Restoring degraded microbiome function with self-assembled communities
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FEMS Microbiology Ecology, 96, 2020, fiaa225
doi: 10.1093/femsec/fiaa225
Advance Access Publication Date: 5 November 2020
Minireview
MINIREVIEW
Restoring degraded microbiome function with
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self-assembled communities
Carlos Fernando Gutierrez1,† , Janeth Sanabria1,‡ , Jos M. Raaijmakers2,3,‡ and
Ben O. Oyserman2,4, *
1
Environmental Microbiology and Biotechnology Laboratory, Engineering School of Environmental and Natural
Resources, Engineering Faculty, Universidad del Valle, 760032, Cali, Colombia, 2 Department of Microbial
Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB, Wageningen, The
Netherlands, 3 Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands and
4
Bioinformatics Group, Wageningen University, Droevendaalsesteeg 1,6708 PB, Wageningen, The Netherlands
∗
Corresponding author: Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB, Wageningen,
The Netherlands. Tel: +31 (0)317 47 34 00; E-mail: BenOyserman@gmail.com
One sentence summary: Microbiomes are essential for healthy ecosystem function, but many microbiomes are severely degraded, and self-assembled
communities of microbes enriched in particular functions provide a scalable approach to restore these degraded functions.
‡
These authors contributed equally to this work.
Editor: Marcus Horn
†
Carlos Fernando Gutierrez Landazuri, http://orcid.org/0000-0002-1542-1751
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.
Keywords: biological nitrogen fixation; biotechnology; biofertilization; microbial ecology; diazotrophs; soil fertilization;
restoration ecology; community assembly
Received: 16 August 2020; Accepted: 3 November 2020
C The Author(s) 2020. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail:
journals.permissions@oup.com.
1
2 FEMS Microbiology Ecology, 2020, Vol. 96, No. 12
A CENTURY OF DEGRADATION: THE IMPACT RESTORING NATURAL POPULATIONS OF
OF SYNTHETIC FERTILIZER AND FREE-LIVING DIAZOTROPHS
ALTERNATIVES Biological nitrogen fixation (BNF), through either symbiotic
The use of synthetic nitrogen (N) for crop fertilization con- or free-living organisms, provides another route for N into
tributes tremendously to the world’s food supply, but the pro- the agroecosystem. For example, synthetic N demand may
duction and application of these fertilizers also contribute to be decreased by intercropping cereals with nodule-forming
a global environmental imbalance. It is estimated that syn- legumes (Brooker et al. 2015; Iannetta et al. 2016). A strength
thetic N is the second most manufactured synthetic product of intercropping is the relatively low cost of entry and scala-
in the world (Elishav et al. 2017), and the Haber–Bosch process bility (Reckling et al. 2020). Another approach to leverage BNF
used to drive this production is estimated to consume 1% of is through biofertilization with either symbiotic or free-living
the global primary energy produced (Cherkasov, Ibhadon and inoculants (Schütz et al. 2018). Research efforts in biofertiliza-
Fitzpatrick 2015). In addition to the high energy cost, N fer- tion are generally small scale and are primarily focused on
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tilizers alter microbiome function, increasing soil respiration screening, characterizing and formulating isolates, and more
and nitrous oxide emissions (Bicer et al. 2017) equivalent to recently synthetic communities (Kong, Hart and Liu 2018; Schütz
1–2.5% of global greenhouse gas emissions (Pfromm 2017), all et al. 2018; Toju et al. 2018). The first commercial biofertilizer
the while reducing soil diversity (Dai et al. 2018) and degrad- was already patented in the 19th century, and there are now
ing the natural community structure and nitrogen-fixing capa- many products on the market (Garcı́a-Fraile, Menéndez and
bilities of soils (Wang et al. 2017; Luo et al. 2018). Paradoxically, Rivas 2015).
