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The significant influx of antibiotics into the environment represents ecological risks and threatens human health. Microbial degradation stands as a highly effective method for reducing antibiotic pollution. This study explored the potential of immobilized microbial consortia to efficiently degrade tetracycline. Concurrently, the suitability of different immobilization materials were assessed, with reed charcoal-immobilized consortia exhibiting the highest efficiency in removing tetracycline (92%). Similarly, wheat-bran-loaded bacterial consortia displayed a remarkable 11.43-fold increase in tetracycline removal compared with free consortia. Moreover, adding the carriers increased the nutrients, while the activities of both intracellular and extracellular catalases increased significantly post-immobilization, thus highlighting this enzyme's crucial role in tetracycline degradation. Finally, analysis of the microbial communities revealed the prevalence of Achromobacter and Parapedobacter, signifying their potential as key degraders. Overall, the immobilized consortia not only hold promise for application in the bioremediation of tetracycline-contaminated environment but also provide theoretical underpinnings for environmental remediation by microorganisms.

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Bacterioruberin is widely used in medicine, food, and cosmetics owing to its prominent characteristics of antioxidants and bioactivities. Bioconversion of methane into bacterioruberin is a promising way to address biomanufacturing substrate costs and greenhouse gas emissions but has not been achieved yet. Herein, this study aimed to upcycle methane to bacterioruberin by microbial consortia. The microbial consortia consist of Methylomonas and Methylophilus capable of synthesizing carotenoids from methane was firstly enriched from paddy soil. Through this microbial community, methane was successfully converted into C50 bacterioruberin for the first time. The bioconversion process was then optimized by the response surface methodology. Finally, the methane-derived bacterioruberin reached a record yield of 280.88 ± 2.94 µg/g dry cell weight. This study presents a cost-effective and eco-friendly approach for producing long-chain carotenoids from methane, offering a significant advancement in the direct conversion of greenhouse gases into value-added products.

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The field of Engineered Living Materials (ELMs) integrates engineered living organisms into natural biomaterials to achieve diverse objectives. Multiorganism consortia, prevalent in both naturally occurring and synthetic microbial cultures, exhibit complex functionalities and interrelationships, extending the scope of what can be achieved with individual engineered bacterial strains. However, the ELMs comprising microbial consortia are still in the developmental stage. In this Review, we introduce two strategies for designing ELMs constituted of microbial consortia: a top-down strategy, which involves characterizing microbial interactions and mimicking and reconstructing natural ecosystems, and a bottom-up strategy, which entails the rational design of synthetic consortia and their assembly with material substrates to achieve user-defined functions. Next, we summarize technologies from synthetic biology that facilitate the efficient engineering of microbial consortia for performing tasks more complex than those that can be done with single bacterial strains. Finally, we discuss essential challenges and future perspectives for microbial consortia-based ELMs.

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Microbial communities are immensely important due to their widespread presence and profound impact on various facets of life. Understanding these complex systems necessitates mathematical modeling, a powerful tool for simulating and predicting microbial community behavior. This review offers a critical analysis of metabolic modeling and highlights key areas that would greatly benefit from broader discussion and collaboration. Moreover, we explore the challenges and opportunities linked to the intricate nature of these communities, spanning data generation, modeling, and validation. We are confident that ongoing advancements in modeling techniques, such as machine learning, coupled with interdisciplinary collaborations, will unlock the full potential of microbial communities across diverse applications.

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Fermentation has been identified as an effective strategy to alter the chemical makeup of tobacco, thereby enhancing its quality. The deliberate introduction of microorganisms can hasten the fermentation process. In this research, microbial consortia harvested from the tobacco surface were utilized to enhance the tobacco quality. This enhancement also elevated several sensory attributes of HnB cigarettes, such as aroma richness, moisture, strength, and reduced irritation, achieving a sensory quality rating of 84.5. This marks a notable improvement compared to the 82 rating of the original, unfermented cigarettes. Untargeted metabolomics analysis revealed a decrease in total polyphenols and unsaturated fatty acids, while the levels of polyacids, alcohols, ketones, furans, and other compounds increased in the fermented tobacco. Additionally, KEGG pathway enrichment analysis indicated that the enhancement in tobacco quality through microbial consortia fermentation is linked to various biological pathways, with pathways related to fatty acid and amino acid degradation playing pivotal roles. The findings of this study will serve as a reference for the commercial production of HnB cigarettes, and the elucidated mechanism offers a theoretical basis for exploring microbial fermentation as a means to improve tobacco quality.

