TY - JOUR
AU - Oyserman, Ben, O
AB - ABSTRACT The natural microbial functions of many soils are severely degraded. Current state-of-the-art technology to restore these functions is through the isolation, screening, formulation and application of microbial inoculants and synthetic consortia. These approaches have inconsistent success, in part due to the incompatibility between the biofertilizer, crop, climate, existing soil microbiome and physicochemical characteristics of the soils. Here, we review the current state of the art in biofertilization and identify two key deficiencies in current strategies: the difficulty in designing complex multispecies biofertilizers and the bottleneck in scaling the production of complex multispecies biofertilizers. To address the challenge of producing scalable, multispecies biofertilizers, we propose to merge ecological theory with bioprocess engineering to produce ‘self-assembled communities’ enriched for particular functional guilds and adapted to a target soil and host plant. Using the nitrogen problem as an anchor, we review relevant ecology (microbial, plant and environmental), as well as reactor design strategies and operational parameters for the production of functionally enriched self-assembled communities. The use of self-assembled communities for biofertilization addresses two major hurdles in microbiome engineering: the importance of enriching microbes indigenous to (and targeted for) a specific environment and the recognized potential benefits of microbial consortia over isolates (e.g. functional redundancy). The proposed community enrichment model could also be instrumental for other microbial functions such as phosphorus solubilization, plant growth promotion or disease suppression. biological nitrogen fixation, biotechnology, biofertilization, microbial ecology, diazotrophs, soil fertilization, restoration ecology, community assembly A CENTURY OF DEGRADATION: THE IMPACT OF SYNTHETIC FERTILIZER AND ALTERNATIVES The use of synthetic nitrogen (N) for crop fertilization contributes tremendously to the world's food supply, but the production and application of these fertilizers also contribute to a global environmental imbalance. It is estimated that synthetic N is the second most manufactured synthetic product in the world (Elishav et al. 2017), and the Haber–Bosch process used to drive this production is estimated to consume 1% of the global primary energy produced (Cherkasov, Ibhadon and Fitzpatrick 2015). In addition to the high energy cost, N fertilizers alter microbiome function, increasing soil respiration and nitrous oxide emissions (Bicer et al. 2017) equivalent to 1–2.5% of global greenhouse gas emissions (Pfromm 2017), all the while reducing soil diversity (Dai et al. 2018) and degrading the natural community structure and nitrogen-fixing capabilities of soils (Wang et al. 2017; Luo et al. 2018). Paradoxically, the increased use of N fertilizers has coincided with a significant global decrease in the efficiency of N use over the last 50 years (Lassaletta et al. 2014). Only 4–14% of synthetic N applied to croplands enters the economy as a downstream final product (Pikaar et al. 2017). Moving forward, increasing N-use efficiency is integral to increased agricultural sustainability (Schils et al. 2018). One approach to reduce the impact of synthetic N is to reduce the energy footprint of its production by increasing the use of renewable resources to drive the Haber–Bosch process (Wang et al. 2018), or by identifying alternative low-energy technologies for synthetic N production (Qiu et al. 2018). However, these approaches do not address the downstream ecological consequences of synthetic N use listed above. Another approach is to increase the use of organic fertilizers (Celestina et al. 2019), and there is considerable demand for the transformation of waste streams into organic fertilizers through composting, fermentation or thermochemical processing (Sudharmaidevi, Thampatti and Saifudeen 2017). However, the use of organic fertilizers has been controversial and restricted because organic fertilizers have also been associated with increased generation of greenhouse gas emissions, heavy metal accumulation/leaching and as a source of emerging contaminants (Moran-Salazar et al. 2016; Lekfeldt et al. 2017; Pan and Chu 2017; Ibekwe, Gonzalez-Rubio and Suarez 2018; Weithmann et al. 2018). The advent of synthetic biology has also offered some possible ‘silver bullets’ that are being investigated intensely. The most drastic approach would be through ‘extreme bioengineering’ of N fixation directly into the plants (Burén, López-Torrejón and Rubio 2018). Although efforts to engineer plants for N fixation have made strides through eukaryotic nitrogenase expression, achieving significant N fixation under field conditions is likely decades away and would require a concerted effort and investment (Good 2018). Furthermore, the ecological consequences of cultivating such plants are completely unknown but would undoubtedly alter global N cycles. Another such ‘silver bullet’ is to engineer symbiont nodules into cereal roots (Rosenblueth et al. 2018). Strategies for engineering rhizobial infection and nodule organogenesis so far remain elusive (Bloch et al. 2020), and again, the ecological