Microbial Co-cultures Research Articles

Exchange of Vitamin B1 and Its Biosynthesis Intermediates Shapes the Composition of Synthetic Microbial Cocultures and Reveals Complexities of Nutrient Sharing.

Microbial communities occupy diverse niches in nature, and community members routinely exchange a variety of nutrients among themselves. While large-scale metagenomic and metabolomic studies shed some light on these exchanges, the contribution of individual species and the molecular details of specific interactions are difficult to track. In this study, we follow the exchange of vitamin B1 (thiamin) and its intermediates between microbes within synthetic cocultures of Escherichia coli and Vibrio anguillarum. Thiamin contains two moieties, 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 4-methyl-5-(2-hydroxyethyl)thiazole (THZ), which are synthesized by distinct pathways using enzymes ThiC and ThiG, respectively, and then coupled by ThiE to form thiamin. Even though E. coli ΔthiC, ΔthiE, and ΔthiG mutants are thiamin auxotrophs, we observed that cocultures of ΔthiC-ΔthiE and ΔthiC-ΔthiG mutants are able to grow in a thiamin-deficient medium, whereas the ΔthiE-ΔthiG coculture does not. Further, the exchange of thiamin and its intermediates in V. anguillarum cocultures and in mixed cocultures of V. anguillarum and E. coli revealed that there exist specific patterns for thiamin metabolism and exchange among these microbes. Our findings show that HMP is shared more frequently than THZ, concurrent with previous observations that free HMP and HMP auxotrophy is commonly found in various environments. Furthermore, we observe that the availability of exogenous thiamin in the media affects whether these strains interact with each other or grow independently. These findings collectively underscore the importance of the exchange of essential metabolites as a defining factor in building and modulating synthetic or natural microbial communities. IMPORTANCE Vitamin B1 (thiamin) is an essential nutrient for cellular metabolism. Microorganisms that are unable to synthesize thiamin either fully or in part exogenously obtain it from their environment or via exchanges with other microbial members in their community. In this study, we created synthetic microbial cocultures that rely on sharing thiamin and its biosynthesis intermediates and observed that some of them are preferentially exchanged. We also observed that the coculture composition is dictated by the production and/or availability of thiamin and its intermediates. Our studies with synthetic cocultures provide the molecular basis for understanding thiamin sharing among microorganisms and lay out broad guidelines for setting up synthetic microbial cocultures by using the exchange of an essential metabolite as their foundation.

Design of stable and self-regulated microbial consortia for chemical synthesis

Microbial coculture engineering has emerged as a promising strategy for biomanufacturing. Stability and self-regulation pose a significant challenge for the generation of intrinsically robust cocultures for large-scale applications. Here, we introduce the use of multi-metabolite cross-feeding (MMCF) to establish a close correlation between the strains and the design rules for selecting the appropriate metabolic branches. This leads to an intrinicially stable two-strain coculture where the population composition and the product titer are insensitive to the initial inoculation ratios. With an intermediate-responsive biosensor, the population of the microbial coculture is autonomously balanced to minimize intermediate accumulation. This static-dynamic strategy is extendable to three-strain cocultures, as demonstrated with de novo biosynthesis of silybin/isosilybin. This strategy is generally applicable, paving the way to the industrial application of microbial cocultures.

Scale-Up of Dark Fermentative Biohydrogen Production by Artificial Microbial Co-Cultures

As a renewable energy carrier, dark fermentative biohydrogen (H2) represents a promising future alternative to fossil fuels. Recently, the limited H2 yield of 4 moles of H2 per mole glucose, the so-called “Thauer limit”, was surpassed by a defined artificial consortium. In this article, we demonstrate the upscaling of this drawing board design, from serum bottles to laboratory scale bioreactors. Our results illustrate that this designed microbial co-culture can be successfully implemented in batch mode, with maximum H2 yields of 6.18 and 4.45 mol mol−1 substrate. Furthermore, we report volumetric H2 productivities of 105.6 and 80.8 mmol H2 L−1 h−1. These rates are higher than for any other dark fermentative H2 production system using a synthetic microbial co-culture applied in batch mode on a defined medium. Our study is an important step forward for the application of artificial microbial consortia in future biotechnology and energy production systems.

