Biotechnological potential and applications of microbial consortia

Journal Pre-proof Biotechnological potential and applications of microbial consortia

Xiujuan Qian, Lin Chen, Yuan Sui, Chong Chen, Wenming Zhang, Jie Zhou, Weiliang Dong, Min Jiang, Fengxue Xin, Katrin Ochsenreither PII:

S0734-9750(19)30200-9

DOI:

https://doi.org/10.1016/j.biotechadv.2019.107500

Reference:

JBA 107500

To appear in:

Biotechnology Advances

Received date:

9 May 2019

Revised date:

13 November 2019

Accepted date:

17 December 2019

Please cite this article as: X. Qian, L. Chen, Y. Sui, et al., Biotechnological potential and applications of microbial consortia, Biotechnology Advances (2019), https://doi.org/ 10.1016/j.biotechadv.2019.107500

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© 2019 Published by Elsevier.

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Biotechnological potential and applications of microbial consortia

Xiujuan Qiana, Lin Chena, Yuan Suib, Chong Chenb, Wenming Zhanga,c, Jie Zhoua,c, Weiliang Donga,c, Min Jianga,c*, Fengxue Xina,c*, Katrin Ochsenreitherd

State Key Laboratory of Materials-Oriented Chemical Engineering,

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a

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College of Biotechnology and Pharmaceutical Engineering,

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Nanjing Tech University, Nanjing, China b

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM),

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c

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2011 College, Nanjing Tech University, Nanjing, China

Nanjing Tech University, Nanjing, China d

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Institute of Process Engineering in Life Sciences, Section II: Technical Biology,

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Karlsruhe Institute of Technology, Fritz-Haber-Weg 4, 76131 Karlsruhe, Germany

*Corresponding author: Fengxue Xin, E-mail: [email protected]. Min Jiang, E-mail: [email protected]. Mailing address: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Puzhu South Road 30#, Nanjing 211800, P. R. China. Tel/Fax: 0086-25-58139927

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Abstract Recent advances in microbial consortia present a valuable approach for expanding the scope of metabolic engineering. Systems biology enable thorough understanding of diverse physiological processes of cells and their interactions, which in turn offers insights into the optimal design of synthetic microbial consortia. Yet, the

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study of synthetic microbial consortia is still in early infancy, facing many unknowns

microbial consortia systems. In this review, we comprehensively

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controllable

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and challenges in intercellular communication and construction of stable and

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discussed the recent application of defined microbial consortia in the fields of human

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health monitoring and medicine exploitation, valuable compounds synthesis, consolidated bioprocessing of lignocellulosic materials and environmental

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bioremediation. Moreover, the outstanding challenges and future directions to

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advance the development of high-efficient, stable and controllable synthetic microbial

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consortia were highlighted.

Key words: Microbial consortia application, Human health supervision, Valuable compounds synthesis, Consolidated bioprocessing, Environment bio-remediation, Microbial consortia construction challenges Introduction Traditional metabolic engineering using axenic cultures has provided great

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opportunities for the production of bulk chemicals and valuable products (Hall and Howe, 2012). However, the inherent exclusiveness to foreign genes, the lack of novel enzymes, the existence of silent pathways and requirements for rigid cultivation conditions always affect bio-product titer and productivity (Bhatia et al. , 2018, Shong et al. , 2012). As a result, the number of successful examples of commercialized

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microbial processes to produce chemicals and fuels cannot fulfill expectations

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(Gustavsson and Lee, 2016), leaving the question of whether the traditional metabolic

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strategy, i.e. construction of “super cell factories” is sufficient for sustainable

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biorefineries.

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Nevertheless, traditional food fermentation processes, such as cheese and soy sauce production are typically carried out by using mixed cultures consisting of

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multiple strains or species (Hanemaaijer et al. , 2015). Moreover, more than 99% of

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microorganisms in the environment cannot be cultured successfully by using

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traditional cultivation technologies (Kaeberlein et al. , 2002). The powerful features of natural microbial consortia inspired researchers to extend synthetic biology from traditional programming of single cells to mixed culture systems construction. As expected, more and more studies related to the exploration of microbial consortia potential, distributing synthetic pathways to different microorganisms within consortia, cell-cell communication mechanism and development of statistical models for microbial consortia systems have been reported recently (Fig. 1) (An et al. , 2019, Roell et al. , 2019, Song et al. , 2014, Wang et al. , 2019b, Zhang and Wang, 2016). The construction of artificial microbial consortia has opened a new horizon in

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synthetic biology in terms of system complexity and functionality. First, these complex consortia create a novel microenvironment for strains, potentially resulting in the activation of silent metabolic pathways, which are not expressed under “normal” cultivation conditions, leading to discovery of novel chemicals, endowing microbial consortia a promising prospect especially in novel drugs development (Netzker et al. ,

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2018). In addition, microbiome behavior in the body is an important factor influence

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human health, as humans are co-evolving with trillions of microbes that inhabit in our

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bodies, which created complex, body-habitat-specific and adaptive ecosystems to

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adapt to the relentlessly changing host physiology (Turnbaugh et al. , 2007). Second,

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microbial consortia employ the approach of “division of labor”, allowing a burden distribution across the population and permitting improved efficiency and more

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complex behavior than monocultures (Hays et al. , 2015, Zhou et al. , 2015). It might

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be easier to optimize the modularized pathway by changing the ratio of constituent

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strains, while a series of molecular engineering with regard to promoters, ribosome binding sites, terminators, and vectors, etc. are needed when using monocultures (Roell et al. , 2019). Third, such microbial communities consist of multiple functional microorganisms, which are more robust towards environmental challenges and possess expanded metabolic capabilities relative to monocultures (McCarty and Ledesma-Amaro, 2018). In this review, recent applications using artificial microbial consortia in the area of human health supervision, valuable compounds synthesis, consolidated bioprocessing for lignocellulose biorefinery and environment bioremediation will be

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comprehensively summarized and discussed, with emphasis on the strategies in synthetic microbial consortia construction, aiming to exploit the promising potential of microbial consortia in more areas. Further, significant challenges and future directions to advance the development of artificial microbial consortia were highlighted.

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Promising trends and potential applications of microbial consortia for human

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health supervision

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The serendipitous discovery of penicillin using microbial consortia of Penicillium

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and Staphylococcus by Alexander Fleming in 1929 was regarded as one of the most

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influential scientific breakthroughs in last century (Bennett and Chung, 2001). Since then, more and more novel chemicals have been discovered especially with the

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development of biotechnology. Table 1 summarized novel chemicals synthesized

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using microbial consortia reported in the last decade. As seen, most of them exhibit

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antimicrobial properties and can only be found in microbial consortia systems. Due to the increasing number of pathogenic antibiotic-resistant strains, the need for research and development of new antibiotic substances is more relevant than ever (Tacconelli et al. , 2018). The unique characteristic of microbial consortia clearly highlights its potential in antibiotic chemicals synthesis. The growing number of available microbial genome sequences showed that many biosynthetic gene clusters, responsible for synthesis of secondary metabolites (SMs) are presumably silent under standard laboratory conditions (Marmann et al. , 2014), e.g. the silent glycopeptide cluster in Amycolatopsis (Spohn et al. , 2014). There is

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evidence that the activation of some silent gene clusters requires the physical presence of other microorganisms (Bertrand et al. , 2014). In order to discover the unrevealed interaction mechanisms behind microbial consortia, a series of investigations from the perspectives of physical contact, chemical communication and gene mutation have been conducted (Fig. 2).