consequences of engineering such interactions are unknown but would likely have global impacts. RESTORING NATURAL POPULATIONS OF FREE-LIVING DIAZOTROPHS Biological nitrogen fixation (BNF), through either symbiotic or free-living organisms, provides another route for N into the agroecosystem. For example, synthetic N demand may be decreased by intercropping cereals with nodule-forming legumes (Brooker et al. 2015; Iannetta et al. 2016). A strength of intercropping is the relatively low cost of entry and scalability (Reckling et al. 2020). Another approach to leverage BNF is through biofertilization with either symbiotic or free-living inoculants (Schütz et al. 2018). Research efforts in biofertilization are generally small scale and are primarily focused on screening, characterizing and formulating isolates, and more recently synthetic communities (Kong, Hart and Liu 2018; Schütz et al. 2018; Toju et al. 2018). The first commercial biofertilizer was already patented in the 19th century, and there are now many products on the market (García-Fraile, Menéndez and Rivas 2015). Generally speaking, the goal of biofertilization efforts is to identify isolates that exhibit the highest ability for a particular function. Once these ‘top performers’ have been identified, they may be mass produced for field application. A meta-analysis of nodule-forming rhizobia highlights a major challenge with this approach: the effectiveness of individual rhizobial isolates is only observed where the indigenous population of rhizobia is low (Thilakarathna and Raizada 2017). One cause of this may stem from fitness trade-offs: isolates that are ‘top performers’ in one environment are maladapted to others (Kaminsky et al. 2019). Indeed, the relationship between the competitiveness of rhizobial isolates that form nodules is often not correlated with their nitrogen-fixing capacity (Bourion et al. 2018). A different meta-analysis highlights another challenge: biofertilizers seem to be most effective when multiple inoculant species are applied (e.g. as co-inoculants) (Schütz et al. 2018), and the efficacy of multispecies inoculants has been shown to improve with increasing diversity (Hu et al. 2016). An alternative approach to biofertilization is soil transplantation, which aims to leverage the function and diversity of one ecosystem (with a desired function) to restore (a subset) these functions in a target ecosystem (Wubs et al. 2016). A classic example of a soil transplantation experiment is the case of disease suppressive soils. This function has been shown to be transferable even at dilutions of 1/1000 from the source soil (Cook and Rovira 1976). However, soil transplantation experiments are not always successful, and may require much higher proportions for crop application (Ma et al. 2020). One of the main drawbacks of soil transplantation is the logistical problems associated with scaling and implementing such large-scale projects. Another important consideration is that scaling soil transplants would put further strain on the natural ecosystems, especially as long-term agricultural practices will require repeated biofertilization. Furthermore, as there are few soil transplantation field studies, the success rate and the mechanisms are relatively unknown. The practical trade-offs between biofertilization approaches may be summarized based on the following characteristics: the complexity of the inoculant and the scalability of the approach (Fig. 1). On one end of the spectrum, ‘top-performing’ isolates are identified and produced at industrial scale. By producing synthetic microbial communities (SynComs), it is possible to increase the complexity of microbial inoculants, and there have been significant inroads in the design and mechanisms of complex SynComs (Finkel et al. 2020). However, there is a trade-off between the complexity and scalability of SynCom production, as each isolate must be produced individually before formulation. On the other end of the spectrum, soil transplantations provide an inoculant with a much higher number of species, but with a trade-off in scalability, as any particular soil is finite. Thus, the current methods for biofertilization represent two extremes of the spectrum in the complexity and scalability of the product. While diverse microbial inoculants are not always better than ‘top performers’, approaches that leverage multiple inoculants often do outperform single isolates (Hu et al. 2016; Schütz et al. 2018), highlighting the need for an intermediate approach that can produce biofertilizers that are complex and scalable (Fig. 1). To address this gap in the production of scalable and complex microbial biofertilizers, we propose a novel approach that leverages ecological theory and process engineering to produce a ‘self-assembled’ community enriched in particular functions related to plant growth promotion, nutrient acquisition or disease suppression, and adapted to a target environment. Figure 1. Open in new tabDownload slide The trade-off between the complexity and scalability of production for isolate-based (●), soil transplant (▲) and self-assembled (▓) biofertilizers. Irrespective of the efficacy of a biofertilizer, a key challenge is the scalability of production. For a single isolate, mass production is relatively