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Solutions in microbiome engineering: prioritizing barriers to

organism establishment
✉
Michaeline B. N. Albright1 , Stilianos Louca2, Daniel E. Winkler3, Kelli L. Feeser 1
, Sarah-Jane Haig 4
, Katrine L. Whiteson 5
,
6
Joanne B. Emerson and John Dunbar1

© The Author(s) 2021

Microbiome engineering is increasingly being employed as a solution to challenges in health, agriculture, and climate. Often
manipulation involves inoculation of new microbes designed to improve function into a preexisting microbial community. Despite,
increased efforts in microbiome engineering inoculants frequently fail to establish and/or confer long-lasting modifications on
ecosystem function. We posit that one underlying cause of these shortfalls is the failure to consider barriers to organism
establishment. This is a key challenge and focus of macroecology research, specifically invasion biology and restoration ecology. We
adopt a framework from invasion biology that summarizes establishment barriers in three categories: (1) propagule pressure, (2)
environmental filtering, and (3) biotic interactions factors. We suggest that biotic interactions is the most neglected factor in
microbiome engineering research, and we recommend a number of actions to accelerate engineering solutions.

The ISME Journal (2022) 16:331–338; https://doi.org/10.1038/s41396-021-01088-5

Microbiome engineering is a rapidly evolving frontier for solutions
to improve human health, agricultural productivity, and climate
management. Microbiome engineering seeks to improve the
function of an ecosystem by manipulating the composition of
microbes. Two major challenges for successful microbiome
engineering are (1) the design of a microbiome with improved
function and (2) the establishment of an improved microbiome in
a recipient system of interest. While multiple articles and reviews
have addressed functional design [1–3], microbiome establish-
ment has received less attention. Here, we propose a strategy to
improve microbiome engineering by focusing on microbial
establishment and leveraging insights from macrobial ecology.
Two general engineering strategies are to manipulate indigen-
ous microbes [4] or to introduce new members [5]. The latter
involves the design and delivery of inoculants (a.k.a., probiotics in
medical and agricultural arenas) and is a rapidly growing
biotechnology sector. In their most general form, both strategies
have been practiced crudely for thousands of years in human
health [6] and agriculture [7]. However, despite current technical
advances, inoculants frequently still fail to establish or confer long-
lasting (months to years) modifications to ecosystem function [8].
We argue that this repeated failure is in part driven by lack of
emphasis on establishment of inoculants.
The problem of organism establishment in recipient ecosystems
is not unique to microbiome engineering; it has roots in
macrobiology, particularly invasion biology and restoration
ecology. We propose that adopting a cross-disciplinary conceptual
framework to identify barriers to organism establishment, and
then prioritizing these barriers through targeted research will
accelerate successful microbiome engineering. In addition,
recognizing differences in terminology and experimental design
within and across disciplines will facilitate research integration
across diverse ecosystems and scales. The components of a more
holistic strategy are discussed below.
CONCEPTUAL FRAMEWORK OF BARRIERS TO ORGANISM
ESTABLISHMENT
Pinpointing and overcoming barriers to inocula establishment are
important research priorities for successful microbiome engineer-
ing. Barriers to establishment have been studied extensively in
both invasion biology and restoration ecology. Invasion biology
aims to understand the mechanisms that promote or deter
invasive species establishment [9]. Restoration ecology aims to
restore beneficial plants, animals, insects, and/or microbial
communities in ecosystems where they have previously been
displaced or depleted [10]. Over time, these fields have produced
numerous theoretical frameworks to explain successful species
establishment [9, 11]. The overlapping foundational theory in
these fields presents an opportunity for cross-disciplinary synergy
with microbiome engineering. Although the goals of microbiome
engineering and restoration/invasion ecology are often different
(e.g., establishment of a new microorganism, consortium, and/or
set of functional properties in microbiome engineering vs.
prevention of establishment and/or removal of invading species
and preservation of native biota in restoration/invasion ecology),
the value in a cross-disciplinary approach to microbiome
engineering that considers restoration/invasion ecology is in
identifying emergent properties of the ecosystem that can
facilitate or inhibit establishment. Furthermore, microbial ecology