Using OMICS technologies to analyze the mechanisms of synthetic microbial co-culture systems: a review

In recent years, the interaction mechanisms underpinning the synthetic microbial co-culture systems have gained increasing attention due to their potentials in various biotechnological applications. Exploration of the inter-species mechanisms underpinning the synthetic microbial co-culture system could contribute to a better understanding of the theoretical basis to further optimize the existing co-culture systems, and design new synthetic co-culture system for large-scale application. OMICS technologies such as genomics, transcriptomics, proteomics, and metabolomics could analyze the biological processes in a high throughput manner. Multi-omics analysis could achieve a "global view" of various members in the microbial co-culture systems, which presents opportunities in understanding synthetic microbial consortia better. This article summarizes recent advances in understanding the mechanisms of synthetic microbial co-culture systems using omics technologies, from the aspects of metabolic network, energy metabolism, signal transduction, membrane transport, stress response, community stability and structural rationality. All these findings could provide important theoretical basis for future application of the microbial co-culture systems with the aids of emerging biotechnologies such as synthetic biology and genome editing.

Developing Microbial Co-Culture System for Enhanced Polyhydroxyalkanoates (PHA) Production Using Acid Pretreated Lignocellulosic Biomass.

In the growing polymer industry, the interest of researchers is captivated by bioplastics production with biodegradable and biocompatible properties. This study examines the polyhydroxyalkanoates (PHA) production performance of individual Lysinibacillus sp. RGS and Ralstonia eutropha ATCC 17699 and their co-culture by utilizing sugarcane bagasse (SCB) hydrolysates. Initially, acidic (H2SO4) and acidified sodium chlorite pretreatment was employed for the hydrolysis of SCB. The effects of chemical pretreatment on the SCB biomass assembly and its chemical constituents were studied by employing numerous analytical methods. Acidic pretreatment under optimal conditions showed effective delignification (60%) of the SCB biomass, leading to a maximum hydrolysis yield of 74.9 ± 1.65% and a saccharification yield of 569.0 ± 5.65 mg/g of SCB after enzymatic hydrolysis. The resulting SCB enzymatic hydrolysates were harnessed for PHA synthesis using individual microbial culture and their defined co-culture. Co-culture strategy was found to be effective in sugar assimilation, bacterial growth, and PHA production kinetic parameters relative to the individual strains. Furthermore, the effects of increasing acid pretreated SCB hydrolysates (20, 30, and 40 g/L) on cell density and PHA synthesis were studied. The effects of different cost-effective nutrient supplements and volatile fatty acids (VFAs) with acid pretreated SCB hydrolysates on cell growth and PHA production were studied. By employing optimal conditions and supplementation of corn steep liquor (CSL) and spent coffee waste extracted oil (SCGO), the co-culture produced maximum cell growth (DCW: 11.68 and 11.0 g/L), PHA accumulation (76% and 76%), and PHA titer (8.87 and 8.36 g/L), respectively. The findings collectively suggest that the development of a microbial co-culture strategy is a promising route for the efficient production of high-value bioplastics using different agricultural waste biomass.

Economic comparison between conventional aquaponics and FLOCponics systems

Biofloc-based fish production involves a dense microbial co-culture that supports nutrient cycling, generally reducing the amount of feed utilized and the need for mechanic and biological filters. These technical benefits of biofloc technology can lower the costs compared to traditional intensive techniques such as recirculating aquaculture systems (RAS). The RAS is conventionally adopted to integrated fish-plant production in aquaponics systems, and recent studies demonstrated the potential for integrating biofloc technology and hydroponics production in FLOCponics systems. The present study compared the economic viability for integrated agri-aquaculture production of tilapia juveniles and lettuce in FLOCponics (FP) and conventional aquaponics (AP) systems. The study was developed as an investment analysis for the commercial production of tilapia juveniles and lettuce in Brazil under four different scenarios. Two scenarios considered previous experimental results of marketable lettuce in both systems, i.e., 37% of total lettuce production would be marketable in FP (scenario 1) and 98% marketable lettuce in AP (scenario 2). The other two scenarios (using FP and AP systems) considered a hypothetical production in which all the lettuce produced have the marketable quality (scenario 3 and 4, respectively). The economic viability indicators calculated were: Internal Rate of Return (IRR); Net Present Value (NPV); PayBack (PB); Discounted PayBack (DPB); Benefit/Cost ratio (B/C) and Discounted Benefit/Cost ratio (DB/DC). The results showed that the production of Nile tilapia juveniles and lettuce in AP (scenario 2) is profitable, but the 37% marketable lettuce from FP (scenario 1) is not. Both hypothetical scenarios 3 and 4 were identified as profitable. A break-even point was calculated in scenario 1 (FP) to assess the quantities of plants that should be visually suitable for marketing to make such an investment project economically profitable. It was estimated that 68.7% of the lettuces produced should have desirable visual marketable characteristics to make FP feasible. The results show that investing in research to evaluate a multi-cycle production to prove the stability of long-term FLOCponics projects is highly recommended since the financial analysis indicates that FLOCponics could be financially feasible.

