Synthetic Microbial Consortium Research Articles

Richness and antagonistic effects co-affect plant growth promotion by synthetic microbial consortia

Plant-related microbial communities are essential to plant growth and development, and empirical evidence suggests that inoculation with a synthetic microbial consortium can enhance plant performance; however, to manipulate the functions of these communities, we require a better understanding of the influencing factors. Here, we used the biodiversity ecosystem function (BEF) framework to specifically test the impact of community richness (1, 2, 4, or 8 strains per community) and antagonistic intensity on plant growth promotion in two kinds of nutritional environments. We found that increasing the richness of microbes can improve the accumulation of plant biomass, revealing that diversity effects were stronger than single-strain effects. The intensity of antagonism between highly diverse microbes may have a consistent negative effect on plant growth in comparison with the effects of communities with the same richness; intriguingly, this negative effect did not change positive BEF relationships. These results suggested that high diversity in the synthetic microbial consortia harboured stable and effective plant growth promotion capability and, in turn, also increased susceptibility to the negative effects of antagonism among microbial strains.

Mg, K-containing microparticle: A possible active principle of a culture extract produced by a microbial consortium.

A synthetic microbial consortium called Effective Microorganisms (EM) consists mainly of photosynthetic bacteria, lactic acid bacteria and yeast. Various effects of EM∙XGOLD, a health drink produced by EM, on life cycle of Dictyostelium discoideum were described previously. Here, we report our attempt to identify the active principle, termed EMF, that brought about the observed effects. Throughout the purification processes, the presence of the active principle was monitored by promoted fruiting body formation. By liquid-liquid separation the activity was recovered in aqueous phase, which, after concentration, was further subjected to reverse-phase column chromatography. No activity was detected in any eluant, while almost all the activity was recovered in residual insoluble material. The application of conventional organic chemistry procedures to the residual fraction did not lead to any informative results. Acid treatment of the insoluble material produced air bubbles, suggesting it to be composed of some inorganic carbonate. Viewed under scanning electronmicroscope, the residue revealed spherical particles of μm size range. Energy Dispersive X-ray (EDX) Spectroscopy pointed to the existence, on the surface of the particles, of magnesium and, to a certain extent, of potassium. In separate experiments, acid treatment and alkali neutralization of EM∙XGOLD completely wiped out the stimulatory activity of fruiting body formation. These lines of evidence indicate these Mg, K-containing microparticles to be an active principle of EM culture extract. How these particles exert their effect is currently under intensive investigation.

Immobilization of Microbial Consortium for Glutaric Acid Production from Lysine

AbstractGlutaric acid is an important C5 platform compound for the synthesis of nylon polymers. However, its production titer and cost are limited by the need for expensive cofactors addition as well as the difficulty of reusing microbial catalysts. Herein, to address these problems, two regeneration systems each for NAD(P)+ and α‐ketoglutaric acid were introduced into a five‐enzyme cadaverine pathway for a whole‐cell biocatalytic approach. We demonstrated the efficient conversion of lysine to glutaric acid in a synthetic microbial consortium manner, producing 73.2 g/L glutaric acid with a 74 % molar yield in a 5‐L bioreactor scale. Moreover, with a colloidal chitin‐based immobilization, the engineered microbial consortium exhibited good stability and reusability in a five‐cycle bioconversion. This study provides an efficient microbial platform for glutaric acid production by developing a cheaper and universal cell immobilization approach.

Effect of culture conditions on the performance of lignocellulose-degrading synthetic microbial consortia