the increased use of N fertilizers has coincided with a signifi- Generally speaking, the goal of biofertilization efforts is to
cant global decrease in the efficiency of N use over the last 50 identify isolates that exhibit the highest ability for a particular
years (Lassaletta et al. 2014). Only 4–14% of synthetic N applied function. Once these ‘top performers’ have been identified, they
to croplands enters the economy as a downstream final prod- may be mass produced for field application. A meta-analysis
uct (Pikaar et al. 2017). Moving forward, increasing N-use effi- of nodule-forming rhizobia highlights a major challenge with
ciency is integral to increased agricultural sustainability (Schils this approach: the effectiveness of individual rhizobial isolates
et al. 2018). is only observed where the indigenous population of rhizobia
One approach to reduce the impact of synthetic N is to is low (Thilakarathna and Raizada 2017). One cause of this may
reduce the energy footprint of its production by increasing stem from fitness trade-offs: isolates that are ‘top performers’
the use of renewable resources to drive the Haber–Bosch pro- in one environment are maladapted to others (Kaminsky et al.
cess (Wang et al. 2018), or by identifying alternative low-energy 2019). Indeed, the relationship between the competitiveness of
technologies for synthetic N production (Qiu et al. 2018). How- rhizobial isolates that form nodules is often not correlated with
ever, these approaches do not address the downstream eco- their nitrogen-fixing capacity (Bourion et al. 2018). A different
logical consequences of synthetic N use listed above. Another meta-analysis highlights another challenge: biofertilizers seem
approach is to increase the use of organic fertilizers (Celestina to be most effective when multiple inoculant species are applied
et al. 2019), and there is considerable demand for the transfor- (e.g. as co-inoculants) (Schütz et al. 2018), and the efficacy of mul-
mation of waste streams into organic fertilizers through com- tispecies inoculants has been shown to improve with increasing
posting, fermentation or thermochemical processing (Sudhar- diversity (Hu et al. 2016).
maidevi, Thampatti and Saifudeen 2017). However, the use of An alternative approach to biofertilization is soil transplan-
organic fertilizers has been controversial and restricted because tation, which aims to leverage the function and diversity of
organic fertilizers have also been associated with increased one ecosystem (with a desired function) to restore (a subset)
generation of greenhouse gas emissions, heavy metal accu- these functions in a target ecosystem (Wubs et al. 2016). A clas-
mulation/leaching and as a source of emerging contaminants sic example of a soil transplantation experiment is the case
(Moran-Salazar et al. 2016; Lekfeldt et al. 2017; Pan and Chu of disease suppressive soils. This function has been shown to
2017; Ibekwe, Gonzalez-Rubio and Suarez 2018; Weithmann be transferable even at dilutions of 1/1000 from the source soil
et al. 2018). (Cook and Rovira 1976). However, soil transplantation experi-
The advent of synthetic biology has also offered some possi- ments are not always successful, and may require much higher
ble ‘silver bullets’ that are being investigated intensely. The most proportions for crop application (Ma et al. 2020). One of the main
drastic approach would be through ‘extreme bioengineering’ of drawbacks of soil transplantation is the logistical problems asso-
N fixation directly into the plants (Burén, López-Torrejón and ciated with scaling and implementing such large-scale projects.
Rubio 2018). Although efforts to engineer plants for N fixation Another important consideration is that scaling soil transplants
have made strides through eukaryotic nitrogenase expression, would put further strain on the natural ecosystems, especially
achieving significant N fixation under field conditions is likely as long-term agricultural practices will require repeated biofer-
decades away and would require a concerted effort and invest- tilization. Furthermore, as there are few soil transplantation
ment (Good 2018). Furthermore, the ecological consequences field studies, the success rate and the mechanisms are relatively
of cultivating such plants are completely unknown but would unknown.
