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BY 4.0 license Open Access Published by De Gruyter Open Access August 30, 2022

Bioremediation of organic/heavy metal contaminants by mixed cultures of microorganisms: A review

  • Xue Li , Chongling Feng EMAIL logo , Min Lei EMAIL logo , Kun Luo , Lingyu Wang , Renguo Liu , Yuanyuan Li and Yining Hu
From the journal Open Chemistry


Although microbial remediation has been widely used in the bioremediation of various contaminants, in practical applications of biological remediation, pure cultures of microorganisms are seriously limited by their adaptability, efficiency, and capacity to handle multiple contaminants. Mixed cultures of microorganisms involve the symbiosis of two or more microorganisms. Such cultures exhibit a collection of the characteristics of each microorganism species or strain, showing enormous potential in the bioremediation of organic or heavy metal pollutants. The present review focuses on the mixed cultures of microorganisms, demonstrating its importance and summarizing the advantages of mixed cultures of microorganisms in bioremediation. Furthermore, the internal and external relations of mixed culture microorganisms were analyzed with respect to their involvement in the removal process to elucidate the underlying mechanisms.

Graphical abstract

1 Introduction

Nowadays, heavy metal pollutants and organic pollutants are the two main pollutants that are present in almost every environment or ecosystem, including wastewater and soil. Despite their essential role in biological metabolic function, once their accumulated concentration exceeds a certain threshold, they can become toxic [1]. Fortunately, with the development of environmental technology, various methods have been put to use and are effective to a certain extent for either organic or heavy metal pollutants.

Supported by the advantages of non-secondary contamination, economic benefits, and the ability to perform in-situ remediation, biological methods have become the mainstream solution for organic or heavy metal pollution. Microorganisms are an effective tool for many processes, such as resource production or food processing, which demonstrates the advantages of microorganisms that have been proved by certain studies [2,3]. In addition, bioremediation is an important role of microorganisms, which targets inorganic and organic compounds, naturally including heavy metals. The acclimatization of microorganisms to different concentrations of pollutants may be a specific factor that contributes to the optimal operation of microorganisms [4,5]. On the one hand, microorganisms benefit from the removal of organic contaminants under heavy metal pressure for the selected strain with heavy metal tolerance [6]. On the other hand, some microorganisms are capable of biotransformation or biodegradation of many organic contaminants and heavy metals present in the environment [7].

Although microorganisms have been widely applied to almost all aspects of development, the research was basically stuck in the application of pure culture microorganisms whose remediation results were usually limited by adaptability, efficiency, and the capacity to handle complex contamination due to the lack of microbial species, not to mention the difficulty of screening, analyzing, maintaining, and retaining pure culture [8,9]. Mixed culture of microorganisms involving the symbiosis of two or more microorganisms that demonstrates the characteristics of all microorganisms is a more efficient tool for bioremediation. In many cases, the mixed culture of microorganisms exhibits a unique capacity for bioremediation that cannot be performed by pure cultures [9,10]. Moreover, secondary pollutants can be produced by many degradation or conversion reactions that cannot be removed by microorganisms of origin; hence, mixed culture microorganisms have to be used. Furthermore, as many reports say that most environments polluted by heavy metal consist of more than one type of metallic element, the mixed culture of microorganisms must be a better tool for the removal of heavy metals compared to the pure culture of microorganisms. Some parts of the mixed culture of microorganisms show a higher efficiency of pollutant removal than pure cultures [1].

2 Applications of mixed culture of microorganisms

Mixed culture of microorganisms has been applied to many fields where pure culture microorganisms could not achieve good results so far. The application of a mixture of microorganisms in bioleaching, removal of pollutants, fermentation, and bioelectrogenesis indicates that the mixed culture of microorganisms is garnering significant interest (Table 1). Based on the main applications of these mixed cultures, the researchers confirmed that these microorganisms can be widely used in the following aspects.