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In terms of chemical communication, microbes can produce some signal

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compounds, such as N-acyl homoserine lactones (AHLs) and small peptides, to

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perform as transcriptional regulators and epigenetic modifiers in a process called

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“quorum sensing” (QS) (Joint et al. , 2002). AHLs are major class of autoinducer

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signal molecules used by gram-negative bacteria for intraspecies QS (Ng and Bassler, 2009). Once AHL concentration in the environment reaches a threshold level, they

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will activate the transcriptional regulator proteins of LuxR family. The LuxR/AHL

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complex can activate the expression of multiple target genes, including those required

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for AHL synthesis (Joint et al. , 2002) (Fig. 2a). Such QS mechanism has been successfully used to engineer spatio-temporally regulated cell-cell communication in Escherichia coli (Basu et al. , 2004), to control the population in microbial consortia by regulating cell growth and death (Balagaddé et al. , 2008, You et al. , 2004), and to reduce the substrate competition between different species (Wang et al. , 2019a). In gram-positive bacteria consortia, small peptides, known as autoinducing peptides (AIPs) are mostly used as QS molecules (Ng and Bassler, 2009, Thoendel and Horswill, 2010). Different from AHLs, these peptides vary in sequence and structure, which are actively exported from the cell by dedicated transporters (Michie et al. ,

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2016). AIPs regulated QS systems typically employ a two-component genetic regulatory mechanism—a membrane-bound AIP-receptor-histidine kinase (HK) and a DNA-binding response regulator (Fig. 2b). Once the critical concentration of AIP is reached in extracellular environment, it will be phosphorylated by the receptor-HK and imported into the cell. The phosphorylated AIP will bind on the target DNA to

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regulate its transcription (Michie et al. , 2016). Beyond the chemical communication

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in the same genus consortia, QS molecules also seem to influence

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prokaryote-eukaryote interactions. For example, orsellinic acid derivatives are usually

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found in a fungal/bacterial mutualism, implying the functional role of orsellinic acid

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in microbial communication (Brakhage and Schroeckh, 2011, Fischer et al. , 2018, Schroeckh et al. , 2009). Owing to these discoveries, it becomes easier to construct a

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communication system by linking the production of QS molecules with the

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corresponding receptors and promoters, ultimately, a controllable microbial

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consortium can be designed.

In addition to freely diffusible QS molecules mentioned in Fig. 2a and 2b, the transport of some signal molecules also needs to be assisted by a special structure. For example, hydrophobic signals such as long-chain AHLs are transported between cells by membrane vesicles (MVs) (Toyofuku, 2019) (Fig. 2c). In some cases, an intimate physical contact of the cell members within a consortium is required to elicit the specific communication. The classical case was the interaction between Aspergillus nidulans with actinomycetes, where archetypal polyketides could be only biosynthesized after physical contact of these two partners in the consortium

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(Schroeckh et al. , 2009) (Fig. 2d). Further gene level analysis indicated that microbial consortia cause a series of gene expression changes in the manner of gene loss, histone modification, and horizontal gene transfer (Fig. 2e). For example, co-culture of Streptomyces clavuligerus with Staphylococcus aureus N315 led to the loss of a large 1.8 Mbp

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plasmid, constituting 21% of the genome content, in S. clavuligerus. Consequently, S.

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clavuligerus acquired the ability to constitutively produce holomycin within this

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microbial consortia system (Charusanti et al. , 2012). Speculatively, the loss of gene

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segment in S. clavuligerus may reduce the metabolic burden during replication and

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gene expression. In return, the silent holomycin synthetic pathway was specifically activated, which is a member of the pyrrhotine class of antibiotics and shows great

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cytotoxicity levels against S. aureus (Oliva et al. , 2001). Bacterium could also alter

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fungal gene expression by inducing histone modification through the main histone

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acetyltransferase complex Saga/Ada (Nützmann et al. , 2011). In general, histone acetylation is linked to transcriptional activation so that regulate gene expression. Moreover, genomic analysis revealed that Rhodococcus 307CO harbors a large DNA segment derived from Streptomyces within the microbial consortia system, leading to the production of new isomeric antibiotics of rhodostreptomycin A and B (Kurosawa et al. , 2008). In fact, horizontal gene transfer between gram-positive and gram-negative bacteria was commonly found, which could be defined as predation and lead to the incorporation of prey DNA by predator (Pérez et al. , 2011). The inter-communication within microbial consortia is a very complex process.

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Currently, the most effective approach to explore the interaction mechanism is through characterizing small of reduced complexities, to detect the specific metabolic interactions or introduce reporter strains, then, to build a representative model library of microbial consortia. Correspondingly, more advanced perceiving and analytical technologies, such as metagenomics, systems biology imaging mass spectrometry,

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high-throughput cultivation should be developed.

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microfluidic technique and flow cytometry, cell isolation and printing, and

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In addition to the antibiotic chemicals discovery, microbial consortia also play

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promising roles in clinical research. Microorganisms that colonize the human body,

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including mucosal and skin environments are at least as abundant as our somatic cells and certainly contain far more genes than our human genome (Gilbert et al. , 2018,

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Lloyd-Price et al. , 2016). The body microbiome behavior is an important factor in

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addition to genetics and environment that influence human health (Turnbaugh et al. ,

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2007). For example, the gut microbiome is now emerging as an important player in personalized medicine, as it enables the breakdown of otherwise indigestible polysaccharides, and is essential for the development and homeostasis of the immune system in the gut to resist against pathogenic bacteria (Chen et al. , 2018). An enhanced understanding of the impact of microbial communities and better elaboration of the exact mechanism of such communities has also become a priority in human microbiome study. Valuable compounds synthesis using microbial consortia through the distribution of metabolic pathways within different strains

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Synthetic biology and metabolic engineering have made great strides in constructing and optimizing metabolic pathways in model microbes, such as E. coli and Saccharomyces cerevisiae for high value product synthesis. Usually, these products can only be synthesized through long or complex metabolic pathways. For example, 35 to 51 steps were required for taxol de novo synthesis from glucose.

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Modification of metabolic pathway in E. coli gave the highest taxadiene titer of 1.02

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g/L, which is the precursor of taxol (Ajikumar et al. , 2010). However, the process

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was still far from industrial manufacturing. In another study, a biosynthetic pathway

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containing 10 genes for the synthesis of dihydrosanguinarine and sanguinarine from

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(R, S)-norlaudanosoline was constructed in S. cerevisiae (Fossati et al. , 2014). This complex biosynthetic pathway only resulted in 1.5% conversion to

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dihydrosanguinarine. Generally, biosynthetic efficiency is significantly reduced when

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multiple genes are simultaneously introduced into a single microorganism, as this will

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cause overwhelming metabolic stresses to chassis. This universal challenge could be potentially overcome through rational design using microbial consortia, which could modularize and segregate one biosynthetic pathway into multiple separate cells. Therefore, each chassis strain can be engineered independently to achieve optimal function of the combined pathway (Zhang and Wang, 2016). Table 2 summarized the most recent progress in valuable compound production. Analyzing these successful attempts according to the types of microorganism recruited in microbial consortia systems, three groups of bacteria-bacteria, eukaryote-eukaryote and bacteria-eukaryote have been categorized, providing specific