undoubtedly alter global N cycles. Another such ‘silver bul- The practical trade-offs between biofertilization approaches
let’ is to engineer symbiont nodules into cereal roots (Rosen- may be summarized based on the following characteristics: the
blueth et al. 2018). Strategies for engineering rhizobial infection complexity of the inoculant and the scalability of the approach
and nodule organogenesis so far remain elusive (Bloch et al. (Fig. 1). On one end of the spectrum, ‘top-performing’ isolates
2020), and again, the ecological consequences of engineering are identified and produced at industrial scale. By producing
such interactions are unknown but would likely have global synthetic microbial communities (SynComs), it is possible to
impacts. increase the complexity of microbial inoculants, and there haveGutierrez et al. 3
polyhydroxyalkanoate (Patel et al. 2009). Thus, there is ample
evidence that not only bioprocess engineering may enrich func-
tionally 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 com-
munities 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
Figure 1. The trade-off between the complexity and scalability of production for select for diazotrophs based on not only what is known about
isolate-based (•), soil transplant () and self-assembled () biofertilizers. Irre- the microbial physiology of diazotrophs but also the target envi-
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spective of the efficacy of a biofertilizer, a key challenge is the scalability of
ronment (soil/rhizosphere) and host (crop). We then consider
production. For a single isolate, mass production is relatively straightforward.
However, as the number of species in an isolate-based inoculum increases, the
how these ecological parameters may be integrated with bio-
ability to scale production decreases, as each isolate must be produced sepa- process engineering and reactor design strategies for the pro-
rately and subsequently formulated together. In the case of soil transplantation, duction of self-assembled communities (Fig. 2).
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 dilut-
ing 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- ECOLOGICAL PARAMETERS FOR COMMUNITY
based approaches, self-assembled communities may be mass produced in a sin- SELF-ASSEMBLY
gle reactor, thus allowing the scalable production. As detailed in this review,
community assembly may be guided toward a desired output through careful To engineer a community-based bioprocess, it is important to
bioprocess engineering and ecological parameterization. Thus, self-assembled recapitulate the ecological conditions that select for the natu-
communities represent a highly scalable approach to produce complex biofertil- ral assembly of a community with a desired function. In the
izers.
case of biofertilizers, this must be taken one step further, as
selection for traits related to survivability in the target soil and
been significant inroads in the design and mechanisms of com- rhizosphere must also be accounted for. With regard to nitro-
plex SynComs (Finkel et al. 2020). However, there is a trade-off gen fixation, the most obvious selective parameter is nitrogen
between the complexity and scalability of SynCom production, limitation. However, what constitutes ‘N-limiting’ is based on
as each isolate must be produced individually before formula- the stoichiometry of various elements and are species specific
tion. On the other end of the spectrum, soil transplantations (Dynarski and Houlton 2018; Inomura et al. 2018; Zheng et al.
provide an inoculant with a much higher number of species, but 2018, 2020). For example, recent work developing a quantita-
with a trade-off in scalability, as any particular soil is finite. Thus, tive model of nitrogen fixation in the heterotrophic bacteria
the current methods for biofertilization represent two extremes Azotobacter vinelandii shows that nitrogen fixation may occur in
of the spectrum in the complexity and scalability of the prod- the presence of ammonium as long as the C:N ratios are suffi-
uct. While diverse microbial inoculants are not always better ciently high at a given O2 availability (Inomura et al. 2018). As a
than ‘top performers’, approaches that leverage multiple inoc- starting guide, C:N ratios in soil typically fall between 8:1 and
ulants often do outperform single isolates (Hu et al. 2016; Schütz 16:1 (Tian et al. 2010). Interestingly, despite the broad range of
et al. 2018), highlighting the need for an intermediate approach soil C:N ratios, a meta-analysis of soil microbial stoichiometry
that can produce biofertilizers that are complex and scalable suggests that soil microbial stoichiometry is uncoupled from
(Fig. 1). To address this gap in the production of scalable and these ratios and a stoichiometry of 60:7:1 C:N:P has been pro-
complex microbial biofertilizers, we propose a novel approach posed for soil microbes (Cleveland and Liptzin 2007). Further-
that leverages ecological theory and process engineering to pro- more, the C:N ratios of leaf litter are much higher and can reach
duce a ‘self-assembled’ community enriched in particular func- between 50:1 and 90:1 (McGroddy, Daufresne and Hedin 2004).
