Introduction

Pure cultures dominate the current industrial bioprocesses; however, they are confronted with challenges due to the increased requirement for higher efficiency of production and fulfillment of more complicated tasks. In nature, 99% microorganisms exist in the form of microbial consortia [1]. Inspired by the omnipresent natural microbial consortia, more attention has been paid on the bioprocess development of artificial ones, which pools different engineered microorganisms in one pot [2,3,4]. However, different from natural microbial communities, which exist mainly for the survival and growth in the environment, the artificial microbial consortia are specifically constructed to broaden the scope of feedstocks, enhance the productivity of target bio-products, etc. [5,6,7].

Diverse microbial communities within the same or different species have been set up to realize more complicated tasks [8,9,10]. In addition to treatment of wastewater, biodegradation of textile azo dye and dispose of contaminated soil, recently, co-cultivation systems were also applied to produce biofuels (bioethanol, biobutanol, biodiesel, etc.), bulk chemicals (lactic acid, 2-keto-l-gulonic acid, etc.) and natural products (alkaloids, polyketides, terpenes, flavonoid, etc.) [11,12,13,14,15,16,17,18,19,20,21]. These artificial microbial consortia interact mutually through the interaction of synergism, commensalism, competition, mutualism, etc. (Fig. 1) [1]. Elaboration of the underlying mechanism in microbial communities, such as the exchange of intermediate metabolites, cell-to-cell electrical connections, communications, etc. would guide the design of artificial microbial consortia and further improve the robustness and stability of the co-cultivation systems [22,23,24,25]. Accordingly, this review summarizes the superiority of co-cultivation systems compared with pure cultures and the most updated advances in artificial microbial consortia for the production of biofuels and chemicals from renewable sources. Nevertheless, further application and development of microbial consortia are still confronted with challenges, such as the uncharacterized microbial interaction mechanisms, etc.

Fig. 1
figure 1

The schematic diagram for interaction modes of artificial microbial consortia. The interaction modes of artificial microbial consortia, including a commensalism, b mutualism, c competition and d parasitism

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Advantages of co-cultivation systems over pure cultures

Compared with pure cultures, co-cultivation systems could broaden the substrate utilization spectra. Lignocellulose is the most abundant sustainable recourses; however, due to the complexity of cellulose-degrading systems, single strain generally can not directly utilize it to synthesize valuable products [26, 27]. In general, two common strategies were developed: one is the incorporation of target product synthesis modules into cellulolytic microbes to achieve product generation from lignocellulose; the other is the introduction of cellulase systems into product-generating microbes (Fig. 2a, b) [28, 29]. However, the long and complex pathways including cellulase secretion and/or product synthesis would burden the metabolic stress and lead to low amounts of product generated [30, 31]. On the contrary, microbial consortia offer a simpler and more efficient approach to achieve this goal through the so-called consolidated bioprocessing (CBP), in which enzymes production, substrate hydrolysis and microbial fermentation are completed in one single reactor. For example, setting up co-cultivation systems including cellulolytic Clostridium sp. and non-cellulolytic Thermoanaerobacter sp. can achieve ethanol production from cellulose through CBP. Argyros et al. [32] set up an artificial C. thermocellum–T. saccharolyticum co-cultivation system, in which organic acids formation pathways were both removed in these two constituent strains. 38 g/L of ethanol was finally produced from 92 g/L of Avicel, which was approximately 80% theoretical maximum, indicating that C. thermocellum could be a cornerstone of a robust cellulolytic platform. On the other hand, the lagged utilization of pentose in both hexose and pentose mixtures is commonly found in most microbes, known as carbon catabolic repression (CCR), when bacteria are exposed to two or more carbon sources [33]. The sequential utilization of component sugars of lignocellulose materials would reduce the whole processes efficiency. Microbial consortia enable to rationally utilize different substrates based on the specific metabolic pathway. A novel binary culture can solve the problem flexibly, in which one could only consume glucose and the other could only consume xylose, shifting the interaction modes from the competition to the commensalism [34].

Fig. 2
figure 2

Comparison between pure cultures and microbial co-culturing systems for butanol production used lignocellulose. Two strategies for achievement of butanol production from lignocellulose via CBP. a the “native cellulolytic strategy”, in which butanol synthetic pathway was introduced into cellulolytic microorganism; b the “recombinant cellulolytic strategy”, in which cellulolytic enzymes were constructed into solventogenic ones. c The strategy for microbial co-culturing systems including lignocellulolytic microorganisms and solventogenic bacteria

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When the biosynthetic pathway of target product is long and complicated, a large number of genes would be heterologously expressed in single strains. Generally, the biochemical properties and expression levels of introduced enzymes vary to a large extent. A single host cell cannot provide the optimal environment to perform the function well for all enzymes, while microbial consortia can provide diversified cellular environments for different enzymes. Especially, when a biosynthetic pathway is composed of both prokaryotic and eukaryotic enzymes, a combination of bacterial and fungal hosts would be highly advantageous over using either host alone [35]. In addition, excessive cellular resources consumption and overwhelming metabolic burden often lead to the impaired growth and/or poor biosynthetic behavior of single host strain [36]. Microbial consortia can reduce this metabolic burden through the strategy of labor division, which not only benefits the growth of individual strains, but also improves the performance of overall bio-production (Fig. 3) [37]. Furthermore, insufficient supply of precursors or excessive accumulation of intermediate products could both influence the end-products generation. In pure culture, the relative expression level of different genes is adjusted through promoter strength, gene copy number, ribosomal binding site etc. [38]. Building microbial consortia is a straightforward way to flexibly balance the biosynthetic strength through changing strain–strain ratios [39].