Table 1

Application of mixed culture microorganisms

Applications Research object Raw material (pollutants) Strains Performance Refs.
Bioleaching Anguran Zinc & Lead Mine (Zanjan, Iran) Pb, Zn, Cd, As and Sb. Iron and sulfur oxidizing moderate thermophilic bacteria. 98.5% of Zn, 98% of Cd, 0.027% of Pb, 7.82% of As, and 8.52% of Sb was dissolved. [11]
Manganese dioxide ore provided by an electrolytic manganese factory of Hunan Province, South Central China Mn. Alicyclobacillus sp. and Sulfobacillus sp. 78% of Mn extraction without pH control to and 99% of Mn with pH control. [12]
Nine acid mine drainage samples from sulfide mines of China Cu. Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Alicyclobacillus spp., and Sulfobacillus spp. 68.89% of maximum copper extraction. [13]
Bioremediation The biodegradation efficiency of natural plant based surfactants against two mixed bacterial cultures. Naphthalene. Degradation carbon compounds bacteria with no biosurfactant producing activity and mining waste water microorganisms. The highest degradation ( >99%) potential was identified with waste water microorganisms mixed culture and having 0.5 mol fraction of Reetha in CTAB-Reetha combination. [14]
Synthetic wastewater. Ammonium. Alcaligenes faecalis No. 4 and L1. The average ammonium removal rate and the average denitrification ratio were 61 mg-N/L/h and 31%, respectively. [15]
136 kg of municipal solid waste (Bryan, College Station, TX). Municipal solid waste. Mesophilic microorganisms. Product concentrations were up to 25 g/L, with productivities up to 1.4 g total acid/(L liquid d), total acid concentration and conversion within 4 and 16%, respectively. [16]
Synthetic wastewater simulating medium strength municipal Cu(ii) Ureolytic bacterium. The maximum removal efficiencies by the UMC was obtained 99% for Cu at pH 5.40. [17]
Fermentation Seed anaerobic sludge collected from a mesophilic UASB reactor for wastewater treatment in a local brewery. Glycerol. Methanothermobacter thermautotrophicus and Thermoanaerobacter spp. The yields of methane and acetate were close to the theoretical yields with 0.74–0.80 mol-methane/mol-glycerol and 0.63–0.70 mol-acetate/mol-glycerol. [18]
Anaerobic mesophilic (37°C) sludge treatment process of Gothenburg’s wastewater treatment plant (Gryaab AB, Sweden). Glycerol. Clostridiaceae (55–57%) and Veillonellaceae (20–21%) High concentrations of 1,3-propanediol/L up to 42 g were achieved in fed-batch mode. [19]
Bioelectrogenesis The wastewater collected from distillery industry (nearby Trichy, India). Sugar cane molasses. Lysinibacillus sphaericus spp. (SN-1 and SN-2) and Bacillus safensis SN-3 The peak power density was observed as 63.8 ± 0.65 mW/m2 at pH 8. [20]

2.1 Bioleaching by mixed culture of microorganisms

Bioleaching is a technique for the recovery of heavy metals from metallic ores, sediment, soil, and sludge contaminated with heavy metals. It is an environmentally cleaner process when compared with physical and chemical processes and rarely emits atmospheric contaminants. Bioleaching operations are relatively simpler and cost-effective. Microorganisms are excellent tools for bioleaching due to their interaction with metal sources for the transformation of organic or inorganic acids (protons), oxidation and reduction reactions, and the excretion of complexing agents. Microscopic structures of microorganisms, such as cell walls and surface appendages, directly contribute to the bioleaching of microorganisms [21]. To date, the pure culture of microorganisms, such as Thiobacillus ferrooxidans, Acidithiobacillus ferrooxidans (AF), and Acidithiobacillus thiooxidans (AT), has been widely used in bioleaching [22,23]. However, the mixed culture of microorganisms has been recently rendered crucial.

The mixed culture of microorganisms has been popularized in bioleaching because mixed metal ions often exist in mines or soils, and pure cultures cannot meet the demands of bioleaching of several elements. The mixed culture of microorganisms has also shown better results [24,25,26]. According to a report by Qiu et al. [27], mixed culture containing the iron-oxidizing AF and the sulfur-oxidizing AT was better than a pure culture of AF or AT in leaching chalcopyrite. The efficacy of copper extraction performed using a mixed culture of microorganisms was higher than that of pure cultures. Besides, AF, AT, Leptosprillum ferrooxidans (LF), and the mixed culture of were used to recover zine in the presence of flotation chemicals. Zinc recovery of 97, 86, 70, and 14.8% and the corresponding zinc extraction values in the absence of flotation chemicals of 74, 64, 61, and 9.5%, respectively, were observed [28]. The results show that mixed culture of microorganisms was a better way for bioleaching compared to pure culture. The increasing usage of bioleaching by mixed culture microorganisms instead of pure culture microorganisms coincides with the aforementioned stipulations and also highlights their potential for the bioremediation of organic or heavy metal pollutants.

The applications of mixed cultures of microorganisms in bioleaching demonstrated that mixed cultures can bioleach the concentrate more rapidly and extensively than pure cultures [29]. This advantage (high efficiency) garnered significant interest, leading to the introduction of mixed cultures in bioremediation. For example, Pan et al. [30] investigated the growth and metal removal by two fungi (Penicillium sp. A1 and Fusarium sp. A19) and their consortium. The authors hypothesized that the differences in resistance to metals between A1 and A19 strains could be attributed to the different detoxification mechanisms of the two fungi. Therefore, the co-inoculation of A1 + A19 resulted in higher resistance and bioaccumulation of multiple metals than individual A1 or A19 strain. Seven microbial consortia were screened from an abandoned mine for resistance to zinc [31], and the best accumulated consortium was tested. The resistance thresholds were higher in consortium than in pure culture.