1
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA. 2Department of Biology, University of Oregon, Eugene, OR, USA. 3United States Geological Survey,
Southwest Biological Science Center, Moab, UT, USA. 4Department of Civil and Environmental Engineering, University of Pittsburgh, Pittsburgh, PA, USA. 5Department of
Molecular Biology and Biochemistry, University of California, Irvine, CA, USA. 6Department of Plant Pathology, University of California, Davis, CA, USA. ✉email: malbright@lanl.gov

Received: 7 January 2021 Revised: 5 August 2021 Accepted: 9 August 2021

Published online: 21 August 2021
 M.B.N. Albright et al.
332
Table 1. Overview of factors impeding organism establishment, potential engineering solutions. A comprehensive source of examples of studies
which illustrate barriers to establishment and solutions is provided in Table S1.
Ecological principle Factor impeding establishment Potential engineering goal
Propagule pressure
• Dose a. Stochastic extinction a. Add a higher dose of the inoculant
b. Density-dependent competitiveness b. Add a lower dose of the inoculant
• Frequency a. Succession makes niche ephemeral a. Add inoculant more frequently
b. Biotic disturbance (i.e., inoculation) is b. Add inoculant at certain timepoint(s)
needed to open a niche
• Delivery mode a. Inoculants do not reach or do not stay in a. Alter delivery mode
intended location b. Increase dose
Environmental filtering
• Disturbance High-turnover of organisms (low a. Persistent delivery of inoculant is necessary
residence time) b. Create a protected physical space
c. Inoculant with characteristics resistant to disturbance
• Niche Breadth Inoculated organism requires a specific a. Engineer inoculant that has a larger niche breadth
resource that is absent b. Pre-adapt inoculant to available environment
c. Add resource specific to inoculant (‘pre-biotic’)
Biotic interactions
• Antagonism via Direct competition exists between resident a. Remove/disturb resident microbes
Competition organisms and inoculants b. Increase “competitive” trait of inoculation (e.g., antibiotic
production, biofilm formation)
c. Both a & b
1234567890();,:

d. Add resources to support the inoculant during establishment

period or beyond
e. Pre-adapt inoculant to available environment
f. Create a protected space
• Antagonism via Antibiotic-producing residents debilitate the a. Make inoculant resistant
Antibiotics inoculant b. Disrupt resident(s) (reduce antibiotic(s) production)
c. Create a protected space
• Antagonism via Predation by resident microbes a. Make the inoculant resistant
Predation b. Remove predators prior to inoculation
c. Create a protected space
• Facilitation Inoculant requires ‘services’ provided by a. Add in an additional organism serving as a ‘keystone’ species to
another organism which is not present modify interactions of the target inoculant and other organisms
and/or modify the environment

can advance foundational theory by overcoming common PRIORITIZING BARRIERS TO ESTABLISHMENT

constraints on the scope and pace of macrobial ecology research,
such as the relatively slow growth rates of macroscopic organisms
and the difficulty of performing multi-species manipulations.
Although microbial studies of factors influencing establishment
are still relatively rare, foundational studies are emerging [12].
A useful theoretical framework for species establishment in
microbiome engineering would identify the abiotic and biotic
levers that engineers could use for successful community
manipulation. Frameworks that vary in granularity have been
developed within invasion biology. At one end of the spectrum,
one synthesis outlined 33 mechanistic hypotheses for establish-
ment success [13]. At the other end of the spectrum, a widely
accepted framework posits that invasion success is mainly
determined by three factors: (1) propagule pressure, (2) environ-
mental filtering, and (3) biotic interactions [14]. Propagule
pressure describes dispersal potential, which determines the
spread of organisms to novel areas, either through natural- or
human-mediated movement. Environmental filtering describes
the compatibility of an organism with a new environment (e.g.,
suitable temperature or moisture range). Biotic interactions
encompass a range of interchanges that can occur between
introduced organisms and residents. Using this three-factor
framework from invasion biology, in the next section we illustrate
some complexity underlying these factors, illustrating the need for
strategic prioritization as a first step to guide microbiome
engineering (Table 1, Table S1).
Potential barrier: propagule pressure
Dose and frequency. Propagule pressure (PP) is a measure of
dispersal used to describe the magnitude (dose) and pattern of
the arrival (frequency) of invasive individuals. PP is one of the
most commonly tested factors in macroorganism invasion biology
and is often linked to invasion success [15]. It also plays a role in
restoration ecology [11]. For example, increased seeding rates can
aid restoration of native plant communities (e.g., [16]), but is not
always effective [17]. Increased PP of a single or a few microbial
invaders may also increase establishment success [18]. However,
independent modeling and experimental work suggest that PP in
multi-species microbial invasions has restricted or minimal
impacts on establishment and community functioning [19, 20].
There are a number of mechanisms by which PP in theory could
influence establishment. Increasing PP can overcome the effects
of ecological drift—random births and deaths that change the
relative abundances of species in a community over time—also
known as demographic stochasticity, which generally increases for
smaller populations such as newly introduced species [21, 22].
Increasing PP can also mitigate the impacts of environmental
stochasticity, unpredictable spatiotemporal fluctuations in envir-
onmental conditions [21]. A higher dose may increase the
likelihood that sufficient inoculum reaches the desired establish-
ment location [23] or may impact density-dependent competi-
tiveness by affecting quorum sensing behaviors either positively
or negatively [24]. The temporal frequency of dose events is an