In this study, we examined a synthetic microbial consortium, composed of two selected bacteria, i.e., Citrobacter freundii so4 and Sphingobacterium multivorum w15, next to the fungus Coniochaeta sp. 2T2.1, with respect to their fate and roles in the degradation of wheat straw (WS). A special focus was placed on the effects of pH (7.2, 6.2, or 5.2), temperature (25 versus 28 °C), and shaking speed (60 versus 180 rpm). Coniochaeta sp. 2T2.1 consistently had a key role in the degradation process, with the two bacteria having additional roles. Whereas temperature exerted only minor effects on the degradation, pH and shaking speed were key determinants of both organismal growth and WS degradation levels. In detail, the three-partner degrader consortium showed significantly higher WS degradation values at pH 6.2 and 5.2 than at pH 7.2. Moreover, the two bacteria revealed up to tenfold enhanced final cell densities (ranging from log8.0 to log9.0 colony forming unit (CFU)/mL) in the presence of Coniochaeta sp. 2T2.1 than when growing alone or in a bacterial bi-culture, regardless of pH range or shaking speed. Conversely, at 180 rpm, fungal growth was clearly suppressed by the presence of the bacteria at pH 5.2 and pH 6.2, but not at pH 7.2. In contrast, at 60 rpm, the presence of the bacteria fostered fungal growth. In these latter cultures, oxygen levels were significantly lowered as compared to the maximal levels found at 180 rpm (about 5.67 mg/L, ~ 62% of saturation). Conspicuous effects on biomass appearance pointed to a fungal biofilm–modulating role of the bacteria.Key points• Coniochaeta sp. 2T2.1 has a key role in wheat straw (WS) degradation.• Bacterial impact shifts when conditions change.• pH and shaking speed are key drivers of the growth dynamics and WS degradation.

Engineering synthetic microbial consortium for efficient conversion of lactate from glucose and xylose to generate electricity

Microbial fuel cell (MFC) is a sustainable electrochemical bioreactor that enables biomass harvesting to produce energy. Shewanella oneidensis, one of the widely used exoelectrogens in MFCs, can only utilize a limited spectrum of carbon sources. To enable the use of abundant carbon sources in MFCs, a lactate-mediated Saccharomyces cerevisiae and S. oneidensis microbial consortium was constructed to utilize 2 g/L xylose or 1 g/L glucose-xylose in generating electricity. The designed system achieved a constant supply of lactate to S. oneidensis, in which the lactate concentration was maintained at 0−0.1 g/L. Meanwhile, the output current promoted the metabolic rate of the carbon sources as well. Finally, the flavin biosynthesis pathway was incorporated into S. oneidensis to enhance the power production of the consortium, and the maximum output power density of 238.7 ± 14.5 mW/m2 was reached. This high-performance power generation could be attributed to the ‘division-of-labor’ between the fermenter and exoelectrogen based on the programmed cooperation and mutualistic interactions among individuals.

Facile bioconversion of vegetable food waste into valuable organic acids and green fuels using synthetic microbial consortium

The production of various organic acids from vegetable waste via a facile and cost-effective method utilizing characterized synthetic microbial consortia is described in this study. Five bacterial species with the ability to produce organic acids from vegetable waste biomass were isolated and identified as Lactobacillus casei, Lactobacillus acidophilus, Bacillus megaterium, Pseudomonas florescence and Escherichia coli. Using these cultures, mixed acid fermentation was developed and was efficient in producing various organic acids. The total organic acids accumulated using optimized fermentation conditions was found to be 72.44±3.43 g L−1. The acetic acid was produced as major acid accumulated up to 25.27±1.26 g L−1, followed by lactic acid 19.11±1.73 g L−1. Efforts were also put forth to check the ability to produce methane by the anaerobic digestion process. Up to 14.97 mL g−1 biomass methane was produced during the anaerobic digestion process. The technology developed in this study is a carbon-neutral process for managing vegetable food waste with economic benefit. The developed technology will have great economic potential and add value to vegetable food waste management.

Multicellular Feedback Control of a Genetic Toggle-Switch in Microbial Consortia

We describe a multicellular approach to control a target cell population endowed with a bistable toggle-switch. The idea is to engineer a synthetic microbial consortium consisting of three different cell populations. In such a consortium, two populations, the Togglers, responding to some reference input, can induce the switch of a bistable memory mechanism in a third population, the Targets, so as to activate or deactivate some additional functionalities in the cells. Communication among the three populations is established by orthogonal quorum sensing molecules that are used to close a feedback control loop across the populations. The control design is validated via in-silico experiments in BSim, a realistic agent-based simulator of bacterial populations.

Consolidated bioprocessing of cellulose to itaconic acid by a co-culture of Trichoderma reesei and Ustilago maydis

BackgroundItaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation.ResultsThe efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation.ConclusionThis study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Eco-Mimicry Opens New Doors for Bioprocess Engineers

The natural world blends microenvironments to create a continuum of spatial niches wherein microbial consortia participate in metabolic exchanges. The capability with which nature selects a consortium to complement a given niche is often taken for granted. Not by Shahab et al. (2020), however, who created a synthetic microbial consortium to mediate a multi-step bioprocess in a single bioreactor system. The concept affords a fair degree of modularity in the choice of biocatalyst and a reasonable level of process control, thus making it an engineering tool.