2.2 Bioremediation by mixed culture of microorganisms

Bioremediation, which uses living organisms, mainly microorganisms (bacteria, fungi, and microalgae) or their processes to degrade or detoxify environmental contaminants, is a cost-effective and environmentally safe method for decontaminating polluted soil and water. It is emerging as an alternative to costly physicochemical remediation technologies. Although traditional pure cultures have achieved some success in bioremediation [32,33], compounds in mixtures are known to interact with biological systems in ways that can greatly alter the toxicity of individual compounds, which significantly reduces the effects of pure cultures. The use of mixed cultures of microorganisms instead of pure cultures has increased dramatically because of their known synergistic metabolism, which improves the efficiency of hydrocarbons and other chemical degradations [34,35].

Moreover, in practical bioremediation applications, microorganisms in microbial cultures have shown more advantages. Docherty et al. [36] reported that the mixed culture of microorganisms can be enriched to degrade chemicals that fail biodegradability assays. This implies that microorganisms can process by-products in consort; the pure culture of single microorganisms does not exhibit sufficient properties or functions for the disposal of pollutants. According to a report by Ling et al. [37], mixed cultures could save chemicals for pH adjustment compared to the pure culture because some strains can optimize conditions for other primary strains. Even composting, the major process of stabilizing agricultural solid waste and municipal solid waste through the degradation of biodegradable compounds by microbial communities has been adopted as one of the most cost-effective technologies for soil bioremediation. In summary, bioremediation involves the application of mixed cultures. It is reliable for the removal of organic or heavy metal pollutants.

2.3 Resource production by mixed culture of microorganisms

Mixed culture of microorganisms has also been applied for resource production due to the symbiotic relationship between the different strains, resulting in a better quality of production than traditional single inoculation. Chemicals, food, and bioelectricity are the main products of degradation, fermentation, and other reactions performed by mixed culture of microorganisms [38,39,40]. In the process of resource production, mixed culture of microorganisms can not only lead to higher productivity but also significantly influence the removal of pollutants from reactants to exert multiple effects. For example, in the process of bioelectricity, efficient electricity generation and simultaneous treatment of distillery wastewater can be realized [20], and a mixed culture of acid-resistant and thermotolerant yeasts is a potential way to produce single-cell protein(animal feed) and simultaneous chemical oxygen demand removal under extreme operating conditions [41].

Compared to pure culture fermentation, mixed culture fermentation forms a rather mature and well-studied technology for converting organic waste into valuable biofuels and chemicals, such as biogas, bioplastics, hydrogen, and acetate; consequently, it offers the benefit of environmental protection combined with energy and resource recovery [42]. In summary, mixed cultures of microorganisms would be developed in the era of sustainable development, owing to their benefits in the process of resource production.

3 Advantages of mixed culture of microorganisms compared to those of pure cultures

Mixed culture microorganisms had many benefits what make them superior to pure culture and turn into growing in popularity among bioremediation instead of pure cultures for instance, adaptability, efficiency, and capacity of handling multiple contamination (Figure 1).

Figure 1 
               Advantages of mixed culture in bioremediation compared to pure culture.
Figure 1

Advantages of mixed culture in bioremediation compared to pure culture.

3.1 High efficiency of mixed culture of microorganisms

During the long period of the evolution of bioremediation technology, efficiency has always been the most important factor that researchers have sought to optimize. Improving the efficiency of bioremediation would undoubtedly improve the results of bioremediation. Pure culture strains, which are widely used in bioremediation, only function with a narrow range of substrates and operating conditions with relatively lower efficiency [43]. Mixed cultures of microorganisms demonstrate superior efficiency for bioremediation applications (Table 2).

Table 2

The high efficiency of mixed culture microorganisms compared to pure culture

Contaminants Strains Pure culture efficiency Mixed culture efficiency Refs.
Oily sludge Stenotrophomonas acidaminiphila, Bacillus megaterium, Bacillus cibi, Pseudomonas aeruginosa, and Bacillus cereus 91.7, 89.0, 89.7, 86.7, and 88.4% of saturated fraction 90.7% of saturated fraction [44]
33.2, 39.6, 64.3, 39.5, and 40.3% of aromatic fraction. 51.8% of aromatic fraction
4-Nitroaniline Acinetobacter sp., Citrobacter freundii, and Klebsiella oxytoca. 35–57% degradation of 4-nitroaniline after 72 h. 92% degradation of 4-nitroaniline in 48 h, followed by complete degradation at 72 h. [45]
Pesticide The white-rot fungus Coriolus versicolor and bacterium from activated sludge sample collected from Shibaura wastewater treatment plant, Tokyo, Japan. Aldicarb, atrazine, and alachlor dropped by 79, 81, and 59% within 7 days by fungus-only culture. Aldicarb, atrazine, and alachlor dropped by 82, 77, and 67% within 7 days by mixed fungus–bacteria cultures. [46]
Dropped by 17, 0, and 35% within 7 days by an non-acclimated bacterial culture.