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experience for more artificial microbial consortium construction.In bacteria-bacteria consortia systems, E. coli was the most commonly used chassis. Zhang et al. (2015b) designed a novel E. coli-E. coli co-culture for the production of cis, cis-muconic acid (MA) and 4-hydroxybenzoic acid (4HB), which both are important platform intermediates for value-added compounds, such as adipic acid, terephthalic acid,

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muconic acid and vanillyl alcohol (Sengupta et al. , 2015, Wang et al. , 2018b). In this

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study, a first E. coli was used for conversion of glucose into the intermediate

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3-dehydroshikimic acid (DHS), which was assimilated and subsequently converted to

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MA or 4HB by the second E. coli strain. To eliminate carbon source competition

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between these two strains, the phosphotransferase system in the first strain was removed, and the xylose isomerase gene xylA, which catalyzes the inter-conversion of

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D-xylose and D-xylulose, was deleted in the second strain. The resulting consortium

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could consume xylose and glucose simultaneously. By using this strategy, the

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limitations of high-level intermediate secretion and low-efficiency of sugar mixture utilization were successfully overcome. This principle has also been used for the production of cadaverine, the desired raw material of bio-polyamides, from a mixture of glucose and glycerol (Wang et al. , 2018a), and the production of glycosides from a mixture of glucose and xylose (Liu et al. , 2018a). Furthermore, through accommodating the upstream and downstream pathways into two independent E. coli strains, Zhang et al. constructed a microbial consortia system, in which glycerol was used as the sole carbon source to support the growth of both strains as well as MA production. The E. coli-E. coli co-culture can be used for complex biosynthesis

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pathway engineering in the context of sole carbon source, regardless of the growth competition between these two strains (Zhang et al. , 2015a). By assembling engineered 4-vinylphenol or 4-vinylcatechol producer modules with cyanidin-3-O-glucoside producer recombinant cell, Akdemir et al ( 2019) first reported the synthesis of important red wine pigments pyranoanthocyanins using E.

the traditional extraction method from plants. Moreover, synthetic E.coli

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stable than

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coli co-cultures. Using this approach, the pyranoanthocyanins production are more

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consortia systems have been designed for more and more valuable compounds

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production, including ester compounds like caffeoylmalic acid (Li et al. , 2018),

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terpene compounds like α-pinene (Niu et al. , 2018), polyphenol compounds like resveratrol (Camacho-Zaragoza et al. , 2016), resveratrol glucosides (Thuan et al. ,

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2018b), amino acid derivative like 3-amino-benzoic acid (3AB) (Zhang and

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Stephanopoulos, 2016), cadaverine (Sgobba et al. , 2018), flavonoids compounds like

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apigetrin (Thuan et al. , 2018a), glycoside compounds like salicylate 2-O-b-D-glucoside (Ahmadi et al. , 2016), and monolignols (Chen et al. , 2017) etc. In addition to E. coli, the engineered co-culture of Gluconobacter oxydans and Ketogulonicigenium vulgare was also developed for one-step production of 2-keto-L-gulonic acid (2-KGA), which is the precursor of vitamin C (Wang et al. , 2016). Compared with bacteria-bacteria consortia systems, the attempts of eukaryote-eukaryote were seldom reported. A representative case is the biosynthesis modules of monacolin J and its derivative lovastatin, a very important natural product

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used in anti-hypercholesterolemia pharmaceuticals (Campbell and Vederas, 2010), can be assembled into two Pichia pastoris strains. The microbial consortia cangenerate 250.8 mg/L of lovastatin and 593.9 mg/L of monacolin J from methanol (Liu et al. , 2018b). Compared to the monoculture, the biosynthesis capability was improved by 2.2-fold for lovastatin and 13.4% for monacolin J.

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Not only limited to the cooperation between prokaryotes, cross-species consortia

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between bacteria and eukaryotes have also been developed for biosynthesis of

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complex valuable products. For example, Rodríguez-Bustamante et al. (2005) isolated

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a microbial consortium consisting of the yeast Trichosporon asahii and the bacterium

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Paenibacillus amylolyticus, where T. asahii was responsible for cleaving lutein to β-ionone, and P. amylolyticus reduced β-ionone into 7,8-dihydro-β-ionone and

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7,8-dihydro-β-ionol derivatives, which are the compounds present in tobacco aroma

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note. In another cross-species co-culture of engineered E. coli and S. cerevisiae, 2

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mg/L of oxygenate taxane (potent chemotherapy agent) was produced by using glucose as the sole carbon source, although neither of them can produce the paclitaxel precursor (Zhou et al. , 2015). In this process, the engineered E. coli strain was responsible for upstream de novo biosynthesis of taxadiene, which was then converted to oxygenated taxanes by S. cerevisiae, in which a cytochrome P450 module was efficiently expressed. However, ethanol produced by S. cerevisiae significantly inhibited the growth of E. coli and the production of taxanes. Hence, to better control the population allocation of these two strains, a mutualistic carbon-source method was employed, where xylose was consumed and converted to acetate by E. coli, and then

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acetate was further metabolized to oxygenated taxanes by S. cerevisiae. After the genetic modification, a high level of oxygenated taxanes production of 33 mg/L was finally obtained. Recently, Zhang et al. (2017) constructed a cross-culture between E. coli and S. cerevisiae for improving the efficiency of naringenin production, which is a value-added natural product and widely dispersed in the citrus plant of rutaceae

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family. Specifically, the endogenous tyrosine pathway was introduced into E. coli for

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high level production of tyrosine, which was subsequently converted into naringenin

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by a downstream engineered S. cerevisiae. As a result, 21.16 mg/L of naringenin was

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finally produced from xylose, showing an 8-fold increased titer compared to that of

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yeast monoculture. Such systems have also been designed to exchange tyrosine and arginine between engineered yeasts.

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Beyond the interaction of two microorganisms, poly-culture consortia containing

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three or more strains were also successfully exploited for constructing a long tandem

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or parallel synthetic pathway . For example, an engineered polyculture containing E.

coli, Bacillus subtilis and Shewanella oneidensis has been designed for microbial power generation (Liu et al. , 2017). In this process, E. coli firstly digested glucose to produce lactate, which was used as carbon source and electron donor. Subsequently, lactate was converted to riboflavin by B. subtilis as an electron shuttle. Finally, S. oneidensis acted as the exoelectrogen to generate electricity. In return, S. oneidensis oxidized lactate to produce acetate, which served as carbon source for E. coli and B. subtilis. Consequently, all three species formed a cross-feeding microbial consortium, which performed “better together” for energy generation. Under optimized condition,

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11 mM of glucose was converted to stable electricity output of ~550 mV for more than 15 days. The potential of modular co-culture engineering can address the challenges associated with complex and non-linear biosynthetic pathways. By employing mixed carbon substrate utilization to minimize upstream carbon flux competition, an efficient microbial consortia system of three E.coli strains was

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constructed for non-linear rosmarinic acid synthesis, which exhibited 38-fold

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improvement in rosmarinic acid production compared to that of the mono-culture (Li

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et al. , 2019). Another polyculture example was the de novo synthesis of anthocyanin,

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an important health-promoting pigment (Jaakola, 2013). In this study, 15 enzymatic

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steps involved in the production of phenylpropanoic acids, flavanones, flavan-3-ols, and anthocyanins were divided into 4 independent E. coli strains respectively,

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realizing the first heterologous production of flavan-3-ols (Jones et al. , 2017). This

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study represents the most complex synthetic consortia to date, providing a new

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paradigm in metabolic engineering for reconstituting the extensive metabolic pathways in non-native hosts. Although remarkable progress in terms of valuable compounds synthesis using microbial consortia has been achieved, it remains a great challenge to realize industrial applications using such multispecies cultures . Except for the modular strain selection and compromised cultivation conditions optimization, which will be discussed in the future perspectives, more practical questions should be considered: ① As discussed in the first section, the physical contact of different microorganisms will cause the production of some secondary chemicals, which brings more

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difficulties in controlling the microorganisms population structure, and whether the induced secondary chemicals will affect the natural chemicals quality should be taken into account; ②In contrast to single cell factories, consortia

each species within microbial

will interact with each other through the interchange of substrates or

intermediates, and how to improve the efficiency of substrates or intermediates

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transfer between cells is the key to construct a highly efficient microbial consortium.