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The wide metabolic diversity of microalgae, their fast growth rates, and low-cost production make these organisms highly promising resources for a variety of biotechnological applications, addressing critical needs in industry, agriculture, and medicine. The use of microalgae in consortia with bacteria is proving valuable in several areas of biotechnology, including the treatment of various types of wastewater, the production of biofertilizers, and the extraction of various products from their biomass. The monoculture of the microalga Chlamydomonas has been a prominent research model for many years and has been extensively used in the study of photosynthesis, sulphur and phosphorus metabolism, nitrogen metabolism, respiration, and flagellar synthesis, among others. Recent research has increasingly recognised the potential of Chlamydomonas-bacteria consortia as a biotechnological tool for various applications. The detoxification of wastewater using Chlamydomonas and its bacterial consortia offers significant potential for sustainable reduction of contaminants, while facilitating resource recovery and the valorisation of microalgal biomass. The use of Chlamydomonas and its bacterial consortia as biofertilizers can offer several benefits, such as increasing crop yields, protecting crops, maintaining soil fertility and stability, contributing to CO2 mitigation, and contributing to sustainable agricultural practises. Chlamydomonas-bacterial consortia play an important role in the production of high-value products, particularly in the production of biofuels and the enhancement of H2 production. This review aims to provide a comprehensive understanding of the potential of Chlamydomonas monoculture and its bacterial consortia to identify current applications and to propose new research and development directions to maximise their potential.

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Introduction: The aim of the research was to demonstrate the efficiency of microorganisms and the effectiveness of biodegradation techniques on Low-density polyethylene (LDPE) plastics. The research question was: What is the efficiency of LDPE-degrading microorganisms and the effectiveness of biodegradation techniques? Methods: The systematic review was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Articles were obtained from Scopus, Web of Science (WOS), Embase, and Google Scholar. The DeCS/Mesh search terms were: Low-density polyethylene, efficiency, biodegradation, microbial consortia, fungi, bacteria. Inclusion criteria were: scientific articles that included bacteria, fungi, and microbial consortia reported as LDPE degraders that report the percentage of weight loss; articles published from January 2010 to October 2022, and publications in Spanish and English with open access. Exclusion criteria were: studies that do not report gravimetry, the biodegradation time of LDPE, and the genus or species of the polyethylene-degrading microorganism. Results: Out of 483 studies found, 50 were included in this Systematic Review (SR). The most frequent study techniques were scanning electron microscopy (SEM), gravimetry, and fourier transform infrared spectroscopy (FTIR), and in the case of microorganisms, the most studied belonged to the genus Pseudomonas, Bacillus, and Aspergillus. Regarding the isolation place, the most frequent mentioned in the reviewed articles were landfill soil and sanitary landfill soil. The efficiency of LDPE-degrading microorganisms was higher in bacteria such as Enterobacter spp., Pantoea spp., Pseudomonas spp., Escherichia coli, and Bacillus spp., which obtained a range of DE of 9.00-70.00%, 24.00-64%, 1.15 - 61.00%, 45.00%, and 1.5-40% with DT of 4-150, 120, 4-150, 30, and 30-120 days, respectively; in the case of fungi, the main microorganisms are Neopestalotiopsis phangngaensis, Colletotrichum fructicola, and Thyrostroma jaczewskii with efficiencies of 54.34, 48.78, and 46.34%, in 90 days, respectively; and the most efficient microbial consortia were from Enterobacter spp. and Pantoea sp. with 38.00 - 81.00%, in 120 days; and, Pseudomonas protegens, Stenotrophomonas sp., B. vallismortis and Paenibacillus sp. with 55. 00 - 75.00% in 120 days. Conclusions: The most efficient microorganisms in LDPE degradation are Enterobacter spp., Pantoea spp., Pseudomonas spp., Escherichia coli, and Bacillus spp.; in fungi Neopestalotiopsis phangngaensis, Colletotrichum fructicola, and Thyrostroma jaczewskii; and in microbial consortia, those formed by Enterobacter spp. and Pantoea sp., and that of P. protegens, Stenotrophomonas sp., B. vallismortis and Paenibacillus sp.; and the most effective techniques used in LDPE biodegradation are SEM, gravimetry, and FTIR.