tions related to plant growth promotion, nutrient acquisition or These ratios may be used as a starting point, but currently lit-
disease suppression, and adapted to a target environment. tle is known about how C:N ratios impact the selection of dif-
The use of bioreactors provides many additional levels of ferent diazotroph lineages, nor the outcome of selection when
real-time control on environmental and biological parameters different ratios are interplayed between other parameters such
such as pH, gas flux, temperature, growth rate and form, and as oxygen.
may thus be used to recapitulate the ecological parameters Another major selective parameter for diazotrophs is oxygen.
required to drive the assembly of communities with even highly Broadly speaking, diazotrophs may be classified as anaerobic,
specialized metabolisms (Imachi et al. 2020). The improvements microaerophilic and aerobic. This broad range of oxygen toler-
that bioreactors provide over traditional plate-based techniques ance is surprising given that nitrogenase, the enzyme respon-
have long been recognized as a powerful tool to investigate iso- sible for nitrogen fixation, is irreversibly inhibited by oxygen.
lated rhizosphere diazotrophs (Fritzsche, Ueckert and Niemann Therefore, all diazotrophs regardless of their life history must
1991; Ueckert, Huckfeldt and Fendrik 1995; Liu et al. 2017). How- employ mechanisms to maintain anaerobic conditions for a
ever, there are only few examples of bioreactors used to enrich functional nitrogenase (Gallon 1981). However, even within a
communities under simulated rhizospheric conditions (Peacock species, the level of sensitivity and responses to oxygen are
et al. 2014; Xiao et al. 2018), and none investigating rhizospheric rather plastic and adaptive (Dingler and Oelze 1985). Indeed, the
diazotroph communities. Despite this, free-living diazotrophs scavenging mechanisms employed are quite costly, and because
are commonly enriched in other community-based biotechnolo- of this, nitrogen fixation is believed to be most efficient under
gies such as wastewater treatment (Welz et al. 2018; Ospina- microaerophilic conditions as shown in A. vinelandii (Inomura
Betancourth et al. 2020) or for the production of the bioplastic et al. 2018) and Crocosphaera watsonii (Großkopf 2012). Thus, on4 FEMS Microbiology Ecology, 2020, Vol. 96, No. 12
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Figure 2. 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.
the one hand, by finely tuning bioreactor oxygen concentrations, The availability of soil carbon was once thought to be a limi-
a highly specialized subset of diazotrophs may be selected for. tation for free-living diazotrophs; however, it is now known that
Conversely, by engineering conditions with varied concentra- plants transfer around 20–50% of the total assimilated carbon
tions of oxygen through time, space or both, diazotroph commu- into the soil to recruit microbes using root exudates (Haichar
nities that may invade various oxygen niches in the soil may be et al. 2014). Indeed, the quantity and quality of root exudation
enriched. Spatially, such niches may range from microaerophilic is now considered an important functional trait associated with
in the endosphere or rhizoplane where respiration is likely high- high growth rates and N acquisition (Guyonnet et al. 2018). Up
est, to much higher concentrations of oxygen further away from to 90% of root exudate carbon may be taken up by microbial
the roots. Temporally, oxygen concentrations likely follow cyclic biomass (Smercina et al. 2019). The current state of the art in
diurnal rhythms based on the photosynthetic status of the plant. the quantity and quality of root exudates is by studies imple-
Such patterns may be mimicked by careful selection of reac- menting axenic systems (Kuijken et al. 2015). From such systems,
tor type and operational parameters as described in subsequent it is clear that organic acids and sugars are the primary com-
sections. ponents of root exudates, but the concentrations and make-up
Many other factors exist that are related to the efficiency are both species specific and context dependent (Johnson et al.