Pretreatment and Detoxification of Acid-Treated Wood Hydrolysates for Pyruvate Production by an Engineered Consortium of Escherichia coli.

The biorefinery concept makes use of renewable lignocellulosic biomass to produce commodities sustainably. A synthetic microbial consortium can enable the simultaneous utilization of sugars such as glucose and xylose to produce biochemicals, where each consortium member converts one sugar into the target product. In this study, woody biomass was used to generate glucose and xylose after pretreatment with 20% (w/v) sulfuric acid and 60-min reaction time. We compared several strategies for detoxification with charcoal and sodium borohydride treatments to improve the fermentability of this hydrolysate in a defined medium for the production of the growth-associated product pyruvate. In shake flask culture, the highest pyruvate yield on xylose of 0.8g/g was found using pH6 charcoal-treated hydrolysate. In bioreactor studies, a consortium of two engineered E. coli strains converted the mixture of glucose and xylose in batch studies to 12.8 ± 2.7g/L pyruvate in 13h. These results demonstrate that lignocellulosic biomass as the sole carbon source can be used to produce growth-related products after employing suitable detoxification strategies.

Sustainable production of FAEE biodiesel using the oleaginous yeast Yarrowia lipolytica.

Fatty acid ethyl esters (FAEEs) are fatty acid‐derived molecules and serve as an important form of biodiesel. The oleaginous yeast Yarrowia lipolytica is considered an ideal host platform for the production of fatty acid‐derived products due to its excellent lipid accumulation capacity. In this proof‐of‐principle study, several metabolic engineering strategies were applied for the overproduction of FAEE biodiesel in Y. lipolytica. Here, chromosome‐based co‐overexpression of two heterologous genes, namely, PDC1 (encoding pyruvate decarboxylase) and ADH1 (encoding alcohol dehydrogenase) from Saccharomyces cerevisiae, and the endogenous GAPDH (encoding glyceraldehyde‐3‐phosphate dehydrogenase) gene of Y. lipolytica resulted in successful biosynthesis of ethanol at 70.8 mg/L in Y. lipolytica. The engineered Y. lipolytica strain expressing the ethanol synthetic pathway together with a heterologous wax ester synthase (MhWS) exhibited the highest FAEE titer of 360.8 mg/L, which is 3.8‐fold higher than that of the control strain when 2% exogenous ethanol was added to the culture medium of Y. lipolytica. Furthermore, a synthetic microbial consortium comprising an engineered Y. lipolytica strain that heterologously expressed MhWS and a S. cerevisiae strain that could provide ethanol as a substrate for the production of the final product in the final engineered Y. lipolytica strain was created in this study. Finally, this synthetic consortium produced FAEE biodiesel at a titer of 4.8 mg/L under the optimum coculture conditions.

Biotransformation of 2,4-dinitrotoluene in a phototrophic co-culture of engineered Synechococcus elongatus and Pseudomonas putida.

SummaryIn contrast to the current paradigm of using microbial mono‐cultures in most biotechnological applications, increasing efforts are being directed towards engineering mixed‐species consortia to perform functions that are difficult to programme into individual strains. In this work, we developed a synthetic microbial consortium composed of two genetically engineered microbes, a cyanobacterium (Synechococcus elongatus PCC 7942) and a heterotrophic bacterium (Pseudomonas putida EM173). These microbial species specialize in the co‐culture: cyanobacteria fix CO2 through photosynthetic metabolism and secrete sufficient carbohydrates to support the growth and active metabolism of P. putida, which has been engineered to consume sucrose and to degrade the environmental pollutant 2,4‐dinitrotoluene (2,4‐DNT). By encapsulating S. elongatus within a barium–alginate hydrogel, cyanobacterial cells were protected from the toxic effects of 2,4‐DNT, enhancing the performance of the co‐culture. The synthetic consortium was able to convert 2,4‐DNT with light and CO2 as key inputs, and its catalytic performance was stable over time. Furthermore, cycling this synthetic consortium through low nitrogen medium promoted the sucrose‐dependent accumulation of polyhydroxyalkanoate, an added‐value biopolymer, in the engineered P. putida strain. Altogether, the synthetic consortium displayed the capacity to remediate the industrial pollutant 2,4‐DNT while simultaneously synthesizing biopolymers using light and CO2 as the primary inputs.

Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium.

Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

Long-range temporal coordination of gene expression in synthetic microbial consortia.

Synthetic microbial consortia have an advantage over isogenic synthetic microbes because they can apportion biochemical and regulatory tasks among the strains. However, it is difficult to coordinate gene expression in spatially extended consortia because the range of signaling molecules is limited by diffusion. Here, we show that spatiotemporal coordination of gene expression can be achieved even when the spatial extent of the consortium is much greater than the diffusion distance of the signaling molecules. To do this, we examined the dynamics of a two-strain synthetic microbial consortium that generates coherent oscillations in small colonies. In large colonies, we find that temporally coordinated oscillations across the population depend on the presence of an intrinsic positive feedback loop that amplifies and propagates intercellular signals. These results demonstrate that synthetic multi-cellular systems can be engineered to exhibit coordinated gene expression using only transient, short-range coupling among constituent cells.

Synthetic microbial consortium with specific roles designated by genetic circuits for cooperative chemical production

Synthetic microbial consortia consisting of microorganisms with different synthetic genetic circuits or divided synthetic metabolic pathway components can exert functions that are beyond the capacities of single microorganisms. However, few consortia of microorganisms with different synthetic genetic circuits have been developed. We designed and constructed a synthetic microbial consortium composed of an enzyme-producing strain and a target chemical-producing strain using Escherichia coli for chemical production with efficient saccharification. The enzyme-producing strain harbored a synthetic genetic circuit to produce beta-glucosidase, which converts cellobiose to glucose, destroys itself via the lytic genes, and release the enzyme when the desired cell density is reached. The target chemical-producing strain was programmed by a synthetic genetic circuit to express enzymes in the synthetic metabolic pathway for isopropanol production when the enzyme-producing strain grows until release of the enzyme. Our results demonstrate the benefits of synthetic microbial consortia with distributed tasks for effective chemical production from biomass.

Application of independent immobilization in benzo[a]pyrene biodegradation by synthetic microbial consortium.

Bioaugmentation is an effective approach to remove the benzo[a]pyrene (BaP) from the environment, while its effect depends on the functional stability of the inoculated microorganisms. The aim of this study is to develop an approach on reducing the mutual exclusion of bacteria in the synthetic consortium in BaP degradation. Eight BaP-degrading bacterial strains were isolated from an enrichment with BaP as the sole carbon source. Two strains of Cupriavidus spp. exhibited greater degradation capacity (3.02-3.30 mg L-1 day-1) and selected as the "good degraders" in the synthetic consortia. Because of the mutual exclusion, the BaP-degradation capacity was reduced (1.47-1.77 mg L-1 day-1) when the other strains were added into "good degraders" through directly mixing the inocula. This mutual exclusion was mitigated through independent immobilization, in which the strains were embedded in sodium alginate before constructing the consortium. The consortium constructed by independent immobilization exhibited comparable BaP-degradation capacity with the high efficient strains. Therefore, the independent immobilization can be an advanced approach in functional consortium synthesis.

Engineering Microbial Consortia for High-Performance Cellulosic Hydrolyzates-Fed Microbial Fuel Cells.

