014 g g DW-1 and 23°C resulted in 0.030 ± 0.003 g g DW-1 (−75.4%). In conclusion, a low light intensity (36–80 µmol photons m-2 s-1) in combination with warm temperature (30°C–34°C) is optimal to obtain cultures with high pigment contents and high biomass concentrations in a batch culture.

Waste Polyurethane Foams as Biomass Carriers in the Treatment Process of Domestic Sewage with Increased Ammonium Nitrogen Content.

In order to understand the mechanisms of microbial growth on waste polyurethane sponge materials, their effectiveness as biomass carriers in domestic sewage with increased ammonium nitrogen content treatment was assessed. Comparative experiments were carried out in microreactors under steady conditions of batch culture, which allowed for an assessment of different carriers, in the form of flexible foams, rigid foams, and flexible foams placed in full casings. In the studies conducted in continuous cultures, biomass carriers selected in batch culture were used as fillings in the column model. The structure of the microbial community inhabiting the spongy material was determined and the pollutant-removing process from real domestic sewage was assessed. Analyzes using the Illumina sequencing technique allowed for demonstrating that Nitrosomonas and Nitrospira were the predominant nitrifiers in the biomass carrier in the form of waste polyurethane foams (PUF). It was found that anammox bacteria, the presence of which-as unidentified Planctomycetes-was confirmed in the polyurethane sponge material, were also responsible for the high removal of N-NH4+. Burkholderia and Sphingopyxis phyla were identified as the dominant denitrifying bacteria involved in the treatment of domestic sewage with increased content of ammonium nitrogen. The biomass carrier in the form of waste PUF placed additionally in full casings proved to be more beneficial for the proliferation of bacteria involved in nitrification and denitrification processes. On the other hand, waste foams without casings proved to be more suitable for the growth of microorganisms known to perform partial denitrification and may accumulate nitrites (Staphylococcus, Dokdonella). Additionally, the presence of Devosia and Pseudonocardia, which participated in the phosphorus removal process, was found in the waste PUR foams.

Microfluidics for long-term single-cell time-lapse microscopy: Advances and applications.

Cells are inherently dynamic, whether they are responding to environmental conditions or simply at equilibrium, with biomolecules constantly being made and destroyed. Due to their small volumes, the chemical reactions inside cells are stochastic, such that genetically identical cells display heterogeneous behaviors and gene expression profiles. Studying these dynamic processes is challenging, but the development of microfluidic methods enabling the tracking of individual prokaryotic cells with microscopy over long time periods under controlled growth conditions has led to many discoveries. This review focuses on the recent developments of one such microfluidic device nicknamed the mother machine. We overview the original device design, experimental setup, and challenges associated with this platform. We then describe recent methods for analyzing experiments using automated image segmentation and tracking. We further discuss modifications to the experimental setup that allow for time-varying environmental control, replicating batch culture conditions, cell screening based on their dynamic behaviors, and to accommodate a variety of microbial species. Finally, this review highlights the discoveries enabled by this technology in diverse fields, such as cell-size control, genetic mutations, cellular aging, and synthetic biology.

Mesostats-A multiplexed, low-cost, do-it-yourself continuous culturing system for experimental evolution of mesocosms.

Microbial experimental evolution allows studying evolutionary dynamics in action and testing theory predictions in the lab. Experimental evolution in chemostats (i.e. continuous flow through cultures) has recently gained increased interest as it allows tighter control of selective pressures compared to static batch cultures, with a growing number of efforts to develop systems that are easier and cheaper to construct. This protocol describes the design and construction of a multiplexed chemostat array (dubbed "mesostats") designed for cultivation of algae in 16 concurrent populations, specifically intended for studying adaptation to herbicides. We also present control data from several experiments run on the system to show replicability, data illustrating the effects of common issues like leaks, contamination and clumps, and outline possible modifications and adaptations of the system for future research.