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In addition, the low concentration of some intermediate metabolites

will bring

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difficulties for partner microorganism to sense and capture them efficiently. Therefore,

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some versatile fermentation equipments should be designed to enhance the substrates

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or intermediates transfer within a microbial consortium system; ③ Microbial community systems are dynamic and it is difficult to achieve long-term production

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stability (Roell et al. , 2019). Thus, it is premature to judge the potential of a microbial

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consortium from a laboratory perspective. Therefore, more experiments in large scale

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and especially long-term cultivation processes should be conducted. As stability, controllability, safety and costs are key elements in assessment of an industrial production process, more attention should be paid to these aspects in later experiment design.

Consolidated bioprocessing for lignocellulose biorefinery using microbial consortia Lignocellulose is the most abundant renewable resource in the world, which is generally considered as a promising feedstock for the production of biofuels and

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bio-based chemicals. However, converting recalcitrant biomass into biochemicals is a complex process compromising many steps, which convergently accommodates the production of saccharolytic enzymes, hydrolysis of carbohydrate components to sugars, and fermentation of hexose and pentose sugars (Klein‐Marcuschamer et al. , 2012). Nevertheless, separating these tasks will cause inevitably high costs and long

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processing periods. Recently, consolidated bioprocessing (CBP), which combines the

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production of lignocellulose degrading enzymes, lignocellulose hydrolysis and

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microbial fermentation in one step has been considered as an economical and efficient

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approach to produce desired products from polysaccharides (Olson et al. , 2012, Xin

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et al. , 2017). Previous CBP reviews addressed fermentative production of chemicals with recombinant lignocellulosic microbes (Fig. 3a) (Jang et al. , 2012, Yang et al. ,

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2015, Zhang et al. , 2011), or expression of lignocellulose degrading enzymes in

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non-lignocellulosic microbes (Fig. 3b) (Edwards et al. , 2011, Favaro et al. , 2015,

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Hasunuma and Kondo, 2012). However, each strategy possesses a considerable challenge due to the assembly of differential models into a single synthetic chassis (Del Vecchio et al. , 2018). Moreover, it is difficult to find a suitable microorganism with the capability to produce all desired lignocellulose degrading enzymes (van Zyl et al. , 2013). Activities of heterologously produced lignocellulose degrading enzymes in current CBP microorganisms remained at only several hundred filter paper unit (FPU)/L (den Haan et al. , 2015, Guo et al. , 2018, Singh et al. , 2018), which were notably lower than that from native cellulolytic fungi, e.g. Trichoderma reesei, a well-known cellulolytic fungus, in which tens of thousands FPU/L can be achieved.

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A promising alternative is microbial consortia-based bioprocessing, which consists of multiple microorganisms with complementary metabolic functions, so that difficult tasks can be divided across a diverse subset of microorganisms. Especially, co-cultivating of lignocellulose degrading strains with chemical producing microbes exhibits superior advantages (Fig 3c). When a stable microbial consortium of

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wild-type T. reesei and wild-type Lactobacilli sp. were co-cultivated, 19.8 g/L of

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lactic acid corresponding to 85.2% of the theoretical maximum yield was obtained

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from non-detoxified steam-pretreated beech wood (Shahab et al. , 2018). Buzzini et al.

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designed a microbial co-culture of wild-type Debaryomyces castellii and wild-type

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Rhodotorula glutinis for the production of carotenoids from oligosaccharides and dextrins from corn syrup. D. castellii was applied to hydrolyze the raw substrate to

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maltose and glucose, which was subsequently converted to carotenoids by R. glutinis.

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Finally, 8.2 mg/L of carotenoid was produced from corn syrup in the fed-batch

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co-culture system (Buzzini, 2001). Through using a symbiotic consortia of the engineered S. cerevisiae and the cellulolytic bacterium Actinotalea fermentans, methyl halide production was achieved from unprocessed biomass, such as switchgrass, corn stover, sugar cane bagasse and poplar (Bayer et al. , 2009). Moreover, Sgobba et al. (2018) established an E. coli-Corynebacterium glutamicum mutualism consortium, which realized the production of L-lysine and its derivatives directly from starch. In detail, α-amylase from S. griseus was heterologously introduced into starch-negative E. coli, allowing it to hydrolyze starch as carbon source. The generated glucose from starch by E. coli was then used to feed C.

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glutamicum. In return, C. glutamicum provided lysine for lysine auxotrophic E. coli to facilitate its growth. The mutualism of each species guaranteed the stable cooperation of this microbial consortia system. Particularly, the major contribution of microbial consortia CBP was applied for bioenergy production, such as bioethanol, biobutanol, microbial lipid and H2 (Table 3).

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For bioethanol production, Patle and Lal demonstrated that a microbial community of

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Zymomonas mobilis and Candida tropicalis could transform enzymatically

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hydrolyzed lignocellulosic biomass into ethanol with a high yield of 97.7% (Patle and

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Lal, 2007). A polyculture of T. reesei, S. cerevisiae and Scheffersomyces stipitis could

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achieve cellulolytic enzyme production, hexose conversion and pentose sugar utilization in one pot, realizing ethanol de novo synthesis from slurry dilute acid

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pretreated wheat straw without detoxification (Brethauer and Studer, 2014). Similarly,

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more microbial consortia CBP systems for bioethanol, biobutanol and isobutanol

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production among bacteria-bacteria, fungi-bacteria and fungi- fungi co-culture have been designed (Minty et al. , 2013, Patle and Lal, 2007, Suriyachai et al. , 2013, Zuroff et al. , 2013). For acetone-butanol-ethanol (ABE) production, Valdez-Vazquez et al. developed a consortium composed of Clostridium beijerinckii 10132 and Clostridium cellulovorans 35296, which could produce ABE from biologically pretreated wheat straw, resulting in 23.3 g/L of total ABE with 3.7 g/L of ethanol, 14.2 g/L of butanol and 5.4 g/L of acetone (Valdez-Vazquez et al. , 2015). In parallel, Wen et al. (2014) constructed a stable symbiotic consortium through co-culturing a cellulolytic, anaerobic, butyrate-producing mesophile of C. cellulovorans 743B with a

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non-cellulolytic, solventogenic bacterium of C. beijerinckii NCIMB 8052, realizing solvents production from alkali extracted deshelled corn cobs (AECC). Under optimized conditions, the microbial consortium degraded 68.6 g/L of AECC and produced 11.8 g/L of solvents (2.6 g/L of acetone, 8.3 g/L of butanol, and 0.9 g/L of ethanol) in less than 80 h, with

17.2% solvents conversion rate. Further genetic

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modification of this consortium improved the solvent production to 22.1 g/L from

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83.2 g/L of lignocellulose hydrolysate, and AECC degradation was increased by

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21.3%, while solvents titer was enhanced by 87.2% (Wen et al. , 2017). In terms of

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microbial lipid production, Papone et al. (2016) reported a co-culture of microalgae

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Chlorella sp. KKU-S2 and yeast Toluraspora globosa YU5/2. A maximum biomass concentration of 6.90 g/L with a lipid concentration of 0.33 g/L was obtained using

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sugarcane molasses as the substrate. Encouraged by the advantages of co-culture

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systems over pure monocultures, many attempts have also been made in biogas and

al. , 2012).