straightforward. However, as the number of species in an isolate-based inoculum increases, the ability to scale production decreases, as each isolate must be produced separately and subsequently formulated together. In the case of soil transplantation, an immensely complex and diverse inoculum is available; however, the trade-off is that any given source soil is finite, and thus, scaling must be achieved by diluting the soil. Eventually, a dilution is reached in which the complexity of the soil is so diluted that the beneficial aspects of the soil are lost. In contrast to isolate-based approaches, self-assembled communities may be mass produced in a single reactor, thus allowing the scalable production. As detailed in this review, community assembly may be guided toward a desired output through careful bioprocess engineering and ecological parameterization. Thus, self-assembled communities represent a highly scalable approach to produce complex biofertilizers. Figure 1. Open in new tabDownload slide The trade-off between the complexity and scalability of production for isolate-based (●), soil transplant (▲) and self-assembled (▓) biofertilizers. Irrespective of the efficacy of a biofertilizer, a key challenge is the scalability of production. For a single isolate, mass production is relatively straightforward. However, as the number of species in an isolate-based inoculum increases, the ability to scale production decreases, as each isolate must be produced separately and subsequently formulated together. In the case of soil transplantation, an immensely complex and diverse inoculum is available; however, the trade-off is that any given source soil is finite, and thus, scaling must be achieved by diluting the soil. Eventually, a dilution is reached in which the complexity of the soil is so diluted that the beneficial aspects of the soil are lost. In contrast to isolate-based approaches, self-assembled communities may be mass produced in a single reactor, thus allowing the scalable production. As detailed in this review, community assembly may be guided toward a desired output through careful bioprocess engineering and ecological parameterization. Thus, self-assembled communities represent a highly scalable approach to produce complex biofertilizers. The use of bioreactors provides many additional levels of real-time control on environmental and biological parameters such as pH, gas flux, temperature, growth rate and form, and may thus be used to recapitulate the ecological parameters required to drive the assembly of communities with even highly specialized metabolisms (Imachi et al. 2020). The improvements that bioreactors provide over traditional plate-based techniques have long been recognized as a powerful tool to investigate isolated rhizosphere diazotrophs (Fritzsche, Ueckert and Niemann 1991; Ueckert, Huckfeldt and Fendrik 1995; Liu et al. 2017). However, there are only few examples of bioreactors used to enrich communities under simulated rhizospheric conditions (Peacock et al. 2014; Xiao et al. 2018), and none investigating rhizospheric diazotroph communities. Despite this, free-living diazotrophs are commonly enriched in other community-based biotechnologies such as wastewater treatment (Welz et al. 2018; Ospina-Betancourth et al. 2020) or for the production of the bioplastic polyhydroxyalkanoate (Patel et al. 2009). Thus, there is ample evidence that not only bioprocess engineering may enrich functionally diverse diazotrophs (Bowers, Reid and Lloyd-Jones 2008; Reid, Bowers and Lloyd-Jones 2008) but also such an approach is generalizable and may be used to produce self-assembled communities with other functions of agro-ecological interest such as phosphorus (P) solubilization and chitin degradation (Cretoiu et al. 2013), or the simultaneous selection of multiple functions (Oyserman et al. 2017). To root our perspective, we use nitrogen fixation as a target function for restoration. We first discuss the key ecological parameters that must be recapitulated in order to select for diazotrophs based on not only what is known about the microbial physiology of diazotrophs but also the target environment (soil/rhizosphere) and host (crop). We then consider how these ecological parameters may be integrated with bioprocess engineering and reactor design strategies for the production of self-assembled communities (Fig. 2). Figure 2. Open in new tabDownload slide A conceptual overview of the design, production and application of self-assembled communities. First, the ecology (in green) of a system must be investigated. A target host and environment are identified, as well as the desired function(s) to be enriched. Based on the target environment, host and function, and the desired end formulation, operational parameters are determined and other bioprocess engineering considerations are made, such as the choice of the carrier media (in blue). The final formulation, level of diversity and function produced are dependent on the operational parameters. For example, a P-solubilizing and nitrogen-fixing community may be enriched using N- and P-limited media on a P-rich mineral fertilizer as a carrier medium (in purple). With increased understanding of the ecology of both target system and bioprocess design, increasingly tailored and effective