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alternative route to enhance dosage while also addressing
uncertain timing of niche access.
Delivery mode. Delivery mode may influence establishment by
affecting dispersal range or dose viability. If the delivery mode is
insufficient, inoculants may not reach the intended location of
establishment or may be debilitated when they arrive. Parallels in
plant invasion biology and restoration ecology include natural
seed coats that enhance plant dispersal range by animal vectors
[25] or artificial seed coatings that prevent desiccation or delay
germination [26]. Delivery modes in microbiome engineering
include direct addition of inoculants as free cells in liquids,
lyophilized cells on solids (e.g., on animal feed or on seed
surfaces), or protected cells (e.g., within seeds or gel beads) [27]. A
delivery mode may be chosen to enhance the probability of
microbial inoculant establishment based on environmental con-
ditions and/or the traits of an inoculant [8].
Potential barrier: environmental filtering
Environmental filtering (EF) refers to the selection of organisms
that are compatible with the existing environment or are able to
rapidly modify local conditions to fit requirements. Incompatible
immigrants become extinct. EF can limit invasive plant establish-
ment (e.g., [28]) and often explains restoration failures (e.g., [11]).
Invasion success is expected to increase if invaded environments
match those of a species optimum range [29]. In restoration,
environmental filters are commonly manipulated to facilitate
establishment of target restoration species [30] and/or prevent
invader establishment [31]. An example of this phenomenon
among microbes is the role of host specificity in microbial invasion
success [32]. In both macro and microbiology, the most broadly
successful invaders tend to have larger habitat ranges than non-
successful invaders [33], which typically corresponds to tolerance
of diverse conditions. EF conventionally includes abiotic (physical,
chemical) and biotic factors (e.g., interactions with resident
species). However, for engineering purposes it is useful to separate
biological interactions (BI) from other modes of EF when
considering barriers to establishment and potential solutions,
because BI are generally harder to control for than abiotic factors.
When EF is a barrier to microbiome engineering, either the
expected longevity of engineering or the environmental condi-
tions must be adjusted. Below, we summarize two aspects of EF to
consider for microbiome engineering: niche availability and
disturbance.
Niche availability. A lack of niche space for an inoculant is an
obvious barrier to establishment. Understanding the temporal and
spatial distributions of niche availability may be key to tailoring
inoculant establishment [34]. Identification of a target niche space
for inoculants through in situ strain characterization and
subsequent isolation has led to success in a number of
bioaugmentation efforts [35] and more recently, high-
throughput phenotyping assays have been used [36]. If the target
niche for an inoculant exists but is already filled, displacing the
resident competitors may be required (e.g., [37]; see Biotic
Interactions).
Alternatively, a niche can be created. Niche availability can be
manipulated in some cases by altering the abundance of a single
or multiple resource(s), as is done with prebiotics [4]. In host-
associated communities, the host can provide a substrate to
recruit health-promoting taxa, as illustrated by oligosaccharides in
human breast milk that foster the growth of Bifidobacterium
infantis strains in infant guts [38]. Similarly, there is increasing
evidence that plant roots secrete metabolites to shape rhizo-
sphere microbial composition [39]. Prebiotics can also be used to
create novel niches for exotic inoculants, resulting in a combined
pre- and probiotic known as a synbiotic. For example, porphyran
—a marine polysaccharide—was used to establish an exogenous
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M.B.N. Albright et al.
333