Rare and localized events stabilize microbial community composition and patterns of spatial self-organization in a fluctuating environment

Spatial self-organization is a hallmark of surface-associated microbial communities that is governed by local environmental conditions and further modified by interspecific interactions. Here, we hypothesize that spatial patterns of microbial cell-types can stabilize the composition of cross-feeding microbial communities under fluctuating environmental conditions. We tested this hypothesis by studying the growth and spatial self-organization of microbial co-cultures consisting of two metabolically interacting strains of the bacterium Pseudomonas stutzeri. We inoculated the co-cultures onto agar surfaces and allowed them to expand (i.e. range expansion) while fluctuating environmental conditions that alter the dependency between the two strains. We alternated between anoxic conditions that induce a mutualistic interaction and oxic conditions that induce a competitive interaction. We observed co-occurrence of both strains in rare and highly localized clusters (referred to as “spatial jackpot events”) that persist during environmental fluctuations. To resolve the underlying mechanisms for the emergence of spatial jackpot events, we used a mechanistic agent-based mathematical model that resolves growth and dispersal at the scale relevant to individual cells. While co-culture composition varied with the strength of the mutualistic interaction and across environmental fluctuations, the model provides insights into the formation of spatially resolved substrate landscapes with localized niches that support the co-occurrence of the two strains and secure co-culture function. This study highlights that in addition to spatial patterns that emerge in response to environmental fluctuations, localized spatial jackpot events ensure persistence of strains across dynamic conditions.

Cyclohumulanoid Sesquiterpenes Induced by the Noncompetitive Coculture of Phellinus orientoasiaticus and Xylodon flaviporus.

Microbial cocultivation has been applied as a strategy to induce the biosynthesis of specialized metabolites. However, most previous studies have focused on competitive interactions between test strains. During our LC-MS-based chemical screening of randomized cocultures of Basidiomycetous fungi, we discovered that the coculture of Phellinus orientoasiaticus (Hymenochaetaceae) and Xylodon flaviporus (Schizoporaceae) induces multiple metabolites, although they did not show any competitive morphology. Targeted isolation yielded three new sesquiterpenes (1-3) along with five known analogues (4-8). The structures of the isolates were determined by MS and NMR experiments as well as electronic circular dichroism analysis. LC-MS analysis suggested that cyclohumulanoids of illudane-, sterpurane-, and tremulane-type scaffolds (1-7) were produced by P. orientoasiaticus, whereas a drimane-type sesquiterpene (8) was produced by X. flaviporus. None of the isolates exhibited antifungal activity or cytotoxicity, and compounds 1-7 exhibited NO production of LPS-treated RAW276.4 cells in a range of 15.9% to 38.0% at 100 μM.

Induction of Isochromanones by Co-Cultivation of the Marine Fungus Cosmospora sp. and the Phytopathogen Magnaporthe oryzae.