of nitrogen fixation at a molecular level. For example, while 1996; Kravchenko et al. 2003). Advances in methodology continue
the primary nitrogenases are molybdenum dependent (Mo- to put this exciting field into focus, such as the role of primary
nitrogenase), alternative vanadium- or iron-dependent nitro- and secondary metabolites in shaping the microbiome (Szobos-
genases may also be used to fix nitrogen (V-nitrogenase and zlay, White-Monsant and Moe 2016; Girkin et al. 2018; Stringlis
Fe-nitrogenase, respectively) when molybdenum is limiting et al. 2018) and the use of split root systems to investigate how
(Smercina et al. 2019). While these alternative nitrogenases are microbial inoculation alters root exudate profiles (Korenblum
less efficient under most conditions, V-nitrogenase appears to be et al. 2020). Therefore, the selection of dominant carbon sources
more efficient under low temperatures (Bellenger et al. 2014). As and dosing of secondary metabolites matching the host plant
with the case above with determining nitrogen limitation, what are important controls that will impact the diversity and func-
constitutes molybdenum deficiency is also dependent on the tion of self-assembled communities (Fig. 2). In particular, a key
environmental context, as P availability may play a large inter- focus here should be investigating how biotic/abiotic stresses
acting role, especially when both nutrients are limiting (Reed, alter root exudates, and how such a ‘cry for help’ may be lever-
Cleveland and Townsend 2013). Indeed, P availability in itself can aged to select communities adapted to them (Berendsen et al.
also be a limiting factor of nitrogen fixation (Reed et al. 2007), 2018). For example, addition of caffeic acid, a phenolic root exu-
and therefore it is not surprising that many diazotrophs tested date and important intermediate in the biosynthesis of lignin,
for biofertilization are also phosphorus solubilizers (Schütz has been used to target the isolation of endophytic diazotrophs
et al. 2018). Thus, by applying selective pressures matching (Adachi, Nakatani and Mochida 2002). A recent review has sum-
target soil characteristics such as trace element and nutri- marized the numerous formulations of N-free media that have
ent stoichiometry, scalable production of pre-adapted biofertil- been to isolate diazotrophs (Baldani et al. 2014), and experiments
izers may be achieved decoupled from limitations of enrich- using artificial root exudates may also serve as an initial guide
ing these communities directly in the soil (e.g. seasonality) (Baudoin, Benizri and Guckert 2003). Altering these formulations
(Fig. 2). to match soil chemistry and crop exudate specifics may serve asGutierrez et al. 5
the ecological basis for selection of self-assembled diazotrophic The level of diversity that develops within a community
communities (Fig. 2). 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
BIOPROCESS ENGINEERING FOR COMMUNITY
flow of the media, such as in a soil column reactor or trickle
SELF-ASSEMBLY
bed reactor. In such systems, influent concentrations are spa-
In the previous section, we detailed how environmental param- tially altered through the flow of the reactor and thus organ-
eters such as C:N:P ratios, oxygen concentrations, trace ele- isms at the beginning generally experience high concentrations
ments, primary carbon sources and secondary metabolites may of influent substrates, whereas organisms at the end of the pro-
be used to steer diazotrophic community assembly. In this sec- cess are exposed to low concentrations of influent substrates,
tion, we explore how, given these parameters, different reac- but high concentrations of waste products. In these systems, it is
tor types (attached versus suspended growth), mode of opera- common for diverse organisms carrying out differential biogeo-
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tion (batch, fed batch or continuous) and process parameters chemical processes to establish through the column (Wery et al.