Bacteriodetes strain in mice guts [40] and xenobiotic compounds
have been applied to support microbes of interest in industrial
fermentations [41]. Continual delivery of an exotic resource can
enable long-term stability of a “specialist” organism [42].
Conversely, short-term delivery of an exogenous resource may
aid ‘generalist’ microbial inoculants that have a wider niche
breadth but need a resource supplement during the transitional
establishment period [43]. An alternative to manipulating niche
availability is to exploit microbial inoculants that modify the
environment, constructing their own niche [44] or a niche for
other inoculants ([45], see Biotic Interactions).
Disturbance. Disturbance, here defined as perturbation of
physical (e.g., structure, flow rates, temperature) or chemical
(e.g., nutrients, pH, oxygen) properties of the environment, has the
potential to increase species turnover. Consequently, disturbance
is either a useful tool to displace unwanted residents [46]; see
Biotic Interactions) or a barrier that impedes inoculant establish-
ment. In highly disturbed environments, long-term establishment
may be an unrealistic goal and persistent delivery of inoculants
may be unavoidable. For example, in phyllospheres, soils, or rivers,
‘washouts’ of microbes may occur from heavy or frequent
precipitation events or other physical disruptions [47]. Periodic
chemical disturbances, such bile secretion in the gut [48] or
application of pesticides in soils [49] can also impact inoculant
establishment. Providing inoculants with an enduring physical
haven that allows continuous dispersal into surrounding space
may overcome disturbance barriers, as seen in some macrobial
and microbial systems [16, 50]. This strategy is enhanced when
microbial inoculants possess superior abilities for attachment [51],
biofilm formation [52], or stress resistance [53].
Potential barrier: biotic interactions
The variety of BI is complex but can be broadly categorized as
either antagonistic or facilitative [54]. Within these categories,
interactions can be described as direct or indirect in relation to an
organism of interest. For example, antagonistic direct interactions
include mechanisms such as predation or competition, while
antagonistic indirect interactions include environmental modifica-
tions like antibiotic production or pH changes may adversely
affect competitors and non-competitors alike. In macrobial
ecology, the impacts of direct effects have been more commonly
considered e.g., [55], but research on indirect effects is increasing
e.g., [56]. There is also growing recognition that the number or
complexity of interactions may affect establishment in macrobial
[57] and microbial systems [58]. In simple systems with cultured
microbes, antagonistic interactions are abundant and are a driving
force in community assembly and stability [59]. Antagonism also
plays a role in functioning of more complex microbial systems [60]
and may be used in microbiome engineering [61]. Like antagon-
ism, facilitative interactions can also be direct or indirect. For
example, the presence of a keystone species that provides a
resource supporting an inoculant is direct facilitation, whereas a
general modification of the environment (e.g., a change in soil pH)
that benefits an inoculant is indirect facilitation. The distinction
between direct and indirect BI is important for microbiome
engineering because preoccupation with direct interactions can
overlook important indirect barriers and solutions.
Antagonism via simple competition. The complexity of competi-
tion can be categorized by the mechanisms organisms use to
capture growth limiting resources. In the simple case, competition
involves differences in search capability (i.e., motility) and/or
resource capture efficiency (e.g., nutrient uptake via transporters).
Increasing the number of competitors for a growth-limiting
substrate may reduce the ability of an inoculant to capture a
sufficient quota of resource to survive [62]. A general trend
observed in multiple ecosystems is that increased resident
 M.B.N. Albright et al.
334