Microbial co-cultivation is a promising approach for the activation of biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. As part of our project aiming at the discovery of marine-derived fungal agrochemicals, we previously used four phytopathogens as model competitors in the co-cultivation of 21 marine fungal strains. Based on comparative untargeted metabolomics analyses and anti-phytopathogenic activities of the co-cultures, we selected the co-culture of marine Cosmospora sp. with the phytopathogen Magnaporthe oryzae for in-depth chemical studies. UPLC-MS/MS-based molecular networking (MN) of the co-culture extract revealed an enhanced diversity of compounds in several molecular families, including isochromanones, specifically induced in the co-culture. Large scale co-cultivation of Cosmospora sp. and M. oryzae resulted in the isolation of five isochromanones from the whole co-culture extract, namely the known soudanones A, E, D (1-3) and their two new derivatives, soudanones H-I (4-5), the known isochromans, pseudoanguillosporins A and B (6, 7), naphtho-γ-pyrones, cephalochromin and ustilaginoidin G (8, 9), and ergosterol (10). Their structures were established by NMR, HR-ESIMS, FT-IR, electronic circular dichroism (ECD) spectroscopy, polarimetry ([α]D), and Mosher’s ester reaction. Bioactivity assays revealed antimicrobial activity of compounds 2 and 3 against the phytopathogens M. oryzae and Phytophthora infestans, while pseudoanguillosporin A (6) showed the broadest and strongest anti-phytopathogenic activity against Pseudomonas syringae, Xanthomonas campestris, M. oryzae and P. infestans. This is the first study assessing the anti-phytopathogenic activities of soudanones.

Co-elicitation of lignocelluloytic enzymatic activities and metabolites production in an Aspergillus-Streptomyces co-culture during lignocellulose fractionation.

Lignocellulose, the most abundant biomass on Earth, is a complex recalcitrant material mainly composed of three fractions: cellulose, hemicelluloses and lignins. In nature, lignocellulose is efficiently degraded for carbon recycling. Lignocellulose degradation involves numerous microorganisms and their secreted enzymes that act in synergy. Even they are efficient, the natural processes for lignocellulose degradation are slow (weeks to months). In this study, the objective was to study the synergism of some microorganisms to achieve efficient and rapid lignocellulose degradation. Wheat bran, an abundant co-product from milling industry, was selected as lignocellulosic biomass. Mono-cultures and co-cultures involving one A.niger strain fungi never sequenced before (DSM 1957) and either one of three different Streptomyces strains were tested in order to investigate the potentiality for efficient lignocellulose degradability. Comparative genomics of the strain Aspergillus niger DSM 1957 revealed that it harboured the maximum of AA, CBM, CE and GH among its closest relative strains. The different co-cultures set-up enriched the metabolic diversity and the lignocellulolytic CAZyme content. Depending on the co-cultures, an over-expression of some enzymatic activities (xylanase, glucosidase, arabinosidase) was observed in the co-cultures compared to the mono-cultures suggesting a specific microbial cross-talk depending on the microbial partner. Moreover, metabolomics for each mono and co-culture was performed and revealed an elicitation of the production of secondary metabolites and the activation of silent biosynthetic cluster genes depending on the microbial co-culture. This opens opportunities for the bioproduction of molecules of interest from wheat bran.

Unraveling the Association between Metabolic Changes in Inter-Genus and Intra-Genus Bacteria to Mitigate Clubroot Disease of Chinese Cabbage

Clubroot disease caused by the obligate parasite Plasmodiophora brassicae is a serious threat to cabbage production worldwide. Current clubroot control primarily relies on a fungicide, but this has a negative impact on the environment and the use of a single biocontrol agent cannot efficiently control the disease. Thus, the combined application of different biocontrol agents has been proposed as a promising alternative. In this study, we used bacterial biocontrol agents as a co-culture (inter-genus and intra-genus) and mono-culture to mitigate the clubroot disease of Chinese cabbage. We evaluated their biocontrol effect and plant growth promoter (PGP) traits in in vitro and in vivo experiments. This study revealed that the inter-genus bacterial co-culture significantly suppresses the incidence of clubroot disease and enhances plant growth compared with intra-genus and mono-culture. In pairwise interaction, we observed that Bacillus cereus BT-23 promotes the growth of Lysobacter antibioticus 13-6 (inter-genus bacterial co-culture), whereas L. capsici ZST1-2 and L. antibioticus 13-6 (intra-genus microbial co-culture) are antagonists to each other. Furthermore, a total of 5575 metabolites, 732 differentially expressed metabolites (DEMs), and 510 unique metabolites were detected through the LC-MS/MS technique in the bacterial co-culture. The number of unique metabolites in inter-genus bacterial co-culture (393 metabolites) was significantly higher than in the intra-genus bacterial co-culture (117 metabolites). Further analysis of DEMs showed that the DEMs were mainly involved in four kinds of metabolism pathways, i.e., carbohydrate metabolism, amino metabolism, nucleotide metabolism, and metabolism of cofactors and vitamins. The contents of some secondary metabolites with biocontrol activity and plant growth-promoting functions were increased in inter-genus bacterial co-culture, indicating that inter-genus bacterial co-culture has a solid potential to suppress clubroot disease. We conclude that the inter-genus bacterial interaction changes the community metabolism and improves several secondary metabolites functions with respect to disease control and PGP ability.