The high efficiency of mixed cultures of microorganisms can be attributed to the multiple metabolic capacities and the synergetic effects between the association members, and the mechanisms through which the degrading bacteria benefits from synergistic interactions can be complex. A species can remove toxic metabolites from the preceding species, which might otherwise hamper microbial activity. It is also possible for other species to degrade compounds that the former species can only partially degrade, thereby promoting co-metabolism processes [44]. As a major part of bioremediation, the biodegradation process becomes much more efficient and much faster with the use of mixed culture owing to several factors: (1) the metabolic intermediate of one bacterium can be utilized by another for efficient degradation; (2) the system becomes much faster from a bioremediation point of view; and (3) suitable trapping methods may be employed for efficient degradation. The aforementioned analysis presented by Das and Dash [47] may explain the reason for the high efficiency. Hence, the reason why mixed culture microorganisms had the higher efficiency may be due to the better handling capacity of mixed cultures toward multiple stresses owing to the variation in tolerance, toxicity response, growth inhibitions, and contaminant sequestration mechanisms of different strains. Biomass productivities of individual strains and consortiums also played an important role in improving bioremediation performance. When biomass productivities of individual strains and their consortium were compared under different conditions, all three strains were well represented in the consortium. Furthermore, the inoculum ratio of different strains may greatly influence the bioremediation efficiency [48], and the right inoculum ratio would lead to the highest efficiency.

3.2 Adaption of mixed culture of microorganisms

Adaptation is another restriction of practical applications of microorganisms. Extreme temperature, pressure, ionic strength, pH, and even the shortage of nutrient supply are all parts of the adaption. Extremophiles are microbes that can adapt to extreme conditions. Interest in extremophilic microorganisms increased significantly once it was realized that the survival of these microorganisms depended on novel enzymes and biochemical pathways that could be used in biotechnological applications, such as the food, pharmaceutical, and detergent industries, and especially in bioremediation [49]. Mixed cultures of microorganisms are commonly employed because pure cultures are difficult to maintain in large-scale units, and mixed cultures can easily adapt to environmental changes. Moreover, mixed cultures of microorganisms exhibit better adaptation, primarily demonstrated via the following properties: viability under extreme environmental conditions, tolerance to high concentrations of contaminants, and bioenergy in the absence of nutrients. Jahid et al. [50] used a mixed culture of the biofilm of Salmonella typhimurium and cultivable indigenous microorganisms on lettuce. With complex interactions between cell populations forming biofilms, certain microorganisms first colonize/attach to the surface, and other microbes then follow to form biofilms that enhance the resistance of their sessile cells to cold oxygen plasma. Moreover, they eventually internalize to a significantly greater degree than monocultures. A mixed culture of mesophilic iron- and sulfur-oxidizing microorganisms, including AF, AT, and LF, was used for the removal of sulfur and ash from fine-grained high pyritic sulfur coals under conditions involving moderately high temperature (35°C) and extreme acidity (pH = 1) [51], demonstrating good performance. These studies revealed that mixed cultures of microorganisms were able to perform bioremediation at high temperatures. The high tolerance of mixed cultures can be attributed to metabolic changes, mainly affecting the expression of proteins related to carbohydrate metabolism and promoting the accumulation of higher amounts of molecular chaperones associated with increased survival at extreme temperatures. In addition, a mixed culture consisting of five phenol-degrading strains was developed to degrade up to 1,000 mg/L of phenol concentration and achieve a maximum phenol degradation of 98.93% at the initial condition [52]. From the report, it is obvious that under such high concentration conditions, greater stability, complete mineralization, and increased metabolism demonstrated by mixed culture contribute to enhanced tolerance. This property of mixed culture can be attributed to alternative strains that promote tolerance to contaminants by forming opportune metabolites and/or changing the intracellular amino acid pool.