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The microbial biotransformation of hexachlorocyclohexane (HCH) by novel anaerobic microbial consortia enriched from sediments of an industrial effluent channel and the river Ravi in Pakistan was examined. The anaerobic consortia were capable of biotransforming α-, ß-, γ-, and δ-HCH through reductive dichloroelimination, resulting in the formation of benzene and monochlorobenzene. Concerning γ-HCH biotransformation by the channel and river cultures, isotopic fractionations for carbon (εC) were - 5.3 ± 0.4 (‰) and - 10.6 ± 1.2 (‰), while isotopic fractionations for chlorine (εCl) were - 4.4 ± 0.4 (‰) and - 7.8 ± 0.9 (‰), respectively. Furthermore, lambda values (Λ), representing the correlation of δ13C and δ37Cl fractionation, were determined to be 1.1 ± 0.1 and 1.3 ± 0.1 for γ-HCH biotransformation, suggesting a reductive dichloroelimination as the initial step of HCH biotransformation in both cultures. Amplicon sequencing targeting the 16S rRNA genes revealed that Desulfomicrobium populations were considerably increased in both cultures, indicating their possible involvement in the degradation process. These findings suggest that Desulfomicrobium-like populations may have an important role in biotransformation of HCH and novel anaerobic HCH-degrading microbial consortia could be useful bioaugmentation agents for the bioremediation of HCH-contaminated sites in Pakistan.

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Growing environmental concerns and the need to adopt a circular economy have highlighted the importance of waste valorization for resource recovery. Microbial consortia-enabled biotechnologies have made significant developments in the biomanufacturing of valuable resources from waste biomass that serve as suitable alternatives to petrochemical-derived products. These microbial consortia-based processes are designed following a top-down or bottom-up engineering approach. The top-down approach is a classical method that uses environmental variables to selectively steer an existing microbial consortium to achieve a target function. While high-throughput sequencing has enabled microbial community characterization, the major challenge is to disentangle complex microbial interactions and manipulate the structure and function accordingly. The bottom-up approach uses prior knowledge of the metabolic pathway and possible interactions among consortium partners to design and engineer synthetic microbial consortia. This strategy offers some control over the composition and function of the consortium for targeted bioprocesses, but challenges remain in optimal assembly methods and long-term stability. In this review, we present the recent advancements, challenges, and opportunities for further improvement using top-down and bottom-up approaches for microbiome engineering. As the bottom-up approach is relatively a new concept for waste valorization, this review explores the assembly and design of synthetic microbial consortia, ecological engineering principles to optimize microbial consortia, and metabolic engineering approaches for efficient conversion. Integration of top-down and bottom-up approaches along with developments in metabolic modeling to predict and optimize consortia function are also highlighted. ONE-SENTENCE SUMMARY: This review highlights the microbial consortia-driven waste valorization for biomanufacturing through top-down and bottom-up design approaches and describes strategies, tools, and unexplored opportunities to optimize the design and stability of such consortia.