A comprehensive investigation into the production of gamma-aminobutyric acid by Limosilactobacillus fermentum NG16, a tuna gut isolate

Abstract Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is widely known to have multiple physiological functions. The present study aimed to investigate the cultivation parameters for GABA production by a lactic acid bacteria (LAB) strain isolated from a tuna gut sample. Among 60 tuna gut LAB, only 7 Limosilactobacillus fermentum isolates, i.e. NG01, NG12, NG13, NG14, NG16, NG23, and NG27, were capable of GABA fermentation, with NG16 being the most potent GABA producer. The GABA production by isolate NG16 was therefore thoroughly characterised. The optimal batch culture conditions for GABA production were an initial cell density of 5×106 CFU mL−1, a monosodium glutamate concentration of 2%, an initial pH of 7, a fermentation temperature of 35 °C, and an incubation time of 96 h. Under this cultivation conditions, NG16 produced a maximum GABA yield of 25.52 ± 0.41 mM.

Effects of País grape marc inclusion in high and low forage diets: ruminal fermentation, methane production and volatile fatty acids

The objective of this study was to evaluate the effects of the inclusion of País grape marc (GM) in high (HF) and low forage (LF) ruminant diets on rumen methane (CH4) production and fermentation parameters in an in vitro (batch) culture. Experimental treatments replaced forage for GM at increasing concentrations (0, 10%, 20% dry matter [DM]) in the incubation substrates. Gas production (GP) and CH4 production were measured at 6, 12 and 24 h of incubation and in vitro DM disappearance, volatile fatty acids (VFA) and ammonia nitrogen (NH3-N) were determined at 24 h. The inclusion of 10% and 20% of GM reduced NH3-N concentrations (p < 0.001) by 48% and 58% respectively, and GM20 inclusion reduced BCVFA concentrations (p = 0.049) by 21% for HF and by 11% LF. Inclusion of 20% GM reduced the percentage of CH4 in gas (p = 0.038), CH4 production (p = 0.004) and CH4 yield (p = 0.027), but leads to a reduction of in vitro dry matter disappearance (IVDMD) (p < 0.001), gas production (p < 0.001) and total VFA (p = 0.001). In conclusion, in batch culture conditions, partial substitution of fibre sources by País GM in high and low fibre diets has the potential to reduce NH3-N concentrations and CH4 production and yield with a reduction of IVDMD. HIGHLIGHTS País GM reduces up to 58% in vitro NH3-N rumen concentrations regardless of forage level in the diet. Partial substitution of fibre sources with País GM reduces methane production by up to 15%. Partial substitution of forage sources with País grape marc has the potential to improve livestock systems sustainability.

Growth and pigment production of Monascus purpureus EG and its beneficial effects

Pigments play an important role in the pharmaceutical industry as well as in the food industry. Biological synthesis of pigments has attained more revenue for easy extraction, high growth rate and high yield. The production of pigments by Monascus purpureus EG was investigated in several static batch cultures, the most suitable medium for its yellow, orange, and red extracellular pigment production was yeast glucose medium (YG), while malt extract medium (ME) had maximum production for its intracellular pigment. The effect of some physical factors on growth and pigment production was studied. Direct illumination inhibited growth and pigment production. Antimicrobial activities of pigments were observed against selected Gram-negative (G-ve) and Gram-positive (G+ve) bacteria. The antibacterial effects of red pigment on G- and G+ were highly effective compared to yellow and orange pigments. The extracted pigment was used for the reduction of the aqueous silver nitrate into silver nanoparticles (AgNPs). The biosynthesized AgNPs were structurally characterized using UV-VIS Spectra which showed absorption peaks at 437, 453 and 447 nm for pH values 3.5, 6.5 and 9.5, respectively. The optimum pH for the maximum synthesis of nanoparticles was 6.5. It showed no nitrate reductase activity, but the synthesized AgNPs exhibited strong antimicrobial activity against G+ and G- bacteria.

Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance.

Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

Boosting the acetol production in methanotrophic biocatalyst Methylomonas sp. DH-1 by the coupling activity of heteroexpressed novel protein PmoD with endogenous particulate methane monooxygenase