(hydraulic retention time, solid retention time and particle size 2003). Thus, packed bed and soil column reactors promote spa-
distribution) may be used to further control community assem- tial segregation of microorganisms optimized for specific envi-
bly. We highlight how these parameters may be tuned to obtain ronments.
a desired functionality and level of diversity, and contextual- Microscale spatial heterogeneity may be promoted through
ize these technical aspects with practical decisions related to the developments of biofilm (Stewart and Franklin 2008). In sus-
the production of diverse self-assembled biofertilizers (Fig. 2). pended growth systems, spatial heterogeneity is dependent on
While product consistency will be a major challenge for self- the particle size distribution (i.e. the thickness of the biofilm).
assembled communities, it is important to note that complex As particle size increases in floccular or granular communities,
bioprocesses often have high spatial and temporal habitat het- diffusion through these biofilms contributes to increasing envi-
erogeneity but still can lead to predictable community struc- ronmental heterogeneity. Biofilm formation is an important rhi-
tures (Nielsen et al. 2012). Furthermore, open reactor systems zosphere process and associated with disease suppression (Bais,
operating with high degrees of spatial and temporal heterogene- Fall and Vivanco 2004), and therefore, particle size distribution
ity may still be consistently enriched with the same target taxa and biofilm thickness is likely a key operational parameter in
(∼90% relative abundance), and their growth state highly charac- selecting for self-assembled biofertilizers. Indeed, the choice of
terized (Oyserman et al. 2016a,b). Thus, while there are certainly carrier media may also be done to promote such heterogeneity.
additional challenges to using mixed communities rather than For example, using mineral fertilizers containing insoluble P as
single isolates, there is also a long history of successful inno- a carrier medium in a P-depleted system would favor the forma-
vation in novel reproduceable bioprocesses, as exemplified by tion of a biofilm on this mineral fertilizer and the establishment
the discovery and now widespread implementation of Anam- of two countering gradients of oxygen (into the biofilm) and P
mox (Kuenen 2008). (out of the biofilm).
Broadly speaking, reactors may be categorized into attached In addition to the spatial heterogeneity discussed above, the
growth or suspended growth systems. An attached growth temporal heterogeneity of a process may also be established.
design employs a carrier medium, or packing material, to sup- The simplest design is a batch system, in which influent addition
port biofilm growth. This carrier medium may be chosen based occurs at a single time point, operation occurs until a reaction
on a combination of practical considerations related to biolog- has gone to completion and concentrations of resources vary
ical properties, costs, and downstream application. Essentially, through the time of production. Many industrial bioprocesses
any material may be used as a carrier and can either be fixed or such as beer production and other microbial fermentation prod-
suspended in the reactor (Loupasaki and Diamadopoulos 2013). ucts are operated as batch systems. In this manner, community
Similarly, packed bed columns or soil columns may be used members adapted to different concentrations may be differen-
(Wery et al. 2003; Wei et al. 2016). The choice of carrier medium tially active at the beginning and end of a bioprocess. Such tem-
is important, especially when it is desired to apply the carrier poral heterogeneity may be important for selecting diverse com-
medium containing the biofertilizer applied directly to the soil munities and must be accounted for when considering product
(Akay and Fleming 2012). One advantage of using an attached consistency. However, this heterogeneity may also be exploited
growth system is that existing and emerging agricultural inputs to select the appropriate growth state, such as the production
such as phosphate rocks, struvite, sand, wood chips, saw dust of storage polymers, which has been shown to improve inocu-
and chitinaceous material may be used as a carrier medium lant survivability. Furthermore, as community structure increas-
and hence ‘activated’ with key agro-ecologically relevant func- ingly diverges from the original community structure after inoc-
tions such as phosphorus solubilization, or chitin degradation ulation before reaching stability (Trebuch et al. 2020), by operat-
before their application into the agroecosystem. Another type ing over a single reaction cycle, the community that develops is
of attached growth system that has been used for the produc- not so far removed from the original inoculant. Determining the
tion of biofertilizer isolates is solid-state fermentation (Jin et al. length of batch operation is also necessary. This may be deter-
2019). Solid-state fermentation is an especially alluring process, mined by monitoring growth or substrate use.