Microbial fuel cells (MFCs) are eco-friendly bio-electrochemical reactors that use exoelectrogens as biocatalyst for electricity harvest from organic biomass, which could also be used as biosensors for long-term environmental monitoring. Glucose and xylose, as the primary ingredients from cellulose hydrolyzates, is an appealing substrate for MFC. Nevertheless, neither xylose nor glucose can be utilized as carbon source by well-studied exoelectrogens such as Shewanella oneidensis. In this study, to harvest the electricity by rapidly harnessing xylose and glucose from corn stalk hydrolysate, we herein firstly designed glucose and xylose co-fed engineered Klebsiella pneumoniae-S. oneidensis microbial consortium, in which K. pneumoniae as the fermenter converted glucose and xylose into lactate to feed the exoelectrogens (S. oneidensis). To produce more lactate in K. pneumoniae, we eliminated the ethanol and acetate pathway via deleting pta (phosphotransacetylase gene) and adhE (alcohol dehydrogenase gene) and further constructed a synthesis and delivery system through expressing ldhD (lactate dehydrogenase gene) and lldP (lactate transporter gene). To facilitate extracellular electron transfer (EET) of S. oneidensis, a biosynthetic flavins pathway from Bacillus subtilis was expressed in a highly hydrophobic S. oneidensis CP-S1, which not only improved direct-contacted EET via enhancing S. oneidensis adhesion to the carbon electrode but also accelerated the flavins-mediated EET via increasing flavins synthesis. Furthermore, we optimized the ratio of glucose and xylose concentration to provide a stable carbon source supply in MFCs for higher power density. The glucose and xylose co-fed MFC inoculated with the recombinant consortium generated a maximum power density of 104.7 ± 10.0 mW/m2, which was 7.2-folds higher than that of the wild-type consortium (12.7 ± 8.0 mW/m2). Lastly, we used this synthetic microbial consortium in the corn straw hydrolyzates-fed MFC, obtaining a power density 23.5 ± 6.0 mW/m2.

The Life Cycle of Dictyostelium discoideum Is Accelerated via MAP Kinase Cascade by a Culture Extract Produced by a Synthetic Microbial Consortium

A cellular slime mold, Dictyostelium discoideum, is an amoeboid organism that has a unique life cycle consisting of distinctly separated vegetative and developmental phases. Thus, this organism presents a rare opportunity in which to examine the effects of bioactive substances on separate cellular activities. In this research, we investigated the effect of a culture extract, termed EMXG, produced by a synthetic microbial consortium. EMXG promoted proliferative response of amoeba cells. It further accelerated the developmental phase, leading to the preferred fruiting body formation from fewer cells. Furthermore, EMXG modulated biological rhythm of this organism, that is, interval of oscillation of cAMP level observed in suspension starvation was significantly shortened. Concomitantly, the level of ERKB, a MAP kinase, was found to oscillate in a similar fashion to that of cAMP. Additionally, ErkB-deficient mutant amoeboid cells did not respond to proliferative stimulation by EMXG. These lines of evidence point to a likelihood that MAP kinase cascade is involved and further that ErkB could be the molecular target of EMXG.

Development of a novel methanotrophic process with the helper micro-organism Hyphomicrobium sp. NM3.

Microbial consortia can be more efficient at biological processes than single isolates. The purposes of this study were to design and evaluate a synthetic microbial consortium containing the methanotroph Methylocystis sp. M6 and the helper Hyphomicrobium sp. NM3, and develop a novel methanotrophic process for this consortium utilizing a dialysis membrane. Hyphomicrobium increased the methane-oxidation rate (MOR), biomass and stability at a dilution rate of 0·067day-1 in fed-batch co-culture. qRT-PCR showed that Methylocystis population increased gradually with time, whereas Hyphomicrobium population remained stable despite cell washing, confirming synergistic population interaction. At 0·1day-1 , spiking of Hyphomicrobium effectively increased the methanotrophic activity, after which Hyphomicrobium population decreased with time, indicating that the consortium is optimal at <0·1day-1 . When Hyphomicrobium was grown in dialysis membrane within the bioreactor, MOR increased linearly up to 155·1±1·0mmoll-1 day-1 at 0·067, 0·1, 0·2 and 0·4day-1 , which is the highest observed value for a methanotrophic reactor. Hyphomicrobium sp. NM3 is a promising helper micro-organism for methanotrophs. Hyphomicrobium-methanotroph consortia used concurrently with existing methods can produce an efficient and stable methane oxidation system. This novel methanotrophic process is superior to those previously reported in the literature, and can provide efficient and stable methane oxidation.

Rhizospheric Microbiome Profiling of Capsicum annuum L. Cultivated in Amended Soils by 16S and Internal Transcribed Spacer 2 rRNA Amplicon Metagenome Sequencing.

ABSTRACTRhizospheric microbiomes of Capsicum annuum L. cultivated either conventionally or amended with a synthetic microbial consortium or a root exudate inductor, were characterized by 16S/internal transcribed spacer 2 (ITS2) rRNA amplicon metagenome sequencing. The most abundant taxa found, although differently represented in each treatment, were Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, and Bacilli, as well as Chytridiomycetes and Mortierellomycotina.