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biohydrogen production (Ike et al. , 1999, Laxman Pachapur et al. , 2015, Masset et

Generally, substrate degradation efficiency is still the rate limiting step in CBP. To further improve the performance efficiency of microbial consortia in CBP, the substrate degradation rate should be enhanced to provide higher monosaccharide concentrations. As lignocellulose possesses a very complex structure, a single microbial strain can not efficiently secrete all desirable components of lignocellulose degradation complex. Microbial co-culture was reported to increase the production of cellulases and hemicellulases complex (Kumar et al. , 2008). For example, high

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for A. oryzae in combination with other fungi, in particular with P. chrysosporium (Hu

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et al. , 2011). The overall activity of degrading enzyme in the microbial consortia is

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not the sole sum of activities of individual microorganism species, which in some

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cases exceeded the sum of single incubations.Co-cultivation of these lignocellulose

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degrading consortia with biological detoxification species could further improve the = efficiency of microbial consortia CBP systems (Kuhar et al. , 2015).

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Bioremediation using microbial consortia

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The growing population and anthropogenic activities are constantly threatening

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the ecosystem, such as water quality deterioration, heavy metals pollution and loss of soluble phosphorus, etc (Ahmed et al. , 2019, Campbell-Lendrum and Prüss-Ustün, 2019, Critchell et al. , 2019, Ibrahim et al. , 2016, Walker et al. , 2019). To achieve green and sustainable development, biological technologies using specific microorganisms to degrade various pollutants into non-toxic or less toxic compounds provides an eco-friendly option to remedy the impaired environment(Azubuike et al. , 2016). However, the efficiency for biodegradation and bioremediation of environmental pollutants using single strains is still very low and restricted (Villegas et al. , 2014). Therefore, more and more attention has been shifted towards microbial

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consortia, since their inherent multiple function, robust and adaptable characteristics. In terms of wastewater eutrophication, the main task is to remove the excess elements such as nitrogen (N), phosphorus (P), pollutants and toxins (Mujtaba and Lee, 2016). Current biological processesinclude anaerobic digestion, nitrification and denitrification steps, however, several cycles of these three steps are required to

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achieve the EU legislation accepted nutrient levels, and each step requires several

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tanks and internal recycles of activated sludge, resulting in an overall increase of

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process cost, complexity and energy input (Gonçalves et al. , 2017). Comparatively,

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microalgal consortia (microalgae and bacteria/fungi) provide an efficient approach for

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water remediation. For instance, co-culture of microalgae Scenedesmus dimorphus and nitrifiers enhanced the removal of N and P from wastewater by 3.4 and 6.5 times,

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respectively compared to those using nitrifiers-only (Choi et al. , 2017). Co-culture of

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microalga Chlorella vulgaris and bacterium P. putida also showed higher removal

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efficiency of both nutrients (N and P) and chemical oxygen demand (COD) than each monoculture (Mujtaba et al. , 2017). Using co-culture of Scenedesmus sp. and anaerobic sludge, 89% N removal and 80% P removal from starch wastewater were achieved (Ren et al. , 2015). In the view of symbiotic relationship, microalgae can release organic compounds and O 2 during photosynthesis, which can be used as carbon and energy sources by bacteria/fungi. In return, bacteria/fungi will provide CO2 and growth promoting factors for microalgae, such as vitamins and siderophores (Fig. 4) (Abinandan et al. , 2018, Bordel et al. , 2009). In addition, microalgae can also serve as refuge for bacteria, protecting them from hostile environmental

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conditions (Byappanahalli et al. , 2009, Mandal et al. , 2011, Unnithan et al. , 2014). In addition to wastewater purification, biomass harvested from effluent can also be used as sustainable substrates (or feedstocks) for biofuels or biochemicals production (Bhatia et al. , 2017, Wrede et al. , 2014), and animal feed preparation (Stiles et al. , 2018), which further improving process economy. Despite the obvious advantages of

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microalgae-bacteria/fungi symbiosis systems, the availability of land, sufficient light

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intensities and appropriate temperature still seriously limits its scale-up progress in

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wastewater treatment (Osundeko et al. , 2019). In addition, the recovery and

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processing of microalgae from the cultivation medium are still a challenge, since

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needs a high energy input is needed (Osundeko et al. , 2019). More functional facility and more effective harvesting technology need to be developed to accommodate

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microalgae treatment and decrease cell mass recovery cost.

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Heavy metals, such as zinc and nickel are considered as the most hazardous

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pollutants, because they can affect the structure of nucleic acids and proteins. Currently, it is impossible to adopt a single method to treat heavy-metal-polluted wastewater (Bhatia et al. , 2018). Though various microbes with different metal-reduction potential have been identified (Sathiyanarayanan et al. , 2016, Shafique et al. , 2017), the metal removal capability using monoculture is still restricted, as the compositions in wastewater are too diverse and complicated. Microbial consortia contain various robust metal-degrading microorganisms provides an economical and eco-friendly option for heavy-metal degradation through division of labor. For instance, co-culture of algae Scenedesmus and Pseudokirchneriella was

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able to absorb a mixture of Zn(II) and Ni(II) ions (Kipigroch et al. , 2012). Ilamathi et al. used an alginate-bead immobilized mixed-culture of yeast and different bacteria, P. aeruginosa, B. subtilis and E. coli in a liquid-solid fluidized bed for heavy metal biosorption, which resulted in 84.62%, 67.17%, 49.25% and 61.02% recovery of copper, cadmium, chromium and nickel, respectively (Ilamathi et al. , 2014).

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Azo dyes, the largest chemical class of dyes with the greatest variety of colors are

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commonly released from textile, dyestuff and dyeing industry, which cause serious

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environmental pollution because of their color, biorecalcitrance and potential toxicity

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to animal and human (Senan and Abraham, 2004). Nevertheless, it is very difficult to

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decolorize these dyes by common physical and chemical methods (Khan et al. , 2013). Microbial strains are able to decolorize azo dyes, but the degradation products are

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frequently toxic aromatic amines or metabolites that are even more difficult to

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degrade than the parent dye (Solís et al. , 2012). Microbial consortia treatment

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systems could achieve a higher degree of biodegradation and mineralization due to the synergistic metabolic activities of the microbial community (Shanmugam et al. , 2017). For instance, orange II can be completely decolorized by microbial consortia of Enterobacter cloacae and Enterococcus casseliflavus, while only 10% decolorization rate was obtained by E. cloacae and 23% by E. casseliflavus alone (Chan et al. , 2011). The complete decolorization of scarlet R was accomplished within only 3 h by microbial consortia of Proteus vulgaris and Micrococcus glutamicus, superior to 14 h by P. vulgaris and 20 h by M. glutamicus alone (Saratale et al. , 2009). The decolorization of sulfonated reactive dye Green HE4BD using

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microbial consortia of P. vulgaris and Micrococcus glutamicus is significantly higher than that using the individual microorganisms (Saratale et al. , 2010). In these microbial consortia, individual strains may attack the dye molecule at different positions or even utilize resulting degradation products, such as the toxic aromatic amines produced by partner strains for further decomposition (Saratale et al. , 2011).