Mixed culture could well adapt to the shortage of nutrient supply by not only improving the resource utilization but also providing nutrients for other strains. Pure cultures, in most cases, demonstrate survival difficulties, let alone fulfill the prerequisites for bioremediation. To solve this problem, researchers have tried mixed culture microorganisms. For example, bioremediation with either effective microorganisms (heterotrophic) or microalgae Chlorella sp. (autotrophic) independently requires a continuous supply of aeration to sustain their growth and treatment efficiency. However, when cultured together, naturally, effective microorganisms would produce CO2 and consume O2 while microalgae would perform remediation and vice versa. Hence, necessary prerequisites of individual strains could be ignored owing to the associated relationship between both with respect to respiration and end up with a more robust and economical process with low maintenance [53]. Similarly, in denitrification, employing heterotrophic strains is difficult with expensive external carbon sources, and combination with sulfur-based autotrophic denitrification could lead to both high nitrate removal efficiency and low sulfate production [54].

3.3 Capacity of mixed culture microorganisms to handle multiple contaminants

Mixed cultures can handle multiple contaminants as every single strain of the consortium possesses unique functions. In bioremediation, the complex sources of contaminants are far beyond the limit of pure cultures, not to mention the variations observed in the forms of a single pollutant. There are three main complications of contamination: multiple pollutants, secondary pollution, and microorganism self-pollution (Figure 2).

Figure 2 
                  Three kinds of multiple contaminations and their removal process by mixed culture microorganisms. (a) Multiple pollutants; (b) secondary pollutants; and (c) microorganism self-pollution.
Figure 2

Three kinds of multiple contaminations and their removal process by mixed culture microorganisms. (a) Multiple pollutants; (b) secondary pollutants; and (c) microorganism self-pollution.

First, in many circumstances, the contaminated soil may simultaneously include organic pollutants and inorganic pollutants, which increase the requirements for bioremediation methods. Jiang et al. [55] used Pleurotus cornucopiae and Bacillus thuringiensis FQ1 and the antioxidant responses of P. cornucopiae for combined remediation of Cd–phenanthrene co-contaminated soil. The results showed that the mixed culture not only enhanced mushroom growth (biomass) and Cd accumulation spiked with 200–500 mg/kg phenanthrene but also alleviated oxidative stress caused by co-contamination with relative decreases in lipid peroxidation and enzyme activity due to bacterial inoculation. This indicates that the presence of the bacteria B. thuringiensis FQ1 could effectively reduce the danger posed by the reaction to the main strain P. cornucopiae and maintain the proper reaction. In addition, mixed culture microorganisms can demonstrate a high removal efficiency for almost all kinds of heavy metals in sundry environments [56,57]. Therefore, mixed cultures of microorganisms can easily handle a manifold of pollutants. Next, bioremediation may result in secondary pollution or a more complex situation. Anabaena variabilis was applied to remove 2,4-dinitrophenol (2,4-DNP) and 2-amino-4-nitrophenol (2-ANP), which is a degradation product of 2,4-DNP removal performed by A. variabilis, accumulated in the culture. To solve this problem, Hirooka et al. [58] used a mixed culture of highly potent cyanobacteria to remove 2-ANP. Anabaena cylindrica and A. variabilis completely removed 2,4-DNP, and only a low concentration of 2-ANP was detected compared to that observed with a pure culture of A. variabilis. Lastly, if microbial growth cannot be controlled, the microorganisms themselves could also become pollutants, especially under the condition of eutrophication or in the absence of natural predators. In the treatment of African catfish, researchers used Chlorella sp. to remove nutrients from aquaculture wastewater and found that inoculation exceeding the optimum dosage of 30% decreased the nutrient removal efficiency. Therefore, researchers need to control the population of microalgae (Chlorella sp.) with sewage fungus (Aspergillus niger) and successfully remove the suspended microalgae biomass without producing any harmful bio-degradative enzymes in the process [59].

Once different strains of mixed-culture microorganisms can grow together and make full use of their individual characteristics, their advantages would lead to their wide application in the field of bioremediation, which would remarkably benefit human beings.

4 Mechanisms of mixed cultures of microorganisms and potential for bioremediation

Not all attempts of the applications of mixed cultures could be performed as anticipated. In an attempt to bioleach heavy metal-contaminated sediment [60], researchers were supposed to reach higher metal solubilization ratios with the use of mixed cultures. However, they found that the pure culture of AT resulted in the highest metal solubilization ratios as the acclimation efficiencies of the bacterial cultures are ranked differently, which directly connects with the solubilization ratios. Mixed cultures may influence the acclimation efficiency of pure cultures. This observation implies that bioremediation mechanisms of mixed culture of microorganisms – internal as well as external factors – are pivotal.

The relationships of mixed cultures were more complex than those of pure cultures. Different physiological types of microorganisms coexist fulfilling different ecological roles. They exist either in harmony or antagonistically in constant dynamic variation; hence, it is necessary to understand this competition [61]. Based on the theory of simple competition in a well-mixed continuous culture, if the growth rate relationships are such that one type of organism grows more rapidly than others under all conditions, it will take over as the other is depleted. These studies showed that internal associations and external factors are the two main kernels of mixed culture.