as it uses minimal water and may therefore be more suitable for A sequencing batch reactor is a simple extension of the batch
mimicking soil conditions, or even cycling drought conditions. reactor. To operate a sequencing batch reactor, the reactor must
In contrast, suspended growth reactors do not contain carrier simply undergo consecutive fill and draw cycles in which the
mediums. Growth is suspended within the reactor as either free- biomass is retained but the bulk fluid replaced with new influ-
living cells, or as floccular and granular structures. Suspended ent. In this way, biomass may be accumulated to a desired con-
growth systems may subsequently be applied directly to the centration. The repeated fill and draw cycles may be advanta-
soil or formulated with a natural or synthetic carrier and then geous when enriching communities for dynamic physiologies
applied to the soil (Malusá, Sas-Paszt and Ciesielska 2012). such as polymer cycling and storage (Oyserman et al. 2016b),6 FEMS Microbiology Ecology, 2020, Vol. 96, No. 12
which has been shown to be an important factor in the suc- addressed. Furthermore, bioprocesses such as anaerobic diges-
cessful application of biofertilizers (Okon and Itzigsohn 1995). tion are increasingly operated by small farms (Wang 2014; Imeni
After consecutive cycles, the community structure may take et al. 2020). Thus, advances in the production of self-assembled
on emergent properties such as the formation of floccular and communities for biofertilization not only provide a means to
granular biomass (Jarvis et al. 2005), which may be leveraged in produce highly complex and scalable production of biofertilizers
downstream processes such as harvesting and application. For but also may provide farmers and land managers a new tool in
example, one of the key features of floccular and granular struc- managing soil health at the local level. Currently, the potential
tures is their high content of extracellular polysaccharides and for self-assembled communities as biofertilizers is completely
alginate-like substances (Trebuch et al. 2020), which, again, has unexplored and we hope that outlining the ecological and engi-
been shown to improve biofertilizer viability after application neering considerations here will spur further research on this
(Okon and Itzigsohn 1995). Finally, dual approaches that com- topic.
bine isolates with community based-approaches may also be
successful. For example, starting inoculants may include ‘top-
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performing’ isolate(s), or may be inoculated at high densities ACKNOWLEDGMENTS
after community assembly has already been initiated. Indeed,
The authors thank Joerg Ueckert and Jort Altenburg for construc-
understanding how/when isolates can invade established com-
tive discussion about reactor types. Publication number 7065 of
munities remains a key challenge in microbiome engineering.
the Netherlands Institute of Ecology, NIOO-KNAW.
Such challenges will also have to be addressed with the applica-
tion of self-assembled communities.
FUNDING
CONCLUSIONS AND FUTURE PERSPECTIVES The contributions of BOO and JMR were funded in part by the
Intensive agricultural practices have resulted in the global Dutch Technology Foundation of the Dutch National Science
degradation of microbial functions in soils. Restoring these Foundation (NWO-Toegepaste en Technische Wetenschappen).
microbial communities is important for the future sustainabil- The contributions of CFGL were funded by COLCIENCIAS and
ity of agriculture. Furthermore, restoring microbial function is the Universidad del Valle Convocatoria Interna para Apoyo a la
important when these agricultural systems are converted back Movilidad.
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
AUTHORS’ CONTRIBUTIONS
the scales of industry. However, isolate-based biofertilizers are BOO and CFGL conceived and drafted the manuscript. JSG and
inherently limited in their complexity, even in the context of JMR provided critical feedback and discussion. All authors con-
synthetic communities. On the other hand, soil transplantation tributed critically to the drafts and gave final approval for publi-
experiments are at the other end of the spectrum. While they cation.
are diverse, scaling soil transplantations is logistically not fea-
Conflict of Interest. None declared.
sible 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
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