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Microbial consortia also demonstrate unique capability in degradation of other

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pollutants, like pesticides, antibiotics and other toxins. For example, co-culture of

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Pseudomonas and Staphylococcus was shown to be more efficient in removing phenol

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than their monocultures (Senthilvelan et al. , 2014). Co-culture of Serratia and

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Trichosporon sp. could completely mineralize chlorpyrifos, one of the most widely used organophosphate insecticide (Xu et al. , 2007). Similarly, microbial consortia of

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Klebsiella pneumoniae and Ralstonia sp. showed better tolerance to thiocyanate with

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a degradation rate of 500 mg/L/d. Poly-culture of Arthrobacter sp. NB1, Serratia sp.

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NB2 and Stenotrophomonas sp. NB3 showed enhanced degradation efficiency of nitrobenzene compared with monoculture (Jin et al. , 2012). Moreover, other studies showed that microbial consortia have higher removal efficiencies in polycyclic aromatic hydrocarbons (PAHs) than monocultures (González et al. , 2011, Simarro et al. , 2011).

Future perspectives The application of synthetic microbial consortia in the area of health, chemistry, energy and environment, etc. has prompted the rapid development of biotechnology.

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Taking industrial biotechnology as an example, microbial consortia could synthesize more complex chemicals, which only exist in plants and can not be synthesized by single microorganism (Song et al. , 2014 ). Synthetic microbial consortia could accomplish this complex task through the division of labor. However, these synthetic microbial consortia are still relatively simple, in which usually two or three wild type

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or genetically engineered microorganisms were recruited. In addition, the construction

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of synthetic microbial consortia is still maintained at trial stage and lack of theory

pr

guide, as microorganisms assembly seems random. To construct a more robust, stable

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and controllable microbial consortium, several challenges must be addressed.

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Stable coexistence of different species within one microbial consortium is prerequisite for microbial consortia construction. Microorganisms within a microbial

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consortium often possess different growth features, such as temperature, pH, or

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dissolved oxygen et al. To harmonize the mismatching of growth conditions,

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sequential cultivation mode through multistage conditions regulation is the most commonly used one, in which growth conditions were adjusted based on different strains (Shahab, 2019). However, this would lead to longer fermentation duration and affect final product productivity. Alternatively, more specific bio-materials and facilities could be designed to obtain stable microbial consortia. For example, microcapsule and microfluidic laminar flow techniques can help create a relatively optimal microenvironment for individual cells, and the mechanical separation of each microbial species would not affect microbial growth in a complex microbial consortia system (Wang et al. , 2019, Lindemann et al. , 2016). And also, bioreactor equipped

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with some inlets, on which parceled with gas permeable dense membranes or other nutrient selective permeable membrane can form a gradient cultivation condition, which could meet the requirement for different nutrient of microbial consortia members (Shahab et al. , 2020). A rational population structure and substrates allocation modes are another two

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key factors to achieve an efficient synthetic microbial consortium. Regulation of

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inoculum size and time of different species within microbial consortia is the most

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direct and efficient way to adjust the population structure. However, the substrate

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competition may exist if each species was fed by the same carbon source, and this

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would lead to the uncontrollable growth of each species within microbial consortia. Alternatively, designing a parallel pathway, where the microbial consortia members

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can specifically utilize different carbon sources, and this would efficiently reduce the

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growth competition. For instance, pentose and hexose utilization pathways can be

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metabolically constructed in different species (Zhang et al. , 2015b). This would not only eliminate the substrate competition, but also achieve simultaneous utilization of mixed sugars in lignocellulosic hydrolysate. Another approach is to construct a sequential utilization mode of substrate and intermediate. For example, monosaccharides can be first metabolized into some intermediates, such as acetates by first strain. Then partner strain, which lost the utilization capability of monosaccharides can further convert intermediates to final product (Zhou et al. , 2015). The ratio of these two species could be balanced by the substrate and intermediate ratio. Recently, with the deeper understanding of signal communication

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mechanism between different species of microbial consortia, QS system could also be constructed within microbial consortia. Through the inter-communication of each member, growth related genes could be regulated at different levels to further adjust growth ratio of different species (You et al. , 2004). Efficient mass transfer, including the intermediates, energy and cofactors can

f

improve the product efficiency by using microbial consortia. In contrast to single cell

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factories, intermediates have to be delivered to other partner strains. The complex

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metabolic pathways within all species of microbial consortia made the metabolic

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regulation more difficult. Herein, a spatial organization concept, or named

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3-dimensional (3D) began to show up. In a 3D consortium, microorganisms are well-organized, which will ensure efficient population structure of members with

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lower fitness, enhance the efficient metabolite and signal molecule transfer among

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different cells, and/or improve the overall resilience to environmental stresses

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(Agapakis et al. , 2012, Johns et al. , 2016). In fact, such 3D microbial consortia widely exist in natural environment. A typical example is the anaerobic sludge granule, which is a spatial organization of many species of microorganisms. Acidogens would wrap outside of granules to break down complex organic molecules into acids, which are subsequently transformed to H2 by acetogens in middle layer. H2 and CO 2 produced by these outer layers will be consumed by methanogens in central core (MacLeod et al. , 1990). This spatial organization creates optimal growth environment for different species of microorganisms in these three layers, and realizes efficient mass transfer with orderly arranged microbes.

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To construct synthetic 3D microbial consortia, two strategies including self-organization and artificial-organization can be adopted. Self-organization can immobilize core microorganisms in bacterial formed matrix without the use of synthetic binding agents. One inspirational example is the microbial consortia consisting of bacterium Acetobacter aceti and photosynthetic microalga

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Chlamydomonas reinhardtii (Das et al. , 2016). Within this system, A. aceti produced

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cellulose and acetate mats at the air-water interface when growing in liquid cultures.

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Consequently, C. reinhardtii thrived in an acetate-rich medium and produced oxygen

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for A. aceti. By co-cultivating these two microorganisms, researchers created a

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composite material using cellulose produced in situ by Acetobacter, which offered a matrix to favor the cell growth and entrap microalgae cells (Das et al. , 2016). Another

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example is the widespread existence of biofilms, which is formed by microorganism

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aggregation for surviving harsh environmental conditions (Azeredo et al. , 2017). In

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biofilms, bacteria live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) and organize themselves into a coordinated functional community, in which cells can share nutrients and are sheltered from harmful factors in the environment, such as detergents and antibiotics (Flemming and Wingender, 2010). As for artificial-organization strategy, smart devices or materials can be designed to help form spatial-arranged microbial consortia. Various physical segregation techniques have been developed recently (Wondraczek et al. , 2019). For example, microfluidic and microwell devices have been used to help building microbial communities, where individual species are grown in separated chambers, allowing intermediates to

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exchange freely (Kim et al. , 2008). Strategies using inkjet printing cells and multiphoton 3D printing of gelatin techniques can allow specific members to be arranged in defined geometric patterns, which has been used for microbial communities construction with more complex structures (Connell et al. , 2013, Hays et al. , 2015). Moreover, fiber structure like calcium alginate fibers with one species in

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the core and another in the exterior can also realize strains ordered arrangement with a

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complex consortium system (Kim et al. , 2010). With the increasing understanding of

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systems biology and metabolic dynamics in various microbial consortia, more

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biocompatible and functionalized supporting materials for organizing microbial

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consortia will be developed in the future.