4.1 Internal relations between different strains

Considering that viruses hardly ever played a positive role in bioremediation, and at certain instances, viruses themselves were the objects to be remedied [62,63,64], the interrelations and interactions between different strains of microorganisms discussed here mainly include three forms: mixed culture of fungus, mixed culture of bacteria and fungus, and a mixed culture of bacteria, which revealed the mechanisms of bioremediation by mixed culture microorganisms. In addition, the relationships between different strains were analyzed at a molecular level.

4.1.1 Mixed culture of fungi

In mixed culture of fungi, two or more mycelia in close proximity with each other can interact in different ways. Mutualistic, neutralistic, or competitive interactions are observed between different strains. The fungi can even switch from one type of interaction type to another. Based on the observation of numerous mixed culture experiments performed on solid agar plates (Figure 3) [65], the interactions of two fungi can be mainly classified into four major types: distance inhibition, zone lines, contact inhibition, and overgrowth. However, according to another study, the interaction outcomes of different fungal isolates were characterized as mutual intermingling, partial mutual intermingling, and deadlock at the contact point (Figure 4) [66]. Regardless of the definition adopted here, it is clear that different types of interactions would show different results of bioremediation by mixed culture fungi.

Figure 3 
                     Four major types (distance inhibition, zone lines, contact inhibition and overgrowth) of mixed culture fungus performed on solid agar plates [65].
Figure 3

Four major types (distance inhibition, zone lines, contact inhibition and overgrowth) of mixed culture fungus performed on solid agar plates [65].

Figure 4 
                     Interaction outcomes of different fungal isolates grown on PDA media. (a) Mutual intermingling; (b) partial mutual intermingling; and (c) deadlock at contact point [66].
Figure 4

Interaction outcomes of different fungal isolates grown on PDA media. (a) Mutual intermingling; (b) partial mutual intermingling; and (c) deadlock at contact point [66].

Interactions, symbiosis, competition, and antagonizing can be observed in almost every application of a mixed culture of fungi. However, a large proportion of mixed culture fungi showed conspicuous antagonizing or competition, which seriously influenced the results of bioremediation [67]. Of course, some fungal mixed cultures do not demonstrate the incident antagonizing or can overcome the downside of competition and form a more stable consortium, thereby showing better results [68,69,70]. This implies that the negative relationships among fungal strains can transform into positive relationships or at least a non-confrontational relationship by adjusting nutrients or community structure [71].

As almost all fungi are heterotrophic, the main requirements of the mixed culture of fungi involve a complete usage of oxidized substrates and temporal stability of the composition. Considering the aforementioned concepts, the main criteria in choosing microorganisms for a mixed culture involve substrate specificity and specific growth rate of pure cultures. It also shows that the substrate consumption rate of a mixed culture is not always equal to the sum of consumption rates of individual strains, and in a number of cases, the activity of a mixed culture could be even lower than that of its constituent cultures. This effect can be explained by the competition between different strains of a mixed culture for the most readily utilizable substrate [72]. Once all strains of mixed culture grow normally and rapidly, maintain a balance between their biomass, and stimulate enzyme activity, bioremediation may result as an ideal consequence [73]. Although the interrelations have not been completely understood until now, based on all observations, the biomass, combination of nutrients, enzyme activity which are all key factors contributing to bioremediation results could be gleaned [74].

4.1.2 Mixed cultures of bacteria and fungi

Mixed cultures of bacteria and fungi are another important subtype of mixed cultures. Due to the higher number of microbe species, the connection between bacteria and fungi is naturally more complex. In such a mixed culture, complex mutualistic interactions include the use of fungal hyphae by bacterial cells for protection against soil modifications or access to nutrients. Some of these interactions are key factors, as exemplified by the oxalate-carbonate pathway in soil, which implies a specific interaction between fungi and bacteria [43]. Antagonistic effects or resource competition also occur to a degree that should be evaluated in polluted soil before scaling up the remediation process to a field scale. These phenomena are observed not only under conditions in which antibiotics produced by fungi, such as penicillin, can kill bacteria but also when bacteria inhibit the outgrowth of some fungi, such as lactic acid bacteria obstruct the growth of some common food-spoiling fungi. In a study performed by Agnolucci et al. [75], the use of microbial starter enhanced the biotransformation process, leading to an early increase in the level of bacterial diversity, which could be another reason for the promotion function. In principle, fungi and bacteria play different roles in bioremediation and respond differently to exogenous pressure, even though the whole process of bioremediation can be divided into two steps, and fungi and bacteria finish alternative steps.