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Microplastics are emerging sources of environmental pollutants that are increasingly of concern because of their harmful impacts on aquatic life and thereby humans. Their accumulation in the environment is in direct proportion to global plastic production; their being nondegradable, recalcitrant and of a persistent nature creates an urgent need to address this issue on a global scale. Recent reports have demonstrated the presence of microplastics in marine life, and directly becoming a part of the food chain when seafood is ingested by humans. The repercussions of these studies point to an even larger scale presence of microplastics across varied habitats, which are yet to be sampled. Bioremediation, using various microorganisms such as bacteria, algae and fungi, alone or as consortia or in biofilm form can be used as an effective remediation tool. Genetically modified microorganisms for focused removal of microplastics and metagenomics studies, providing taxonomic details of uncultured organisms, are also expected to provide an additional catalogue of technologies in this field. This review offers a comprehensive overview of microplastic sources, existing technologies for treating microplastics and an in-depth analysis of bioremediation mechanisms, its components, and the results from various studies which provide sufficient clues as to the directions to be chosen to address microplastics pollution and can facilitate and instruct researchers to further investigate the more practical approaches and create new and innovative strategies for advanced remediation of microplastic in the future.

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BACKGROUND: Sponges (phylum Porifera) constantly interact with microbes. They graze on microbes from the water column by filter-feeding and they harbor symbiotic partners within their bodies. In experimental setups, sponges take up symbionts at lower rates compared with seawater microbes. This suggests that sponges have the capacity to differentiate between microbes and preferentially graze in non-symbiotic microbes, although the underlying mechanisms of discrimination are still poorly understood. Genomic studies showed that, compared to other animal groups, sponges present an extended repertoire of immune receptors, in particular NLRs, SRCRs, and GPCRs, and a handful of experiments showed that sponges regulate the expression of these receptors upon encounter with microbial elicitors. We hypothesize that sponges may rely on differential expression of their diverse repertoire of poriferan immune receptors to sense different microbial consortia while filter-feeding. To test this, we characterized the transcriptomic response of two sponge species, Aplysina aerophoba and Dysidea avara, upon incubation with microbial consortia extracted from A. aerophoba in comparison with incubation with seawater microbes. The sponges were sampled after 1 h, 3 h, and 5 h for RNA-Seq differential gene expression analysis. RESULTS: D. avara incubated with A. aerophoba-symbionts regulated the expression of genes related to immunity, ubiquitination, and signaling. Within the set of differentially-expressed immune genes we identified different families of Nucleotide Oligomerization Domain (NOD)-Like Receptors (NLRs). These results represent the first experimental evidence that different types of NLRs are involved in microbial discrimination in a sponge. In contrast, the transcriptomic response of A. aerophoba to its own symbionts involved comparatively fewer genes and lacked genes encoding for immune receptors. CONCLUSION: Our work suggests that: (i) the transcriptomic response of sponges upon microbial exposure may imply "fine-tuning" of baseline gene expression as a result of their interaction with microbes, (ii) the differential response of sponges to microbial encounters varied between the species, probably due to species-specific characteristics or related to host's traits, and (iii) immune receptors belonging to different families of NLR-like genes played a role in the differential response to microbes, whether symbionts or food bacteria. The regulation of these receptors in sponges provides further evidence of the potential role of NLRs in invertebrate host-microbe interactions. The study of sponge responses to microbes exemplifies how investigating different animal groups broadens our knowledge of the evolution of immune specificity and symbiosis.