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Not only limited to cultivation strategies and biomaterials or facilities design,

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more advanced computational analysis tools to predict community behavior should be

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developed (Jones et al. , 2016, Klitgord and Segre, 2010, Lindemann et al. , 2016, Manikandan and Viruthagiri, 2010, Panda et al. , 2010, Park et al., 2011, Sun et al. , 2010, Suriyachai et al. , 2013). For this purpose, Minty et al. (2013) have designed an equation model consisting of 50 parameters to describe and predict the behavior of a symbiotic consortium of E. coli and T. reesei. The sophisticated model allowed the identification of key parameters during isobutanol production from lignocellulosic biomass, as well as in-depth evaluation of the co-culture stability, which marked a significant progress in developing modular co-culture engineering. Moreover, the development of computational tools can also help elaborate the interaction mechanism

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of many natural microbial consortia models, such as methanogenic communities (Stams and Plugge, 2009), the inner life of sludge (Liu et al. , 2002), soil and rhizosphere ecosystems (Kent and Triplett, 2002), and marine environment (Ka and Keerthi, 2017), which will further provide theoretical guidance for construction of synthetic microbial consortia.

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Conclusion

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Synthetic microbial consortia possess several promising characteristics and

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potential over monocultures, which harness the resources and metabolic power of

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multiple microbial strains to meet the requirements of discovery of novel chemicals

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and functional reconstitution of complex pathways. More importantly, microbial consortia offer a viable option to share the undesired metabolic burden among

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different microbial strains and go beyond the limit of the metabolic capacity within a

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single microbial strain. And it also presents a new approach for efficient utilization of

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complex substrates and remediation of the environment. However, many uncertain factors, such as the unclear mechanism of intercellular communications, change of microbial population structure and compromise of cultivation conditions, etc. still exist when synthetic microbial communities were constructed. To address these problems, a comprehensive study focusing on interaction mechanism

of natural

microbial consortia, introduction of functional supporting materials, and development of analytical and predictable computational models should be carried out in the future. Ultimately, high-efficient, stable and controllable synthetic microbial communities will be rationally designed for many intriguing applications in the foreseeable future.

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Acknowledgements This work was supported by National Key Research and Development Program of China (2018YFA0902200), National Natural Science Foundation of China (No. 21978130, No. 31961133017, No.21706125), Jiangsu Province Natural Science Foundation for Youths (BK20170993, BK20170997), the Jiangsu Synergetic

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Innovation Center for Advanced Bio-Manufacture, Jiangsu Key Lab of

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Biomass-based Green Fuels and Chemicals Foundation (JSBEM201908), and Project

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Table 1. Recent studies on mixed cultures identifying novel secondary metabolites. Production in monoculture Yes

(Chagas et al. , 2013)

No

(König et al. , 2013)

Antibacterial and antiprotozoal

No

(Rateb et al. , 2013)

Antibiotic activity

No

(Wang et al. , 2013)

N/A

No

(Bertrand et al. 2014)

N/A (1) Cathepsin K inhibitors

No

(Schroeckh et al. , 2009)

Holomycin (2)

(2) Antimicrobial activity

No

(Charusanti et al. , 2012)

Salinispora arenicola & Emericella sp.

Emericellamides A and B

Antibacterial activities

Yes

(Oh et al. , 2007)

Penicillium pinophilum & Trichoderma harzianum

Secopenicillide C Penicillide, MC-141 and Stromemycin

N/A

No Yes

(Nonaka et al. , 2011)

Microorganisms

Novel product

Activity

Alternaria tenuissima & Nigrospora sphaerica

Stemphyperylenol, alterperylenol

Antifungal activity

Aspergillus fumigates & Streptomyces rapamycinicus

Fumicyclines A, B

Antibiotic activity

A. fumigatus & S. bullii

Ergosterol, diketopiperazine alkaloids

Fusarium tricinctum & Fusarium begoniae

Subenniatins A and B

Trichophyton rubrum & Bionectria ochroleuca

4″-hydroxysulfoxy-2,2″ -dimethylthielavin P

Aspergillus nidulans & Collection of 58 actinomycetes

Orsellinic acid, lecanoric acid F-9775A, F-9775B (1)

Streptomyces clavuligerus & Staphylococcus aureus

rn

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u o

J

f o

o r p

e

r P

Reference

,

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Rhodococcus fascians & Streptomyces padanus

Rhodostreptomycin A and B

Antibiotic activities

No

(Kurosawa et al. , 2008)

A. fumigates & Streptomyces peucetius

Fumiformamide (1) N,N’-((1Z,3Z)-1,4-bis(4-methoxyp henyl)buta-1,3-diene-2,3-diyl) diformamide (2)

(1) Inactive (2) Cytotoxicit

No

(Zuck et al. , 2011)

l a n

J

r u o

f o

r P

e

o r p

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Table 2. Summary of recent progress in valuable compound production applying modular co-culture engineering. Improvement compared to Co-culture

Product

Reference monoculture/other method*

Bacteria-Bacteria

f o

E. coli-E. coli

Muconic acid (MA)

19-fold titer improvement

E. coli-E. coli

Pyranoanthocyanins

more stable than plant extraction

4-hydroxybenzoic acid (4HB) 3-hydroxybenzoic acid (3HB)

E. coli-E. coli E. coli-E. coli E. coli-E. coli

Cadaverine

E. coli-E. coli

Apigetrin

E. coli-E. coli

al

rn

u o

o r p

e

8.6-fold titer improvement

r P

5.3-fold titer improvement

(Zhang, Li, 2015a) (Akdemir et al. , 2019) (Zhang et al. , 2015b) (Zhou et al. , 2019)

3-fold and 2.7-fold higher in cadaverine production and productivity

(Wang et al. , 2018a)

2.1-fold titer improvement

(Thuan et al. , 2018a)

Caffeoylmalic acid

5-fold titer improvement

Salidroside

Over 20-fold titer improvement

Resveratrol glucosides

2.9-fold titer improvement

(Thuan et al. , 2018b)

E. coli-E. coli

Naringenin

1.5-fold titer improvemen

(Ganesan et al. , 2017)

E. coli-E. coli

Bisdemethoxycurcumin (BDMC)

6.28 mg/L in titer, BDMC first de novo production from glucose*

(Fang et al. , 2018)

E. coli-E. coli

Caffeyl alcohol

12-fold improvement

(Chen et al. , 2017)

E. coli-E. coli E. coli-E. coli

J

(Li et al. , 2018) (Liu et al. , 2018a)

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E. coli-E. coli

Muconic acid (MA)

14-fold titer improvement

E. coli-E. coli

3-amino-benzoic acid (3AB)

15-fold titer improvement

E. coli-E. coli

Resveratrol

E. coli-E. coli

Perillyl acetate (POHAc)

E. coli-E. coli

Flavonoids

970-fold titer improvement

(Jones et al. , 2016)

E. coli-E. coli

Pinene

1.9-fold titer improvement

(Niuet al. , 2018)