BackgroundMethylacidiphilum sp. IT6 has been validated its C3 substrate assimilation pathway via acetol as a key intermediate using the PmoCAB3, a homolog of the particulate methane monooxygenase (pMMO). From the transcriptomic data, the contribution of PmoD of strain IT6 in acetone oxidation was questioned. Methylomonas sp. DH-1, a type I methanotroph containing pmo operon without the existence of its pmoD, has been deployed as a biocatalyst for the gas-to-liquid bioconversion of methane and propane to methanol and acetone. Thus, Methylomonas sp. DH-1 is a suitable host for investigation. The PmoD-expressed Methylomonas sp. DH-1 can also be deployed for acetol production, a well-known intermediate for various industrial applications. Microbial production of acetol is a sustainable approach attracted attention so far.ResultsIn this study, bioinformatics analyses elucidated that novel protein PmoD is a C-terminal transmembrane–helix membrane with the proposed function as a transport protein. Furthermore, the whole-cell biocatalyst was constructed in Methylomonas sp. DH-1 by co-expression the PmoD of Methylacidiphilum sp. IT6 with the endogenous pMMO to enable acetone oxidation. Under optimal conditions, the maximum accumulation, and specific productivity of acetol were 18.291 mM (1.35 g/L) and 0.317 mmol/g cell/h, respectively. The results showed the first coupling activity of pMMO with a heterologous protein PmoD, validated the involvement of PmoD in acetone oxidation, and demonstrated an unprecedented production of acetol from acetone in type I methanotrophic biocatalyst. From the data achieved in batch cultivation conditions, an assimilation pathway of acetone via acetol as the key intermediate was also proposed.ConclusionUsing bioinformatics tools, the protein PmoD has been elucidated as the membrane protein with the proposed function as a transport protein. Furthermore, results from the assays of PmoD-heteroexpressed Methylomonas sp. DH-1 as a whole-cell biocatalyst validated the coupling activity of PmoD with pMMO to convert acetone to acetol, which also unlocks the potential of this recombinant biocatalyst for acetol production. The proposed acetone-assimilated pathway in the recombinant Methylomonas sp. DH-1, once validated, can extend the metabolic flexibility of Methylomonas sp. DH-1.

Xanthoepocin, a photolabile antibiotic of Penicillium ochrochloron CBS\xa0123823 with high activity against multiresistant gram-positive bacteria

BackgroundWith the steady increase of antibiotic resistance, several strategies have been proposed in the scientific community to overcome the crisis. One of many successful strategies is the re-evaluation of known compounds, which have been early discarded out of the pipeline, with state-of-the-art know-how. Xanthoepocin, a polyketide widespread among the genus Penicillium with an interesting bioactivity spectrum against gram-positive bacteria, is such a discarded antibiotic. The purpose of this work was to (i) isolate larger quantities of this metabolite and chemically re-evaluate it with modern technology, (ii) to explore which factors lead to xanthoepocin biosynthesis in P. ochrochloron, and (iii) to test if it is beside its known activity against methicillin-resistant Staphylococcus aureus (MRSA), also active against linezolid and vancomycin-resistant Enterococcus faecium (LVRE)—a very problematic resistant bacterium which is currently on the rise.ResultsIn this work, we developed several new protocols to isolate, extract, and quantify xanthoepocin out of bioreactor batch and petri dish-grown mycelium of P. ochrochloron. The (photo)chemical re-evaluation with state-of-the-art techniques revealed that xanthoepocin is a photolabile molecule, which produces singlet oxygen under blue light irradiation. The intracellular xanthoepocin content, which was highest under ammonium-limited conditions, varied considerably with the applied irradiation conditions in petri dish and bioreactor batch cultures. Using light-protecting measures, we achieved MIC values against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which were up to 5 times lower than previously published. In addition, xanthoepocin was highly active against a clinical isolate of linezolid and vancomycin-resistant Enterococcus faecium (LVRE).ConclusionsThis interdisciplinary work underlines that the re-evaluation of known compounds with state-of-the-art techniques is an important strategy in the combat against multiresistant bacteria and that light is a crucial factor on many levels that needs to receive more attention. With appropriate light protecting measures in the susceptibility tests, xanthoepocin proved to be a powerful antibiotic against MRSA and LVRE. Exploring the light response of other polyketides may be pivotal for re-introducing previously discarded metabolites into the antibiotic pipeline and to identify photosensitizers which might be used for (antimicrobial) photodynamic therapies.