The differences between mixed cultures of bacteria and fungi revealed that based on the discrepant ability (resistance or adaptation), the different structures or dominant strains of mixed cultures may generate different results. Almost all bioremediation processes are dynamic, especially those performed utilizing various microorganisms. Hence, it would be more important to control the structure of the mixed culture community. The structural control can improve the community structure and functional diversity of bacteria and fungi. It can effectively inhibit competition within the inoculant microbes and the competition between inoculant agents and indigenous microbes, which was a benefit for the collaborative symbiosis between different microbes, and enhances the growth of dominant microorganisms [76]. Structure control plays an indispensable role in all mixed cultures composed of microorganisms other than bacteria and fungi. However, for mixed cultures of bacteria and fungi, it is particularly apparent due to more intervallic internal discrepancy.

Although efficient mass transfer between microorganisms is a prerequisite for successful metabolic interaction, microbial partners trying to form a close association seem reasonable [77]. As for mixed cultures of bacteria and fungi, on the one hand, according to the competitive exclusion principle, no two species can occupy the same niche in the same environment for very long [78]. However, on the other hand, metabolic potential in the presence of contaminants could be maintained within a wider range. Co-metabolism of mixed culture also significantly influences the biodegradation of emerging trace organic contaminants (EOCs) through co-metabolic activities of both heterotrophic and autotrophic microorganisms. In this process, heterotrophic microorganisms are responsible for the metabolism of EOCs, and autotrophic microorganisms co-metabolize a variety of EOCs via non-specific enzymes. Therefore, from a metabolic perspective, it can be inferred that the ability of bioremediation of fungi with only one metabolic model (heterotrophic) would be greatly improved when mixed with bacteria, especially autotrophic bacteria.

4.1.3 Mixed culture of bacteria

Bacteria are often organized into multicellular populations. One or several bacterial species interact closely and evolve in communities to exploit limited resources in a confined environment to ensure species survival and procure advantages such as access to nutrients, dynamic growth, or increased antibiotic resistance [1]. Typically, bacteria have a smaller body size than fungi; thus, they are easier and more consistent to disperse from local to global scales. Bacteria also demonstrate a wide array of metabolic capabilities. During the colonization of natural environments, bacteria generally show more deterministic assembly patterns, whereas mixed fungal cultures exhibit relatively stochastic assembly patterns due to the strong influence of dispersal and priority effects [79]. Therefore, the allocation of nutrients is the major component influencing the interrelations between bacterial strains. Mechanisms of nutrient uptake by pure and mixed cultures were investigated. One of the two uptake mechanisms (interfacial accession and biosurfactant-mediated hydrocarbon uptake) prevailed depending upon the changes in cell surface hydrophobicity, the extent of emulsification, and associations among strains [80]. The aforementioned three aspects of mixed cultures of bacteria may play a key role in bioremediation. Of course, this does not mean that the mixed culture of bacteria will not be affected by the competition of trophic status between autotrophs and heterotrophs. In contrast, in the report of Unnithan et al. [81], before introducing a mixed culture of bacteria, the trophic status of the water column, the associated nutrient availability, and the physicochemical properties of suspended organic aggregates were considered. Both wastewaters and algal stocks act as sources of bacteria, effectively creating a tripartite system. In fact, bacteria can perform with a variety of nutritional sources. Hence, the lack of specific nutritional sources for bacterial mixed culture is not a significant issue, unlike fungal mixed culture, when applied to bioremediation.

4.1.4 Molecular dynamics

The mixed culture discussed here was a wide range of definitions ranging from double mixed cultures to microbial communities. Analysis of molecular biology for mixed-culture microorganisms could transcend the species limit and present functional relationships. For example, in a mixed culture of Lactobacillus delbrueckii subsp. bulgaricus, the expression of the genes of Streptococcus thermophiles increased, implying that one could detect its mixed culture partner and set up some regulatory responses to its presence [82]. In another study [83], this mixed culture also showed a high expression of genes involved in extracellular polysaccharides’ biosynthesis and secretion, which may facilitate the exchange of metabolites by forcing close proximities or even physical contact between the two species. The increase in gene expression involved in the acquisition and metabolism of polysaccharides was found in other studies because of mixed culture [84]. These results demonstrated that in mixed culture conditions, some species could adjust their gene expression to change their function or co-metabolism capacity.

As for a more complex mixed culture system, DNA/gene analysis of the microbial community plays a very important role and directly influences the functions of the community. In 12 wastewater treatment systems, when FeOB (iron-oxidizing bacteria) and FeRB (iron-reducing bacteria) genes were detected in abundance, ideal results were observed [85]. Hence, setting functions by screening genes in the microbial community may be a remarkable approach. Using the 16S rDNA gene-based method, different microbial communities can be ranked by different functions. Hence, it would be easy to choose a specific community. Moreover, microorganisms form motile swarms, which offer a competitive advantage owing to the combination of genes [86]. Regardless of the type of mixed culture, the internal relationships at the molecular level would greatly influence the efficiency of bioremediation.