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Fusarium wilt of banana (FWB) is the most limiting disease in this crop. The phytosanitary emergency caused by FWB since 2019 in Colombia has required the development of ecofriendly control methods. The aim of this study was to test the effectiveness of microbial-based biofungicides against FWB caused by Fusarium oxysporum f. sp. cubense race 1 (Foc R1) and correlate such effect with plant physiological parameters. Five Trichoderma (T1 to T4 and T9) and four Bacillus (T5 to T8)-based biofungicides were evaluated in pot experiments. In vitro, dual confrontation tests were also carried out to test whether the in vitro effects on Foc growth were consistent with the in vivo effects. While Trichoderma-based T3, T4, and T9, and Bacillus-based T8, significantly reduced the growth of Foc R1 in vitro, Trichoderma-based T1, T3, T4, and T9 temporarily reduced the Foc population in the soil. However, the incidence progress of FWB was significantly reduced by Bacterial-based T7 (74% efficacy) and Trichoderma-based T2 (50% efficacy). The molecular analysis showed that T7 prevented the inner tissue colonization by Foc R1 in 80% of inoculated plants. The T2, T4, T7, and T9 treatments mitigated the negative effects caused by Foc R1 on plant physiology and growth. Our data allowed us to identify three promising treatments to control FWB, reducing the progress of the disease, delaying the colonization of inner tissue, and mitigating physiological damages. Further studies should be addressed to determine the modes of action of the biocontrol agents against Foc and validate the utilization in the field.

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Lignin is an aromatic macromolecule and one of the main constituents of lignocellulosic materials. Kraft lignin is generated as a residual by-product of the lignocellulosic biomass industrial process, and it might be used as a feedstock to generate low molecular weight aromatic compounds. In this study, we seek to understand and explore the potential of ruminal bacteria in the degradation of kraft lignin. We established two consortia, KLY and KL, which demonstrated significant lignin-degrading capabilities. Both consortia reached maximum growth after two days, with KLY showing a higher growth and decolorization rate. Additionally, SEM analysis revealed morphological changes in the residual lignin from both consortia, indicating significant degradation. This was further supported by FTIR spectra, which showed new bands corresponding to the C-H vibrations of guaiacyl and syringyl units, suggesting structural transformations of the lignin. Taxonomic analysis showed enrichment of the microbial community with members of the Dickeya genus. Seven metabolic pathways related to lignin metabolism were predicted for the established consortia. Both consortia were capable of consuming aromatic compounds such as 4-hydroxybenzoic acid, syringaldehyde, acetovanillone, and syringic acid, highlighting their capacity to convert aromatic compounds into commercially valuable molecules presenting antifungal activity and used as food preservatives as 4-hydroxyphenylacetic, 3-phenylacetic, and phenylacetic acids. Therefore, the microbial consortia shown in the present work are models for understanding the process of lignin degradation and consumption in bacterial anaerobic communities and developing biological processes to add value to industrial processes based on lignocellulosic biomass as feedstock.

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Drought affects several plant physiological characteristics such as photosynthesis, carbon metabolism, and chlorophyll content, causing hormonal and nutritional imbalances and reducing nutrient uptake and transport, which inhibit growth and development. The use of bioinoculants based on plant growth-promoting microorganisms such as plant growth-promoting rhizobacteria (PGPR), yeasts, and arbuscular mycorrhizal fungi (AMF) has been proposed as an alternative to help plants tolerate drought. However, most studies have been based on the use of a single type of microorganism, while consortia studies have been scarcely performed. Therefore, the aim of this study was to evaluate different combinations of three PGPR, three AMF, and three yeasts with plant growth-promoting attributes to improve the biochemical, nutritional, and physiological behavior of strawberry plants growing under severe drought. The results showed that the growth and physiological attributes of the non-inoculated plants were significantly reduced by drought. In contrast, plants inoculated with the association of the fungus Claroideoglomus claroideum, the yeast Naganishia albida, and the rhizobacterium Burkholderia caledonica showed a stronger improvement in tolerance to drought. High biomass, relative water content, fruit number, photosynthetic rate, transpiration, stomatal conductance, quantum yield of photosystem II, N concentration, P concentration, K concentration, antioxidant activities, and chlorophyll contents were significantly improved in inoculated plants by up to 16.6%, 12.4%, 81.2%, 80%, 79.4%, 71.0%, 17.8%, 8.3%, 6.6%, 57.3%, 41%, and 22.5%, respectively, compared to stressed non-inoculated plants. Moreover, decreased malondialdehyde levels by up to 32% were registered. Our results demonstrate the feasibility of maximizing the effects of inoculation with beneficial rhizosphere microorganisms based on the prospect of more efficient combinations among different microbial groups, which is of interest to develop bioinoculants oriented to increase the growth of specific plant species in a global scenario of increasing drought stress.