self-assembled communities may be produced. Figure 2. Open in new tabDownload slide A conceptual overview of the design, production and application of self-assembled communities. First, the ecology (in green) of a system must be investigated. A target host and environment are identified, as well as the desired function(s) to be enriched. Based on the target environment, host and function, and the desired end formulation, operational parameters are determined and other bioprocess engineering considerations are made, such as the choice of the carrier media (in blue). The final formulation, level of diversity and function produced are dependent on the operational parameters. For example, a P-solubilizing and nitrogen-fixing community may be enriched using N- and P-limited media on a P-rich mineral fertilizer as a carrier medium (in purple). With increased understanding of the ecology of both target system and bioprocess design, increasingly tailored and effective self-assembled communities may be produced. ECOLOGICAL PARAMETERS FOR COMMUNITY SELF-ASSEMBLY To engineer a community-based bioprocess, it is important to recapitulate the ecological conditions that select for the natural assembly of a community with a desired function. In the case of biofertilizers, this must be taken one step further, as selection for traits related to survivability in the target soil and rhizosphere must also be accounted for. With regard to nitrogen fixation, the most obvious selective parameter is nitrogen limitation. However, what constitutes ‘N-limiting’ is based on the stoichiometry of various elements and are species specific (Dynarski and Houlton 2018; Inomura et al. 2018; Zheng et al. 2018, 2020). For example, recent work developing a quantitative model of nitrogen fixation in the heterotrophic bacteria Azotobacter vinelandii shows that nitrogen fixation may occur in the presence of ammonium as long as the C:N ratios are sufficiently high at a given O2 availability (Inomura et al. 2018). As a starting guide, C:N ratios in soil typically fall between 8:1 and 16:1 (Tian et al. 2010). Interestingly, despite the broad range of soil C:N ratios, a meta-analysis of soil microbial stoichiometry suggests that soil microbial stoichiometry is uncoupled from these ratios and a stoichiometry of 60:7:1 C:N:P has been proposed for soil microbes (Cleveland and Liptzin 2007). Furthermore, the C:N ratios of leaf litter are much higher and can reach between 50:1 and 90:1 (McGroddy, Daufresne and Hedin 2004). These ratios may be used as a starting point, but currently little is known about how C:N ratios impact the selection of different diazotroph lineages, nor the outcome of selection when different ratios are interplayed between other parameters such as oxygen. Another major selective parameter for diazotrophs is oxygen. Broadly speaking, diazotrophs may be classified as anaerobic, microaerophilic and aerobic. This broad range of oxygen tolerance is surprising given that nitrogenase, the enzyme responsible for nitrogen fixation, is irreversibly inhibited by oxygen. Therefore, all diazotrophs regardless of their life history must employ mechanisms to maintain anaerobic conditions for a functional nitrogenase (Gallon 1981). However, even within a species, the level of sensitivity and responses to oxygen are rather plastic and adaptive (Dingler and Oelze 1985). Indeed, the scavenging mechanisms employed are quite costly, and because of this, nitrogen fixation is believed to be most efficient under microaerophilic conditions as shown in A. vinelandii (Inomura et al. 2018) and Crocosphaera watsonii (Großkopf 2012). Thus, on the one hand, by finely tuning bioreactor oxygen concentrations, a highly specialized subset of diazotrophs may be selected for. Conversely, by engineering conditions with varied concentrations of oxygen through time, space or both, diazotroph communities that may invade various oxygen niches in the soil may be enriched. Spatially, such niches may range from microaerophilic in the endosphere or rhizoplane where respiration is likely highest, to much higher concentrations of oxygen further away from the roots. Temporally, oxygen concentrations likely follow cyclic diurnal rhythms based on the photosynthetic status of the plant. Such patterns may be mimicked by careful selection of reactor type and operational parameters as described in subsequent sections. Many other factors exist that are related to the efficiency of nitrogen fixation at a molecular level. For example, while the primary nitrogenases are molybdenum dependent (Mo-nitrogenase), alternative vanadium- or iron-dependent nitrogenases may also be used to fix nitrogen (V-nitrogenase and Fe-nitrogenase, respectively) when molybdenum is limiting (Smercina et al. 2019). While these alternative nitrogenases are less efficient under most conditions, V-nitrogenase appears to be more efficient under low temperatures (Bellenger et al. 2014). As with the case above with determining nitrogen limitation, what constitutes molybdenum deficiency is also dependent on the environmental context, as P availability may play a large interacting role, especially when both nutrients are limiting (Reed, Cleveland and Townsend 2013). Indeed, P availability in itself can also be a limiting factor of nitrogen fixation (Reed et al. 2007), and therefore it is not surprising that many diazotrophs tested for biofertilization are also phosphorus solubilizers (Schütz et al. 2018). Thus, by applying selective pressures matching target soil characteristics such as trace element and nutrient stoichiometry, scalable production of pre-adapted biofertilizers may be achieved decoupled from limitations of enriching these communities directly in the soil (e.g. seasonality) (Fig. 2). The availability of soil carbon was once thought to be a limitation for free-living diazotrophs; however, it is now known that plants transfer around 20–50% of the total assimilated carbon into the soil to recruit microbes using root exudates (Haichar et al. 2014). Indeed, the quantity and quality of root exudation is now considered an important functional trait associated with high growth rates and N acquisition (Guyonnet et al. 2018). Up to 90% of root exudate carbon may be taken up by microbial biomass (Smercina et al. 2019). The current state of the art in the quantity and quality of root exudates is by studies implementing axenic systems (Kuijken et al. 2015). From such systems, it is clear that organic acids and sugars are the primary components of root exudates, but the concentrations and make-up are both species specific and context dependent (Johnson et al. 1996; Kravchenko et al. 2003). Advances in methodology continue to put this exciting field into focus, such as the role of primary and secondary metabolites in shaping the microbiome (Szoboszlay, White-Monsant and Moe 2016; Girkin et al. 2018; Stringlis et al. 2018) and the use of split root systems to investigate how microbial inoculation alters root exudate profiles (Korenblum et al. 2020). Therefore, the selection of dominant carbon sources and dosing of secondary metabolites matching the host plant are important controls that will impact the diversity and function of self-assembled communities (Fig. 2). In particular, a key focus here should be investigating how biotic/abiotic stresses alter root exudates, and how such a ‘cry for help’ may be leveraged to select communities adapted to them (Berendsen et al. 2018). For example, addition of caffeic acid, a phenolic root exudate and important intermediate in the biosynthesis of lignin, has been used to target the isolation of endophytic diazotrophs (Adachi, Nakatani and Mochida 2002). A recent review has summarized the numerous formulations of N-free media that have been to isolate diazotrophs (Baldani et al. 2014), and experiments using artificial root exudates may also serve as an initial guide (Baudoin, Benizri and Guckert 2003). Altering these formulations to match soil chemistry and crop exudate specifics may serve as the ecological basis for selection of self-assembled diazotrophic communities (Fig. 2). BIOPROCESS ENGINEERING FOR COMMUNITY SELF-ASSEMBLY In the previous section, we detailed how environmental parameters such as C:N:P ratios, oxygen concentrations, trace elements, primary carbon sources and secondary metabolites may be used to steer diazotrophic community assembly. In this section, we explore how, given these parameters, different reactor types (attached versus suspended growth), mode of operation (batch, fed batch or continuous) and process parameters (hydraulic retention time, solid retention time and particle size distribution) may be used to further control community assembly. We highlight how these parameters may be tuned to obtain a desired functionality and level of diversity, and contextualize these technical aspects with practical decisions related to the production of diverse self-assembled biofertilizers (Fig. 2). While product consistency will be a major challenge for self-assembled communities, it is important to note that complex bioprocesses often have high spatial and temporal habitat heterogeneity but still can lead to predictable community structures (Nielsen et al. 2012). Furthermore, open reactor systems operating with high degrees of spatial and temporal heterogeneity may still be consistently enriched with the same target taxa (∼90% relative abundance), and their growth state highly characterized (Oyserman et al. 2016a,b). Thus, while there are certainly additional challenges to using mixed communities rather than single isolates, there is also a long history of successful innovation in novel reproduceable bioprocesses, as exemplified by the discovery and now widespread implementation of Anammox (Kuenen 2008). Broadly speaking, reactors may be categorized into attached growth or suspended growth systems. An attached growth design employs a carrier medium, or packing material, to support biofilm growth. This carrier medium may be chosen based on a combination of practical considerations related to biological properties, costs, and downstream application. Essentially, any material may be used as a carrier and can either be fixed or suspended in the reactor (Loupasaki and Diamadopoulos 2013). Similarly, packed bed columns or soil columns may be used (Wery et al. 2003; Wei et al. 2016). The choice of carrier medium is important, especially when it is desired to apply the carrier medium containing the biofertilizer applied directly to the soil (Akay and Fleming 2012). One advantage of using an attached growth system is that existing