Efficient lactic acid production from dilute acid-pretreated lignocellulosic biomass by a synthetic consortium of engineered Pseudomonas putida and Bacillus coagulans

BackgroundLignocellulosic biomass is an attractive and sustainable alternative to petroleum-based feedstock for the production of a range of biochemicals, and pretreatment is generally regarded as indispensable for its biorefinery. However, various inhibitors that severely hinder the growth and fermentation of microorganisms are inevitably produced during the pretreatment of lignocellulose. Presently, there are few reports on a single microorganism that can detoxify or tolerate toxic mixtures of pretreated lignocellulose hydrolysate while effectively transforming sugar components into valuable compounds. Alternatively, microbial coculture provides a simpler and more efficacious way to realize this goal by distributing metabolic functions among different specialized strains.ResultsIn this study, a novel synthetic microbial consortium, which is composed of a responsible for detoxification bacterium engineered Pseudomonas putida KT2440 and a lactic acid production specialist Bacillus coagulans NL01, was developed to directly produce lactic acid from highly toxic lignocellulosic hydrolysate. The engineered P. putida with deletion of the sugar metabolism pathway was unable to consume the major fermentable sugars of lignocellulosic hydrolysate but exhibited great tolerance to 10 g/L sodium acetate, 5 g/L levulinic acid, 10 mM furfural and HMF as well as 2 g/L monophenol compound. In addition, the engineered strain rapidly removed diverse inhibitors of real hydrolysate. The degradation rate of organic acids (acetate, levulinic acid) and the conversion rate of furan aldehyde were both 100%, and the removal rate of most monoaromatic compounds remained at approximately 90%. With detoxification using engineered P. putida for 24 h, the 30% (v/v) hydrolysate was fermented to 35.8 g/L lactic acid by B. coagulans with a lactic acid yield of 0.8 g/g total sugars. Compared with that of the single culture of B. coagulans without lactic acid production, the fermentation performance of microbial coculture was significantly improved.ConclusionsThe microbial coculture system constructed in this study demonstrated the strong potential of the process for the biosynthesis of valuable products from lignocellulosic hydrolysates containing high concentrations of complex inhibitors by specifically recruiting consortia of robust microorganisms with desirable characteristics and also provided a feasible and attractive method for the bioconversion of lignocellulosic biomass to other value-added biochemicals.

Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture

BackgroundQuantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design.ResultsWe present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail.ConclusionsThe method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Comprehensive evaluation for the one-pot biosynthesis of butyl acetate by using microbial mono- and co-cultures

BackgroundButyl acetate has shown wide applications in food, cosmetics, medicine, and biofuel sectors. These short-chain fatty acid esters can be produced by either chemical or biological synthetic process with corresponding alcohols and acids. Currently, biosynthesis of short chain fatty acid esters, such as butyl butyrate, through microbial fermentation systems has been achieved; however, few studies regarding biosynthesis of butyl acetate were reported.ResultsIn this study, three proof-of-principle strategies for the one-pot butyl acetate production from glucose through microbial fermentation were designed and evaluated. (1) 7.3 g/L of butyl acetate was synthesized by butanol-producing Clostridium acetobutylicum NJ4 with the supplementation of exogenous acetic acid; (2) With the addition of butanol, 5.76 g/L of butyl acetate can be synthesized by acetate-producing Actinobacillus succinogenes130z (ΔpflA); (3) Microbial co-culture of C. acetobutylicum NJ4 and A. succinogenes130z (ΔpflA) can directly produce 2.2 g/L of butyl acetate from glucose by using microbial co-culture system with the elimination of precursors. Through the further immobilization of A. succinogenes130z (ΔpflA), butyl acetate production was improved to 2.86 g/L.ConclusionDifferent microbial mono- and co-culture systems for butyl acetate biosynthesis were successfully constructed. These strategies may be extended to the biosynthesis of a wide range of esters, especially to some longer chain ones.