E. coli-E. coli

Salicylate 2-O-β-d-glucoside

G. oxydans-K. vulgare

2-keto-L-gulonic acid (2-KGA)

P. putida-P. putida

2-hydroxybiphenyl (2HBP)

Up to 50% 2HBP formation

Electricity

0.28 g of glucose was converted to electricity (~550 mV) for more than 15 days

(Liu et al. , 2017)

E. coli-E. coli-E. coli Rosmarinic acid

38-fold titer improvement

(Li et al. , 2019)

E. coli-E. coli-E. coli- E. coli P. pastorisP. pastoris

9.5 mg/L of titer, the first report in non-plant host* 55% improvement of monacolin J and 71%improvement of lovastatin from

al

n r u

o J

E. coli-B. subtilis-S. oneidensis

EukaryoteEukaryote

Callistephin Monacolin J, Lovastatin

22.6 mg/L titer, first de novo production from glycerol 3.3-fold titer and ~34-fold productivity improvement

ro

-p

e r P

f o

2.5 g/L titer, higher than the monoculture 89.7% of the theoretical yield, comparable to the conventional two-step fermentation

(Zhang et al. , 2015a) (Zhang and Stephanopoulos, 2016) (Camacho-Zaragoza et al. , 2016) (Willrodt et al. , 2015)

(Ahmadi et al. , 2016) (Wang et al. , 2016) (Martínez et al. , 2016)

(Jones et al. , 2017) (Liu et al. , 2018b)

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methanol Bacteria-Eukaryote

E. coli-S.cerevisiae

Oxygenated taxanes

E. coli-S.cerevisiae

Naringenin

33 mg/L vs non-detected by monoculture 21.16 mg/L of titer, 8-fold titer improvement

f o

*No report using mono-culture de novo synthesis.

l a n

(Zhou et al., 2015) (Zhang et al. , 2017)

o r p

r P

e

r u o

J

Table 3. An overview of microbial consortia mediated consolidated bioprocessing (CBP) systems for bulk chemical production.

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Product

Co-culture strains

Substrate

Achievement

Lactic acid

T. reesei & Lactobacilli sp.

Non-detoxified steam‐ pretreated beech wood

E. coli & C. glutamicum

Starch

19.8 g/L, 85.2% of the theoretical maximum 12.3  mM of L-lysine, 6.8mM of cadaverine and 3.4 mM of L-pipecolic acid

L-lysine, cadaverine and L-pipecolic acid Ethanol

Isobutanol ABE

T. reesei & S. cerevisiae & S. stipitis C. phytofermentans & S. cerevisiae

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wheat straw α-cellulose

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~ 9 g/L, 67% yield

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S. cerevisiae & S. stipitis

Pretreated rice straw

Z. mobilis & C. tropicalis T. reesei & E. coli

Enzymatically hydrolysed lignocellulosic biomass Corn Stover

C. cellulovorans & C. beijerinckii

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Corn cobs (alkali extracted)

C. beijerinckii & C. cellulovorans

Biologically treated wheat straw

C. beijerinckii & C. cellulovorans

Alkali extracted deshelled corn cobs

22 g/L

Reference (Shahab et al. , 2018) (Sgobba et al. , 2018) (Brethauer and Studer, 2014) (Zuroff et al. , 2013)

15.2 g/L, 99% of the theoretical maximum

(Suriyachai et al. , 2013)

52 g/L, 97.7% yield

(Patle and Lal, 2007)

1.88 g/L, 62% of the theoretical maximum 22.1 g/L solvents (4.25 g/L acetone, 11.5 g/L butanol and 6.37 g/L ethanol) 23.3 g/L solvents (3.7 g/L ethanol, 14.2 g/L butanol, and 5.4 g/L acetone) 11.8 g/L solvents (2.64 g/L acetone, 8.30 g/L butanol and

(Minty, Singer, 2013) (Wen et al. , 2017) (Valdez-Vazquez, Pérez-Rangel, 2015) (Wen et al. , 2014)

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0.87 g/L ethanol) Microbial lipid

Chlorella sp. KKU-S2 & T.globosa YU5/2

Sugarcane molasses

(Papone, Kookhunthod, 2016)

0.33g/L

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Figure legends Figure 1. A brief timeline of some of the key milestones in microbial consortia development.

Figure 2. The interaction mechanism models of microbial consortia. (a) Quorum

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sensing in Gram-negative bacteria consortium. In typical Gram-negative LuxIR

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circuits, the LuxI-type protein catalyzes the synthesis of an N-acylhomoserine

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lactones (AHLs) autoinducer (pentagons). Once AHLs reach a threshold level in the

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environment, they will activate the transcriptional regulator proteins of LuxR family.

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The LuxR/AHL complex can activate the expression of multiple target genes, including those required for AHL synthesis. (b) Quorum sensing in Gram-positive

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bacteria consortium. Autoinducing peptides (AIPs) are actively exported from the cell

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and bind to the sensor domain of an extracellular receptor-HK. Once a critical

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concentration of AIP is reached in the environment, it will be phosphorylated by the receptor-HK and imported into the cell, the phosphorylated AIP will bind on the target DNA to regulate its transcription. (c) A model of signal molecule transport using membrane vesicles (MVs). Gram-negative bacteria pinches off of a producing cell from cell surface, which forms a spherical container. The MVs fuse with a receptor cell, delivering hydrophobic signal molecules, subsequently activate the transcription of target genes. (d) The model of physical contact. The typical case is the intimate contact between A. nidulans and actinomycetes, which led to an increased acetylation of histone H3 catalyzed by the Saga/Ada complex. (e) Gene level changes in microbial

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consortia. The mixed culture can cause a serial changes in gene expression in the manner of gene loss, histone modification, and horizontal gene transfer.

Figure 3. Schematic illustrations of different consolidated bioprocessing engineering strategies. (a) The “native” Lignocellulolytic Strategy means recombinant solvents

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synthesis pathway in the plant biomass degrading microorganism, while (b)

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Recombinant Lignocellulolytic Strategy means expression these lignocelluloses

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degrading enzymes in solvents producing microorganisms. (c) Consolidated

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bioprocessing Strategy for lignocellulose biorefinery using microbial consortia, where

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biomass is first degraded into fermentable carbon source catalyzed by degrading enzyme released from cellulosic microorganism, subsequently, the produced

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fermentation carbon source will be utilized by non-cellulosic microorganism to

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produce various valuable chemicals. Main enzymes involved in in cellulose and

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hemicelluloses degradation are illustrated with in different symbols.

Figure 4. Symbiotic relationship between microalgae and bacteria/fungi during wastewater treatment. Microalgae can sustain and drive an engineered consortium with other microorganisms through metabolite exchange, while microalgae can release organic compounds and O 2 during photosynthesis for bacteria/fungi, In return, bacteria/fungi will provide microalgae CO 2 and growth promoting factors. The harvested biomass of microalgae and co-cultured microorganism can further be used as feedstock for biofuel or biochemical production.

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Highlights:  This review comprehensively summarized the distinguished advantages of microbial consortia against monocultures and classified the functional applications of microbial consortia in multiple fields.  Comprehensively discussed the unrevealed interaction mechanisms behind the

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microbial consortia from the perspectives of physical contact, chemical

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communication and gene mutation.

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 Providing guidance and experience in the construction of artificial microbial

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The outstanding challenges and future directions to advance artificial microbial consortia development were highlighted, especially on spatial organization

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schemes design and supporting materials introduction.

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

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consortia systems.