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Increasing evidence shows that microbial synthesis plays an important role in producing high value-added products. However, microbial monoculture generally hampers metabolites production and limits scalability due to the increased metabolic burden on the host strain. In contrast, co-culture is a more flexible approach to improve the environmental adaptability and reduce the overall metabolic burden. The well-defined co-culturing microbial consortia can tap their metabolic potential to obtain yet-to-be discovered and pre-existing metabolites. This review focuses on the use of a co-culture strategy and its underlying mechanisms to enhance the production of products. Notably, the significance of comprehending the microbial interactions, diverse communication modes, genetic information, and modular co-culture involved in co-culture systems were highlighted. Furthermore, it addresses the current challenges and outlines potential future directions for microbial co-culture. This review provides better understanding the diversity and complexity of the interesting interaction and communication to advance the development of co-culture techniques.

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Metabolically engineered microbial consortia can contribute as a promising production platform for the supply of polyamide monomers. To date, the biosynthesis of long-chain α,ω-diamines from n-alkanes is challenging because of the inert nature of n-alkanes and the complexity of the overall synthesis pathway. We combined an engineered Yarrowia lipolytica module with Escherichia coli modules to obtain a mixed strain microbial consortium that could catalyze an efficient biotransformation of n-alkanes into corresponding α,ω-diamines. The engineered Y. lipolytica strain was constructed (YALI10) wherein the two genes responsible for ß-oxidation and the five genes responsible for the overoxidation of fatty aldehydes were deleted. This newly constructed YALI10 strain expressing transaminase (TA) could produce 0.2 mM 1,12-dodecanediamine (40.1 mg/L) from 10 mM n-dodecane. The microbial consortia comprising engineered Y. lipolytica strains for the oxidation of n-alkanes (OM) and an E. coli amination module (AM) expressing an aldehyde reductase (AHR) and transaminase (TA) improved the production of 1,12-diamine up to 1.95 mM (391 mg/L) from 10 mM n-dodecane. Finally, combining the E. coli reduction module (RM) expressing a carboxylic acid reductase (CAR) and an sfp phosphopantetheinyl transferase with OM and AM further improved the production of 1,12-diamine by catalyzing the reduction of undesired 1,12-diacids into 1,12-diols, which further undergo amination to give 1,12-diamine as the target product. This newly constructed mixed strain consortium comprising three modules in one pot gave 4.1 mM (41%; 816 mg/L) 1,12-diaminododecane from 10 mM n-dodecane. The whole-cell consortia reported herein present an elegant "greener" alternative for the biosynthesis of various α,ω-diamines (C8, C10, C12, and C14) from corresponding n-alkanes.

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The persistence of PAHs poses a significant challenge for conventional remediation approaches, necessitating the exploration of alternative, sustainable strategies for their mitigation. This review underscores the vital role of specialized microbial species (nitrogen-fixing, phosphate-solubilizing, and biosurfactant-producing bacteria) in tackling the environmental impact of polycyclic aromatic hydrocarbons (PAHs). These resistant compounds demand innovative remediation strategies. The study explores microbial metabolic capabilities for converting complex PAHs into less harmful byproducts, ensuring sustainable mitigation. Synthesizing literature from 2016 to 2023, it covers PAH characteristics, sources, and associated risks. Degradation mechanisms by bacteria and fungi, key species, and enzymatic processes are examined. Nitrogen-fixing and phosphate-solubilizing bacteria contributions in symbiotic relationships with plants are highlighted. Biosurfactant-producing bacteria enhance PAH solubility, expanding microbial accessibility for degradation. Cutting-edge trends in omics technologies, synthetic biology, genetic engineering, and nano-remediation offer promising avenues. Recommendations emphasize genetic regulation, field-scale studies, sustainability assessments, interdisciplinary collaboration, and knowledge dissemination. These insights pave the way for innovative, sustainable PAH-contaminated environment restoration.