Arsenosugars and arsenolipids are formed simultaneously by the unicellular alga

Environmental context Arsenic is a globally distributed element, occurring in various chemical forms with toxicities ranging from harmless to highly toxic. We conducted 48-h cell culture experiments under batch and continuous conditions using the ubiquitous marine unicellular alga Dunaliella tertiolecta and evaluated the alga’s arsenic metabolome over time. We found that the alga first methylates the inorganic As taken up from the surrounding water, and then further metabolises the intermediate simultaneously into more complex organo-arsenic molecules like sugars and lipids. These time series experiments are valuable pieces in the puzzle of how algae bio-metabolise arsenic, and in our understanding of the global arsenic cycle. Rationale The uptake of arsenate by algae from oceanic waters and its transformation to arsenosugars and arsenolipids is well established, but the biosynthetic pathways remain largely unknown. Methodology We investigated these pathways by using time-series experiments over 48 h to follow the formation of organoarsenic species from arsenate-enriched medium (15 µg As L−1) by the unicellular alga Dunaliella tertiolecta cultured under batch and continuous culture conditions. We used complementary mass spectrometry methods for the determination and quantification of 14 arsenic species; an additional three species could be quantified but remained unidentified. Results The alga rapidly methylated the arsenate to dimethylarsinate (DMA), which then served as the precursor to arsenosugars and arsenolipids; the concentrations of these complex organoarsenicals increased throughout the experiments accompanied by a concomitant reduction in DMA concentrations. The pattern of compounds formed by the alga was similar for both batch and continuous cultures, but the concentrations were 2–3-fold higher in the continuous culture samples and the increases with time were much clearer. Discussion The data suggest that the arsenosugars and the arsenolipids were mostly formed simultaneously from DMA, although there was an indication that the arsenic phospholipids were at least partly also being formed from the arsenosugars. Overall, the data are consistent with a direct biosynthesis of DMA from arsenate by D. tertioleta, and thereafter a non-specific incorporation of DMA into commonly available alga metabolites encompassing various sugars and lipids.

Co-culture of Two Different Cell Lines in a Two-Layer Microfluidic Device.

Microfluidic devices have become a promising alternative approach for cellular co-culture. Many approaches incorporate a semipermeable barrier to physically separate, yet chemically connect, two cell types; however, the majority of these approaches utilize batch culture conditions which can result in nutrient depletion and waste accumulation. This chapter describes an alternative approach that allows for the continuous infusion of media, relieving the constraints of batch culture. The microfluidic device consists of two separate layers: a bottom layer of 3% (w/v) agarose to facilitate chemical diffusion and a top polydimethylsiloxane (PDMS) layer into which four parallel fluidic channels were imprinted. The microfluidic approach allows for facile visualization of cells with light microscopy and the ability to add (or subtract) drugs or biomolecules to interrogate the system or modulate the cellular response. Finally, the approach allows for terminal immunostaining of either (or both) cell types.

Biodegradable, metal-chelating compounds as alternatives to EDTA for cultivation of marine microalgae

Iron (Fe) is an essential nutrient for microalgal metabolism. The low solubility of Fe in oxic aquatic environments can be a growth-limiting factor for phytoplankton. Synthetic chelating agents, such as ethylenediaminetetraacetic acid (EDTA), are used widely to maintain Fe in solution for microalgal cultivation. The non-biodegradable nature of EDTA, combined with sub-optimal bioavailability of Fe-EDTA complexes to microalgae, indicates opportunity to improve microalgal cultivation practices that amplify production efficiency and environmental compatibility. In the present study, the effects of four organic chelating ligands known to form readily bioavailable organic complexes with Fe in natural aquatic environments were investigated in relation to growth and biochemical composition of two marine microalgae grown as live feeds in shellfish hatcheries (Chaetoceros calcitrans and Tisochrysis lutea). Three saccharides, alginic acid (ALG), glucuronic acid (GLU), and dextran (DEX), as well as the siderophore desferrioxamine B (DFB), were compared to EDTA. Organic ligands characterized by weaker binding capacity for cationic metals (i.e., ALG, GLU, DEX) significantly improved microalgal growth and yields in laboratory-scale static batch cultures or bubbled photobioreactors. Maximal microalgal growth enhancement relative to the control (e.g., EDTA) was recorded for GLU, followed by ALG, with 20–35% increase in specific growth rate in the early stages of culture development of C. calcitrans and T. lutea. Substitution of EDTA with GLU resulted in a 27% increase in cellular omega 3-polyunsaturetd fatty acid content of C. calcitrans and doubled final cell yields. Enhanced microalgal culture performance is likely associated with increased intracellular Fe uptake efficiency combined with heterotrophic growth stimulated by the organic ligands. Based upon these results, we propose that replacement of EDTA with one of these organic metal-chelating ligands is an effective and easily implementable strategy to enhance the environmental compatibility of microalgal cultivation practices while also maximizing algal growth and enhancing the nutritional quality of marine microalgal species commonly cultured for live-feed applications in aquaculture.