Fig. 3
figure 3

Illustration of the advantages and challenges of co-cultivation systems

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In pure cultures, most strains have individual suitable conditions for the growth. If cultural conditions changed, the growth and metabolism of strains would be affected. Microbial consortia could endure more changeable environments, providing an important new frontier for industrial production [1]. In microbial consortia, environmental disturbance can be dynamically balanced and regulated due to the coordination and cooperation of different strains. The undesired interference within different pathway modules in host strains would also be reduced [40]. Modular compartmentalization offers a new effective approach to limit negative interaction between pathway modules and improve the biosynthesis performance. Hence, microbial consortia commonly possess higher stability and robustness to environmental perturbations.

Biofuels production by using co-cultivation systems

Bioethanol

As an environmentally friendly and sustainable source, biofuels production including bioethanol, biobutanol and biodiesel has gained considerable interests [41,42,43]. Bioethanol was regarded as one of the most promising biofuels, particularly as a carbon-neutral liquid transportation fuel [44]. Solventogenic yeasts, such as Saccharomyces cerevisiae and some bacteria, such as Thermoanaerobacter species are widely used to produce ethanol [45,46,47]. However, the feedstock spectrum is limited to some starchy-based materials [48]. Compared to grain-derived feedstocks, lignocellulose is a more economically feasible alternative because of its abundance and low cost [49, 50]. An artificial Escherichia coli binary culture was constructed for direct conversion of hemicellulose into ethanol. The final ethanol concentration reached 2.84 g/L, which is 55% of the theoretical yield [51]. In this binary system, one E. coli strain was engineered to hydrolyze hemicellulose to xylooligosaccharides through co-expression of two hemicellulase genes. Xylooligosaccharide-utilizing enzymes were then over-expressed in the other E. coli strain to realize the conversion of xylooligosaccharides into ethanol. This co-cultivation system distributed the metabolic burden through extracellular and intracellular expression of different functional enzymes, resulting in the improved ethanol production over pure cultures. Furthermore, cellulase system can also be built in a microbial consortium. For example, dual-microbe Bacillus/yeast system was developed for cellulosic ethanol production. Recombinant B. subtilis carries eight cellulosomal genes originating from C. thermocellum: one scaffolding protein gene (cipA), one cell-surface anchor gene (sdbA), two exo-glucosidase genes (celK and celS), two endo-glucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). The partner Kluyveromyces marxianus KY3-NpaBGS carries a glucosidase (NpaBGS) gene from rumen fungus. Ultimately, 9.5 g/L of ethanol was produced from 20 g/L of cellulose (Table 1) [52].

Table 1 Biofuels and chemicals production by co-cultivation systems
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Considering the complex of lignocellulose degradation enzymes, co-culturing cellulolytic microorganism with ethanol-producing one is a convenient and flexible approach to produce ethanol from lignocellulose through CBP. Cellulolytic C. thermocellum is a model organism for CBP; however, its application was limited due to the low ethanol yield [53,54,55]. Considering its efficient capability of cellulose degradation, C. thermocellum can be co-cultured with non-cellulolytic Thermoanaerobacter strains (X514 and 39E), which showed high efficiency of ethanol production [56]. The final ethanol production achieved at 7.56 and 6.59 g/L, respectively, which were significantly improved by 194–440%. The labor division is straightforward in this system: C. thermocellum is mainly responsible for cellulolysis, while Thermoanaerobacter sp. takes charge of ethanol production owing to its high-efficient ethanol production capability. The interaction within these two strains was through the exchange of intermediate metabolites. Similarly, a co-cultivation system, in which cellulose hydrolysis and ethanol production were conducted by C. phytofermentans and S. cerevisiae, was set up [

Conclusions

In recent years, construction of co-cultivation systems for biofuels and chemicals production has attracted more and more attention. Not only limited to simply mix the wild strains, co-cultivation has also expanded into synthetic biology. The introduction of synthetic intercellular communication into the cell engineering toolbox will open new frontiers and greatly contribute to the future success of synthetic biology and its applications. Although the production could be improved when using co-cultivation systems, challenges still exist. Currently, studies associated with co-cultivation systems are mainly constricted at the levels of exchange of intermediate metabolites. Other elements of environmental variation, such as energy flux, signal exchange and nutrient cycling are still unknown. Only based on the comprehensive understanding of the interaction among microbes, the improvement of robustness, stability and reproducibility can be further achieved.