Fig. 1 Barriers to organism establishment and a path forward for microbiome engineering. A The current state of research focused on
inoculum establishment. Without quantitative prioritization, potential barriers like the illustrated examples are investigated randomly. B Path
forward to improve establishment success. Prioritization of barriers includes quantifying their relative impact in order to create decision trees
that can simultaneously rank barriers and summarize the potential impact of overcoming each barrier in a multi-factor solution path. Values in
parentheses on the decision tree illustrate an example of percentage impact of a barrier on inoculum establishment, guiding microbiome
engineering investments.

community diversity generally reduces invasibility because diverse
communities leave less free niche space [63, 64]. For microbiome
engineering solutions to overcome a competition barrier might
include creating a protected physical space or adding resources to
support the inoculant during an establishment period [58, 65].
Antagonism via antibiotics. Complex competition involves addi-
tional strategies to undermine competitors. Strategies include
secretion of antibiotics, signaling compounds that adversely affect
the metabolism of other species (e.g., quorum signals or volatile
organic compounds), or siderophores that create new growth
limitations for competitors. Antibiotic production is the best
studied strategy. In pairwise interactions of cultured isolates,
antagonistic antibiotic production occurred in approximately half
of Bacillus [66] and Streptomyces [67] isolates. Antagonistic growth
inhibition tends to increase with phylogenetic similarity of species
[68], presumably because competition is greatest among close
relatives with similar traits, a concept shared across microbial and
macrobial biology. The impact of antibiotics in natural commu-
nities is illustrated by the production of andrimid by marine
bacteria, which inhibits Vibrio cholerae growth [69]. The extent to
which antibiotic production is a barrier to establishment of
inoculants in microbiome engineering remains to be established,
but has been shown in at least one instance [70] and might be
routinely assayed by inhibition assays with filtrates from a resident
community. Active antagonism may also be an engineering tool
for establishment, illustrated by an artificial biocontrol strain used
to remove unwanted bacteria in a community [71].
Antagonism via predation. Another type of antagonistic interac-
tion that may influence the ability of inoculants to establish is
predation. Predation by protists, bacteria, and fungi is likely to be
density dependent, not taxon-specific, although cases of the latter
have been documented [61]. Predation by host-specific viruses
may also impact invader success. Phages vary in host range but
generally display host specificity. While phage are known to
impact the diversity and function of microbial communities [72],
they are still rarely considered when assessing controls on
microbiome succession [73]. Phages may stabilize the co-
existence of competing bacteria by preventing dominance of a
single species—an example of the “kill-the-winner” hypothesis
[74]. In microbiome engineering, establishing inoculants with