Hydrologic Alteration and Enhanced Microbial Reductive Dissolution of Fe(III) (hydr)oxides Under Flow Conditions in Fe(III)-Rich Rocks: Contribution to Cave-Forming Processes.

Previous work demonstrated that microbial Fe(III)-reduction contributes to void formation, and potentially cave formation within Fe(III)-rich rocks, such as banded iron formation (BIF), iron ore and canga (a surficial duricrust), based on field observations and static batch cultures. Microbiological Fe(III) reduction is often limited when biogenic Fe(II) passivates further Fe(III) reduction, although subsurface groundwater flow and the export of biogenic Fe(II) could alleviate this passivation process, and thus accelerate cave formation. Given that static batch cultures are unlikely to reflect the dynamics of groundwater flow conditions in situ, we carried out comparative batch and column experiments to extend our understanding of the mass transport of iron and other solutes under flow conditions, and its effect on community structure dynamics and Fe(III)-reduction. A solution with chemistry approximating cave-associated porewater was amended with 5.0 mM lactate as a carbon source and added to columns packed with canga and inoculated with an assemblage of microorganisms associated with the interior of cave walls. Under anaerobic conditions, microbial Fe(III) reduction was enhanced in flow-through column incubations, compared to static batch incubations. During incubation, the microbial community profile in both batch culture and columns shifted from a Proteobacterial dominance to the Firmicutes, including Clostridiaceae, Peptococcaceae, and Veillonellaceae, the latter of which has not previously been shown to reduce Fe(III). The bacterial Fe(III) reduction altered the advective properties of canga-packed columns and enhanced permeability. Our results demonstrate that removing inhibitory Fe(II) via mimicking hydrologic flow of groundwater increases reduction rates and overall Fe-oxide dissolution, which in turn alters the hydrology of the Fe(III)-rich rocks. Our results also suggest that reductive weathering of Fe(III)-rich rocks such as canga, BIF, and iron ores may be more substantial than previously understood.

A new, reliable, and high-throughput strategy to screen bacteria for antagonistic activity against Staphylococcus aureus

BackgroundAntibiotic-resistant Staphylococcus aureus clones have emerged globally over the last few decades. Probiotics have been actively studied as an alternative to antibiotics to prevent and treat S. aureus infections, but identifying new probiotic bacteria, that have antagonistic activity against S. aureus, is difficult since traditional screening strategies are time-consuming and expensive. Here, we describe a new plasmid-based method which uses highly stable plasmids to screen bacteria with antagonistic activity against S. aureus.ResultsWe have created two recombinant plasmids (pQS1 and pQS3) which carry either gfpbk or mCherry under the control of a S. aureus quorum-sensing (QS) promoter (agrP3). Using this recombinant plasmid pair, we tested 81 bacteria isolated from Holstein dairy milk to identify bacteria that had growth-inhibiting activity against S. aureus and suggest potential explanations for the growth inhibition. The stability test illustrated that pQS1 and pQS3 remained highly stable for at least 24 h in batch culture conditions without selection pressure from antibiotics. This allowed co-culturing of S. aureus with other bacteria. Using the newly developed pQS plasmids, we found commensal bacteria, isolated from raw bovine milk, which had growth-inhibiting activity (n = 13) and quorum-quenching (QQ) activity (n = 13) towards both S. aureus Sa25 (CC97) and Sa27 (CC151). The pQS-based method is efficient and effective for simultaneously screening growth-inhibiting and QQ bacteria against S. aureus on agar media.ConclusionsIt was shown that growth-inhibiting and QQ activity toward pQS plasmid transformants of S. aureus can be simultaneously monitored by observing the zone of growth inhibition and reporter protein inhibition on agar plates. Newly identified antagonistic bacteria and their functional biomolecules are promising candidates for future development of probiotic drugs and prophylactics/therapeutics for bacterial infections including S. aureus. Furthermore, this new approach can be a useful method to find bacteria that can be used to prevent and treat S. aureus infections in both humans and animals.