Resource recovery of lignocellulosic biomass waste into lactic acid - Trends to sustain cleaner production

Biomass waste generation concerns regulatory authorities to develop novel methods to sustain biotransformation processes. Particularly, lactic acid (LA) is a bulk commodity chemical used in diverse industries and holds a growing global market demand. Recently, lignocellulosic waste biomass is preferred for LA bio-production because of its non-edible and inexpensive nature. However, the information about new pretreatment methods for lignocellulosic feedstock, and novel strains capable to produce LA through fermentation is limited. Therefore, this review highlights the advancement of pretreatments methods of lignocellulosic biomass and biotransformation. Herein, we first briefly explored the main sources of lignocellulosic waste biomass, then we explored their latest advances in pretreatment processes particularly supercritical fluid extraction, and microwave-assisted extraction. Approaches for bioconversion were also analyzed, such as consolidated bioprocessing (CBP), simultaneous saccharification and fermentation (SSF), separate hydrolysis fermentation (SHF), among other alternatives. Also, new trends and approaches were documented, such as metagenomics to find novel strains of microorganisms and the use of recombinant strategies for the creation of new strains. Finally, we developed a holistic and sustainable perspective based on novel microbial ecology tools such as next-gen sequencing, bioinformatics, and metagenomics. All these shed light on the needs to culture powerful microbial isolates, co-cultures, and mixed consortia to improve fermentation processes with the aim of optimizing cultures and feedstock pretreatments.

"Microbial Wars" in a Stirred Tank Bioreactor: Investigating the Co-Cultures of Streptomyces rimosus and Aspergillus terreus, Filamentous Microorganisms Equipped With a Rich Arsenal of Secondary Metabolites.

Microbial co-cultivation is an approach frequently used for the induction of secondary metabolic pathways and the discovery of novel molecules. The studies of this kind are typically focused on the chemical and ecological aspects of inter-species interactions rather than on the bioprocess characterization. In the present work, the co-cultivation of two textbook producers of secondary metabolites, namely Aspergillus terreus (a filamentous fungus used for the manufacturing of lovastatin, a cholesterol-lowering drug) and Streptomyces rimosus (an actinobacterial producer of an antibiotic oxytetracycline) in a 5.5-L stirred tank bioreactor was investigated in the context of metabolic production, utilization of carbon substrates and dissolved oxygen levels. The cultivation runs differed in terms of the applied co-culture initiation strategy and the composition of growth medium. All the experiments were performed in three bioreactors running in parallel (corresponding to a co-culture and two respective monoculture controls). The analysis based upon mass spectrometry and liquid chromatography revealed a broad spectrum of more than 40 secondary metabolites, including the molecules identified as the oxidized derivatives of rimocidin and milbemycin that were observed solely under the conditions of co-cultivation. S. rimosus showed a tendency to dominate over A. terreus, except for the runs where S. rimosus was inoculated into the already developed bioreactor cultures of A. terreus. Despite being dominated, the less aggressive strain still had an observable influence on the production of secondary metabolites and the utilization of substrates in co-culture. The monitoring of dissolved oxygen levels was evaluated as a fast approach of identifying the dominant microorganism during the co-cultivation process.

Peculiar Response in the Co-Culture Fermentation of Leuconostoc mesenteroides and Lactobacillus plantarum for the Production of ABE Solvents

Two bacterial strains (CL11A and CL11D) that are capable of ABE fermentation, identified as Leuconostoc mesenteroides and Weissella cibari, were isolated from the soil surrounding the roots of bean plants. Another strain (ZM 3A), identified as Lactobacillus plantarum, which is capable of purely ethanolic fermentation was isolated from sugarcane. Glucose was used as a standard substrate to investigate the performance of these strains in mono—and co-culture fermentation for ABE production. The performance parameters employed in this study were substrate degradation rates, product and metabolite yields, pH changes and microbial growth rates. Both ABE isolates were capable of producing the three solvents but Leuconostoc mesenteroides had a higher specificity for ethanol than Weissella cibari. The co-culturing of Leuconostoc mesenteroides and Lactobacillus plantarum enhanced ethanol production at the expense of both acetone and butanol, and also influenced the final substrate consumption rate and product yield. The experiments indicated the potential of these niche environments for the isolation of ABE-producing microorganisms. This study contributes to the formulation of ideal microbial co-culture and consortia fermentation, which seeks to maximize the yield and production rates of favored products.