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“naïve immunity” may fail owing to lysis by phages in the resident
community [75]. Conversely, predation can be a tool for tailored
removal of residents that impede establishment of desired strains,
by building on concepts from work using phages for biocontrol of
pathogens in humans, on foods, in aquaculture, and on plants
(e.g., [76]).
Facilitation. Stable facilitative interactions between microorgan-
isms are only expected in restricted cases [77]. For example,
environmental conditions can mediate a tradeoff between
facilitation and competition, where harsher environments foster
microbial facilitation [78]. Another view posits that microbial
communities are able to organize into metabolically cohesive
units where consortia with positive feedback loops use resources
in a stable manner and minimize competition through resource
specialization and exclusion of resource generalists [79]. For
microbiome engineering, facilitative interactions could be lever-
aged, for example by creating highly specialized microbial units
where organisms are filling different niches.
Summary
The framework of PP, EF, and BI is a simple way of categorizing a
long list of ecological phenomena that can be barriers (or
engineering tools) for establishment of inocula in resident
communities (Table 1, Table S1). The overview above highlights
a few of the many sub-factors that have been documented in
macrobial ecology [13]. Given the large number and context-
dependence of factors that may impede organism establishment,
ranking barriers at the highest level (PP, EF, and BI) in a decision
tree framework is a critical first step to accelerate microbiome
engineering and avoid random testing of factors (Fig. 1).
Decision trees are increasingly being used in macrobial ecology
to manage species invasions and ecological restoration. For
example, some decision trees prioritize actions based on the
potential severity of invasive species on ecosystem services [80]. In
a restoration ecology examples, decision trees based on seed
dormancy attributes [81] or optimal habitat characteristics for bird
nesting [82] can guide restoration plans. Many decision trees
available to managers in macrobial ecology include a decision-
making step related to establishment (e.g., is there adequate time
for a seedling to establish during its typical growing season?).
Building decision trees in microbiome engineering requires
studies that test the relative importance of factors on establish-
ment success. For example, a recent study found that the relative
importance of PP and BI varied by organism domain, bacteria
versus fungi [19]. Another study found that the impacts of PP
depended on the competitive interactions of residents and
inoculants [18]. Findings from many studies like these must be
averaged to elucidate general rules for decision trees, but such a
pursuit is inefficient without planning for quantitative synthesis.
BREAKING BARRIERS: SYNTHESIS AND RECOMMENDATIONS
Overall, we posit that increasing focus on BI will lead to increased
success in inoculant establishment and improve outcomes in
microbiome engineering (Fig. 1B). While BI are commonly
considered in invasion biology and restoration ecology, manipulat-
ing BI is an under-explored mechanism in microbiome engineer-
ing, likely due to the abundance and complexity of organism
interactions in any given microbiome. Greater success in manip-
ulating early succession ecosystems (e.g., infant compared to adult
guts) [38, 65] may be due in part to their lower complexity, which
would point to the importance of BI. Assessing the number and
type of BI relevant for engineering complex microbiomes is
challenging because of the lack of direct measurement capabilities.
Network reconstruction techniques based on correlated species
abundances are often used to infer interactions (e.g., [83]), but
further development is needed to move beyond speculative
The ISME Journal (2022) 16:331 – 338
M.B.N. Albright et al.
335
inferences [84]. Manipulation of microbial keystone species
deserves attention as a potential solution in microbiome engineer-
ing as this concept has proven to be a powerful strategy in
restoration ecology [85]. Although keystone microbial taxa have
been predicted in co-occurrence networks [86] and linked to
compositional shifts [87], few studies have confirmed the
physiological role of keystone microbes in a community [88].
In order to confirm the role of BI in microbiome engineering
outcomes and refine a decision tree of barriers to organism
establishment we recommend a number of actions. First, greater
attention is needed in microbial research to measure establish-
ment (persistence and/or proliferation) of inocula over long
timescales (e.g., months to years) with whole-community mea-
surement techniques that offer broader insight and more
standardized experimental design and reporting to facilitate
meta-analyses. Second, a careful cross-discipline synthesis—i.e.,
meta-analyses of microbial studies that measure the dependence
of inocula establishment on aspects of BI, as well as PP and EF,
that would illuminate knowledge gaps is needed. We expand on
these concepts in the sections below.
Recommendations for future research
To accelerate microbiome engineering across different applica-
tions, there is a need to target knowledge gaps that are
contingent on experimental design. Gaps range from complexity
of the inoculum to multifactorial manipulation strategies. We
highlight four gaps. (1) To date, most microbial establishment
studies have one or a few inoculant species. In contrast,
transplant of entire microbiomes (i.e., fecal transplants and
activated sludge transplants) is an increasing practice for some
medical and wastewater treatment applications, respectively, yet
knowledge of how these complex invasions impact community
composition and functioning is limited. (2) Often microbiome
engineering studies measure functional changes, not inoculant
establishment. Monitoring inoculants is needed to determine if
failure to achieve desired functional changes is due to lack of