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Metabolic burden is defined by the influence of genetic manipulation and environmental perturbations on the distribution of cellular resources. The rewiring of microbial metabolism for bio-based chemical production often leads to a metabolic burden, followed by adverse physiological effects, such as impaired cell growth and low product yields. Alleviating the burden imposed by undesirable metabolic changes has become an increasingly attractive approach for constructing robust microbial cell factories. In this review, we provide a brief overview of metabolic burden engineering, focusing specifically on recent developments and strategies for diminishing the burden while improving robustness and yield. A variety of examples are presented to showcase the promise of metabolic burden engineering in facilitating the design and construction of robust microbial cell factories. Finally, challenges and limitations encountered in metabolic burden engineering are discussed.

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Bradyrhizobia are the principal symbiotic partner of the leguminous plant and take active part in biological nitrogen-fixation. The present investigation explores the underlying competition among different strains during colonization in host roots. Six distinct GFP and RFP-tagged Bradyrhizobium strains were engineered to track them inside the peanut roots either independently or in combination. The Bradyrhizobium strains require different time-spans ranging from 4 to 21 days post-infection (dpi) for successful colonization which further varies in presence of another strain. While most of the individual strains enhanced the shoot and root dry weight, number of nodules, and nitrogen fixation capabilities of the host plants, no significant enhancement of plant growth and nodulation efficiency was observed when they were allowed to colonize in combinations. However, if among the combinations one strains is SEMIA 6144, the co-infection results in higher growth and nodulation efficiency of the hosts. From the competition experiments it has been found that Bradyrhizobium japonicum SEMIA 6144 was found to be the most dominant strain for effective nodulation in peanut. The extent of biofilm and exopolysaccharide (EPS) production by these isolates, individually or in combinations, were envisaged to correlate whether these parameters have any impact on the symbiotic association. But the extent of colonization, growth-promotion and nitrogen-fixation ability drastically lowered when a strain present together with other Bradyrhizobium strain. Therefore, it is imperative to understand the interaction between two co-inoculating Bradyrhizobium species for nodulation followed by plant growth promotion to develop suitable consortia for enhancing BNF in peanut and possibly for other legumes.

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Soil microbial flora constitutes a highly diverse and complex microbiome on Earth, often challenging to cultivation, with unclear metabolic mechanisms in situ. Here, we present a pioneering concept for the in situ construction of functional microbial consortia (FMCs) and introduce an innovative method for creating FMCs by utilizing phenanthrene as a model compound to elucidate their in situ biodegradation mechanisms. Our methodology involves single-cell identification, sorting, and culture of functional microorganisms, resulting in the formation of a precise in situ FMC. Through Raman-activated cell sorting-stable-isotope probing, we identified and isolated phenanthrene-degrading bacterial cells from Achromobacter sp. and Pseudomonas sp., achieving precise and controllable in situ consortia based on genome-guided cultivation. Our in situ FMC outperformed conventionally designed functional flora when tested in real soil, indicating its superior phenanthrene degradation capacity. We revealed that microorganisms with high degradation efficiency isolated through conventional methods may exhibit pollutant tolerance but lack actual degradation ability in natural environments. This finding highlights the potential to construct FMCs based on thorough elucidation of in situ functional degraders, thereby achieving sustained and efficient pollutant degradation. Single-cell sequencing linked degraders with their genes and metabolic pathways, providing insights regarding the construction of in situ FMCs. The consortium in situ comprising microorganisms with diverse phenanthrene metabolic pathways might offer distinct advantages for enhancing phenanthrene degradation efficiency, such as the division of labour and cooperation or communication among microbial species. Our approach underscores the importance of in situ, single-cell precision identification, isolation, and cultivation for comprehensive bacterial functional analysis and resource exploration, which can extend to investigate MFCs in archaea and fungi, clarifying FMC construction methods for element recycling and pollutant transformation in complex real-world ecosystems.

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