and emerging agricultural inputs such as phosphate rocks, struvite, sand, wood chips, saw dust and chitinaceous material may be used as a carrier medium and hence ‘activated’ with key agro-ecologically relevant functions such as phosphorus solubilization, or chitin degradation before their application into the agroecosystem. Another type of attached growth system that has been used for the production of biofertilizer isolates is solid-state fermentation (Jin et al. 2019). Solid-state fermentation is an especially alluring process, as it uses minimal water and may therefore be more suitable for mimicking soil conditions, or even cycling drought conditions. In contrast, suspended growth reactors do not contain carrier mediums. Growth is suspended within the reactor as either free-living cells, or as floccular and granular structures. Suspended growth systems may subsequently be applied directly to the soil or formulated with a natural or synthetic carrier and then applied to the soil (Malusá, Sas-Paszt and Ciesielska 2012). The level of diversity that develops within a community may be managed by adjusting the level of spatial and temporal heterogeneity. Spatial heterogeneity may be impacted on both macro- and microscale. On the macroscale, heterogeneity may be achieved when the packing material is fixed in relation to the flow of the media, such as in a soil column reactor or trickle bed reactor. In such systems, influent concentrations are spatially altered through the flow of the reactor and thus organisms at the beginning generally experience high concentrations of influent substrates, whereas organisms at the end of the process are exposed to low concentrations of influent substrates, but high concentrations of waste products. In these systems, it is common for diverse organisms carrying out differential biogeochemical processes to establish through the column (Wery et al. 2003). Thus, packed bed and soil column reactors promote spatial segregation of microorganisms optimized for specific environments. Microscale spatial heterogeneity may be promoted through the developments of biofilm (Stewart and Franklin 2008). In suspended growth systems, spatial heterogeneity is dependent on the particle size distribution (i.e. the thickness of the biofilm). As particle size increases in floccular or granular communities, diffusion through these biofilms contributes to increasing environmental heterogeneity. Biofilm formation is an important rhizosphere process and associated with disease suppression (Bais, Fall and Vivanco 2004), and therefore, particle size distribution and biofilm thickness is likely a key operational parameter in selecting for self-assembled biofertilizers. Indeed, the choice of carrier media may also be done to promote such heterogeneity. For example, using mineral fertilizers containing insoluble P as a carrier medium in a P-depleted system would favor the formation of a biofilm on this mineral fertilizer and the establishment of two countering gradients of oxygen (into the biofilm) and P (out of the biofilm). In addition to the spatial heterogeneity discussed above, the temporal heterogeneity of a process may also be established. The simplest design is a batch system, in which influent addition occurs at a single time point, operation occurs until a reaction has gone to completion and concentrations of resources vary through the time of production. Many industrial bioprocesses such as beer production and other microbial fermentation products are operated as batch systems. In this manner, community members adapted to different concentrations may be differentially active at the beginning and end of a bioprocess. Such temporal heterogeneity may be important for selecting diverse communities and must be accounted for when considering product consistency. However, this heterogeneity may also be exploited to select the appropriate growth state, such as the production of storage polymers, which has been shown to improve inoculant survivability. Furthermore, as community structure increasingly diverges from the original community structure after inoculation before reaching stability (Trebuch et al. 2020), by operating over a single reaction cycle, the community that develops is not so far removed from the original inoculant. Determining the length of batch operation is also necessary. This may be determined by monitoring growth or substrate use. A sequencing batch reactor is a simple extension of the batch reactor. To operate a sequencing batch reactor, the reactor must simply undergo consecutive fill and draw cycles in which the biomass is retained but the bulk fluid replaced with new influent. In this way, biomass may be accumulated to a desired concentration. The repeated fill and draw cycles may be advantageous when enriching communities for dynamic physiologies such as polymer cycling and storage (Oyserman et al. 2016b), which has been shown to be an important factor in the successful application of biofertilizers (Okon and Itzigsohn 1995). After consecutive cycles, the community structure may take on emergent properties such as the formation of floccular and granular biomass (Jarvis et al. 2005), which may be leveraged in downstream processes such as harvesting and application. For example, one of the key features