New trends in bioprocesses for lignocellulosic biomass and CO2 utilization

Innovation and technology are seen today as key points in the transition to a greener and more sustainable economy. In this sense, the development of new processes able to result in reduced emissions and levels of CO2 in the atmosphere has been considered essential to support this transition and, at the same time, promote economic growth. Several techno-economic and life cycle assessment studies have shown promising data when sugars derived from lignocellulosic biomass are used as carbon source for fermentation processes. The use of CO2 as carbon source for fermentation is another smart concept for reusing this molecule while minimizing its emissions to the atmosphere. However, the large-scale implementation of biomass-based and CO2-based processes still require significant research efforts to result in robust and cost-competitive technologies. This paper discusses some promising approaches able to advance this research area including potential strategies for process intensification (enzymatic hydrolysis using high solid loading, simultaneous saccharification and fermentation, fermentation with downstream process integration), robust microbial strains for application in biomass-based and CO2-based bioprocesses, microbial co-cultivation systems, greener technologies for lignocellulosic biomass fractionation, among others. A critical evaluation of sustainability aspects including techno-economic and life cycle assessment is also provided. Overall, this study contributes with information on innovative trends able to advance the development of greener bioprocesses.

A Multi–Membrane System to Study the Effects of Physical and Metabolic Interactions in Microbial Co-Cultures and Consortia

Continuous cell-to-cell contact between different species is a general feature of all natural environments. However, almost all research is conducted on single-species cultures, reflecting a biotechnological bias and problems associated with the complexities of reproducibly growing and controlling multispecies systems. Consequently, biotic stress due to the presence of other species remains poorly understood. In this context, understanding the effects of physical contact between species when compared to metabolic contact alone is one of the first steps to unravelling the mechanisms that underpin microbial ecological interactions. The current technologies to study the effects of cell-to-cell contact present disadvantages, such as the inefficient or discontinuous exchange of metabolites when preventing contact between species. This paper presents and characterizes a novel bioreactor system that uses ceramic membranes to create a “multi-membrane” compartmentalized system whereby two or more species can be co-cultured without the mixing of the species, while ensuring the efficient sharing of all of the media components. The system operates continuously, thereby avoiding the discontinuities that characterize other systems, which either have to use hourly backwashes to clean their membranes, or have to change the direction of the flow between compartments. This study evaluates the movement of metabolites across the membrane in co-cultures of yeast, microalgae and bacterial species, and monitors the movement of the metabolites produced during co-culturing. These results show that the multi-membrane system proposed in this study represents an effective system for studying the effects of cell-to-cell contact in microbial consortia. The system can also be adapted for various biotechnological purposes, such as the production of metabolites when more than one species is required for such a process.

Utilizing a tyrosine exporter to facilitate 4-hydroxystyrene biosynthesis in an E. coli-E. coli co-culture

4-hydroxystyrene is an important commodity chemical with recognized industrial values. In this study, 4-hydroxystyrene biosynthesis was achieved using engineered E. coli. An amino acid exporter protein PhpCAT was utilized in combination with a previously developed biosensor-assisted cell selection mechanism to strengthen the biosynthesis of the pathway intermediate tyrosine. When incorporated into an E. coli-E. coli co-culture, the exporter also improved tyrosine’s mass transfer between the co-culture members to facilitate the streamlined 4-hydroxystyrene bioproduction. It was shown that the resulting co-culture produced 298 mg/L 4-hydroxystyrene from 5 g/L glucose, which is 96% higher than the control co-culture without the exporter. The achieved yield of 0.06 g/g glucose, to our knowledge, is the highest among previous studies on microbial biosynthesis. Our finding demonstrates that the utilization of the exporter protein for a pathway intermediate is an effective methodology for enhancing biosynthesis performance of microbial co-cultures.