4.2 Impact of external factors

The process of removal of organic or heavy metal pollutants is bidirectional instead of unidirectional, and the pollutants could also have effects on the mixed cultures of microorganisms. Moreover, the effects ate more complicated than those observed on pure cultures. For organic pollutants such as oil, the difference between phases may lead to differences in the capability of the mixed cultures for the removal of total petroleum hydrocarbons due to constant aeration provided during shaking in the slurry phase rather than in the solid phase [87]. In addition, organic components would affect the activity of microorganisms, especially enzymes, which may alter the degradation ability of organic pollutants [88]. Heavy metal pollutants with highly toxic or ecotoxic properties can not only force cells to change their morphology but also cause DNA damage to microorganisms, which is negatively correlated with their treatment capability [89].

Interactions between microorganisms in the mixed cultures and external factors of their environment are also difficult to disentangle as they influence one another in complex ways. For example, external factors influence the structure and function of the mixed culture of microorganisms by imposing selection pressures, regulating the supply of substrates, and removing products. Mixed cultures of microorganisms, in turn, influence environmental conditions via reactions of the metabolic products with components of the surrounding environment [90]. This observation demonstrates an interplay between microorganisms in the mixed culture and external conditions. Space, carbon source, salinity, and even metal speciation could affect bioremediation results.

All living creatures require a certain space to maintain basic growth and development, except for microorganisms. Moreover, mixed cultures often demand a more suitable allocation of space due to species diversity. A previous study demonstrated that regardless of whether the culture pattern was pure or mixed, the consortium was not sustainable. A study performed by Bučková et al. [78] also supported this hypothesis. In addition to space, the carbon source is another key external factor that can significantly influence the enzyme activity of mixed cultures of microorganisms instead of nitrogen source, sulfur source, hydrolytic enzymes, or inducers. However, this effect was not crucial to the results observed in the aforementioned studies. In the process of bioremediation, enzymes produced by microorganisms play an important role in improving the results by stimulating enzyme activity. Salinity is known to influence the performance of biological wastewater treatment plants. Its impact on the mixed culture of fungi has been ignored for a long time. However, its effects on bacteria have been studied extensively. The diversity of mixed culture is increased as salinity is increased up to a certain limit, which significantly improves treatment results. Otherwise, the diversity would decrease, resulting in opposite effects [91]. Many external factors have not been elucidated, but if optimal bioremediation is to be achieved by using mixed cultures of microorganisms, external factors cannot be ignored.

5 Conclusion

Mixed culture, consisting of various strains, possesses a natural advantage, which leads to a diversity of metabolites. Three major advantages were summarized: high efficiency, adaptation, and capacity to handle multiple contaminations from the applications of mixed culture microorganisms demonstrate the significant benefits of its usage. Multiple metabolic capacities, extremophiles, and structures contribute to its advantages and show superiority to pure culture. Moreover, some mixed culture microorganisms possess more than one advantage to further improve the results of bioremediation so long as a stable microbial community can be established.

However, how can these advantages be obtained by mixed-culture microorganisms? The mechanisms can be classified with respect to two aspects: interrelations between strains and external factors. On the one hand, interrelations of mixed cultures of fungi, mixed cultures of fungi and bacteria, and mixed culture of bacteria involve different mechanisms underlying various types of interactions, metabolism types, supply of nutrients, enzymatic activity, etc., which render the characteristics of mixed cultures compatible with complex remediation requirements. On the other hand, external factors may also influence the results of bioremediation such as space, carbon source, and salinity. Although some mechanisms of mixed culture microorganisms have been identified, a clearer understanding of these mechanisms remains to be investigated. A microflora consisting of a number of microorganisms undergoing a long duration period of adaptation and symbiosis can possess systematic functions with high stabilities and have thus considerably high potential in intensifying the effect of phytoremediation.

  1. Funding information: This work was financially supported by the National Natural Science Foundation of China (Nos. 51308076 and 52000013), Natural Science Foundation of Hunan of China (2020JJ4643), and the Research Project of Education Department of Hunan Province of China (18B406).

  2. Author contributions: Xue Li and Chongling Feng: writing – original draft, Min Lei and Kun Luo: investigation, Lingyu Wang: validation, Renguo Liu and Yuanyuan Li: supervision and conceptualization, and Yining Hu: project management.

  3. Conflict of interest: The authors have no conflicts of interest to declare.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


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Received: 2022-04-15
Revised: 2022-06-22
Accepted: 2022-08-03
Published Online: 2022-08-30

© 2022 Xue Li et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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