of floccular and granular structures is their high content of extracellular polysaccharides and alginate-like substances (Trebuch et al. 2020), which, again, has been shown to improve biofertilizer viability after application (Okon and Itzigsohn 1995). Finally, dual approaches that combine isolates with community based-approaches may also be successful. For example, starting inoculants may include ‘top-performing’ isolate(s), or may be inoculated at high densities after community assembly has already been initiated. Indeed, understanding how/when isolates can invade established communities remains a key challenge in microbiome engineering. Such challenges will also have to be addressed with the application of self-assembled communities. CONCLUSIONS AND FUTURE PERSPECTIVES Intensive agricultural practices have resulted in the global degradation of microbial functions in soils. Restoring these microbial communities is important for the future sustainability of agriculture. Furthermore, restoring microbial function is important when these agricultural systems are converted back into natural systems. Current methods for biofertilization are limited in their diversity and scalability. On the one hand, the production of isolate-based biofertilizers may take advantage of the scales of industry. However, isolate-based biofertilizers are inherently limited in their complexity, even in the context of synthetic communities. On the other hand, soil transplantation experiments are at the other end of the spectrum. While they are diverse, scaling soil transplantations is logistically not feasible because natural soils are a limited natural resource that may also be depleted. To address these deficiencies, we present a novel approach to produce functional, diverse and scalable biofertilizers by merging bioprocess engineering and ecological theory. There are many microbial functions that could be targeted by such an approach, and ecological selection is often already applied to screen isolates with these characteristics. For example, to identify microbes with ability to degrade 1-aminocyclopropane-1-carboxylic acid (ACC), isolates are often grown with ACC as a sole nitrogen source. Such selective strategies can be directly applied to select for self-assembled communities enriched in such functions. In this manner, communities with enhanced abilities for phosphorus solubilization, siderophore production, chitin degradation and many other agro-ecologically relevant traits may be targeted. Interestingly, BNF organisms often have many of these functions, and therefore, it can be hypothesized that selecting for key functions such as BNF may act as a ‘Trojan Horse’, whereby many desired traits accompany the selection of a key function. Selection may also take a deliberately modular approach and select for compatibility between community members with different functions (Oyserman, Medema and Raaijmakers 2018). For example, a carrier medium composed of recalcitrant insoluble phosphorus in nitrogen-free media would select for phosphorus-solubilizing and nitrogen-fixing organisms. Thus, by moving beyond classic isolate-based approaches and leveraging ecological theory merged with bioprocess engineering, it is likely that current challenges related to soil health may be addressed. Furthermore, bioprocesses such as anaerobic digestion are increasingly operated by small farms (Wang 2014; Imeni et al. 2020). Thus, advances in the production of self-assembled communities for biofertilization not only provide a means to produce highly complex and scalable production of biofertilizers but also may provide farmers and land managers a new tool in managing soil health at the local level. Currently, the potential for self-assembled communities as biofertilizers is completely unexplored and we hope that outlining the ecological and engineering considerations here will spur further research on this topic. ACKNOWLEDGMENTS The authors thank Joerg Ueckert and Jort Altenburg for constructive discussion about reactor types. Publication number 7065 of the Netherlands Institute of Ecology, NIOO-KNAW. FUNDING The contributions of BOO and JMR were funded in part by the Dutch Technology Foundation of the Dutch National Science Foundation (NWO-Toegepaste en Technische Wetenschappen). The contributions of CFGL were funded by COLCIENCIAS and the Universidad del Valle Convocatoria Interna para Apoyo a la Movilidad. AUTHORS’ CONTRIBUTIONS BOO and CFGL conceived and drafted the manuscript. JSG and JMR provided critical feedback and discussion. All authors contributed critically to the drafts and gave final approval for publication. Conflict of Interest None declared. REFERENCES Adachi K , Nakatani M, Mochida H. Isolation of an endophytic diazotroph, Klebsiella oxytoca, from sweet potato stems in Japan . Soil Sci Plant Nutr . 2002 ; 48 : 889 – 95 . Google Scholar Crossref Search ADS WorldCat Akay G , Fleming S. 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TI - Restoring degraded microbiome function with self-assembled communities
JF - FEMS Microbiology Ecology
DO - 10.1093/femsec/fiaa225
DA - 2020-11-26
UR - https://www.deepdyve.com/lp/oxford-university-press/restoring-degraded-microbiome-function-with-self-assembled-communities-Empulo0kGm
VL - 96
IS - 12
DP - DeepDyve
ER -