establishment or instead, to attenuation of desired functions in
established inoculants. (3) More insight into the temporal
dynamics of functional changes is needed. It is common practice
to assess function at only one or two timepoints. Furthermore, the
time interval between manipulation and functional assessment
varies from hours to weeks. In many studies functional measure-
ments occur immediately following probiotic intervention and
longer-term impacts are not assessed, limiting insights into
barriers to establishment. (4) It is likely that multiple factors
simultaneously contribute to the failure or success of species
establishment (e.g., see [18]). Thus, multifactorial experiments
that test the interactive effects of establishment barriers (or
engineering solutions) are needed and may be especially helpful
in building decision trees.
Translating among fields
Prioritizing establishment barriers through cross-discipline synth-
esis depends on the capacity to find related knowledge that is
obscured by discipline-specific jargon. For example, medical
research on microbiome engineering typically uses the term
“probiotics” [89], while agricultural studies use the terms “micro-
bial inoculants”, “plant growth promoting microbes” (PGPM’s),
“biocontrol agents”, plant “biostimulants” or “probiotics” [90]. To
facilitate cross-discipline synthesis, we compiled some key
concepts, terms, and definitions (Table S2)—a step towards
making related knowledge findable, accessible, interoperable,
and reusable (FAIR), as recommended by the National Microbiome
Data Collaborative (NMDC; [91]).
Experimental design
Some standardization of experimental design features (e.g.,
independent and dependent variables, replication, testing scale,
 M.B.N. Albright et al.
336
and temporal sampling) can also facilitate synthesis. At present,
wide variation in experimental design [92] impedes this goal. For
example, functional changes in the gut microbiome might be
assessed in some studies qualitatively from statements of
symptom relief among patients [93], whereas other studies may
quantify changes in the concentration of a specific analyte [94].
Microbial community composition is often reported in a
qualitative way; whereas variance partitioning would facilitate
quantification of probiotic impacts among studies. Furthermore,
use of positive controls (mock communities) and/or internal
standards in amplicon sequencing for taxonomic profiling can
improve the quantitative insights of this common and relatively
low-cost measurement technique [95]. Including a link in
publications to an easily accessible data table summarizing
experimental design can also facilitate data syntheses within
and across disciplines. A database of searchable studies that
include the experimental design and results with permanent
identification records and rich contextual metadata (somewhat
analogous to clinicalstudies.info.nih.gov/) would greatly accelerate
progress. Publicly accessible genomic repositories are a prime
example of how data aggregation and standardized formatting
across disparate studies is leading to groundbreaking discoveries
[96]. Data attribution guidelines that give researchers credit for
datasets used in subsequent work [96, 97] is another useful step.
CONCLUSIONS
Microbiome engineering is a rapidly expanding field. There are
notable cases of success in microbiome engineering for human
health, in particular in the infant gut [38, 65], bioremediation [98],
wastewater engineering [99], and agriculture [100]. However,
inoculants often fail to establish or to modify ecosystem
functioning over significant time periods [8]. Dispersal, environ-
mental, and biotic barriers to organism establishment likely
contribute to failures. Given the complex suite of possible barriers,
developing a decision tree to prioritize barriers is a top priority to
guide engineering. This priority may be aided by cross-disciplinary
synthesis because disparate fields are tackling similar challenges.
However, to leverage research and unite findings across fields
there is a need to recognize differences in terminology and to
standardize reporting of tested factors and magnitude of effects.
Lastly, increased attention to the types of experiments performed
and extended time-course measurements of inoculant establish-
ment will provide insights that accelerate successful microbiome
engineering across a range of applications.
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ACKNOWLEDGEMENTS
This work was supported by a Los Alamos National Laboratory Postdoc Fellowship
20180746PRD3 to MBNA., by SFA grant 2018LANLF255 from the U.S. Department of
Energy Office of Biological and Environmental Research to JD. SL was supported by a
startup grant from the University of Oregon. JBE was supported by USDA National
Institute of Food and Agriculture (NIFA) grant number 2021-67013-34815-0 and
USDA NIFA Hatch project number CA-D-PPA-2464-H. SJH was supported by a
National Science Foundation Rapid Response Research (RAPID) grant, grant number
1929843. We thank reviewers for their helpful comments and suggestions on earlier
versions of the manuscript.
AUTHOR CONTRIBUTIONS
MBNA and JD wrote the first draft of the manuscript. SL, DEW, KLF, SJH, KLW, JBE, JD,
and MBNA contributed to ideas and manuscript editing.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41396-021-01088-5.
Correspondence and requests for materials should be addressed to M.B.N.A.
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© The Author(s) 2021
The ISME Journal (2022) 16:331 – 338
Table S1. Some examples of relevant literature across fields pertaining to establishment
barriers and solutions.
Ecological Principle
Propagule Pressure (PP)
Dose/Frequency
Delivery Mode
Field
Invasion biology
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Invasion biology
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Relevant Literature
Catford et al. 2009, Colautti
et al. 2006, Lockwood et al.
2005, Simberloff et al. 2009,
Von Holle and Simberloff
2005, Wittmann et al. 2014
Corbin and Holl 2012, Funk et
al. 2012, Hulvey et al. 2017,
Schantz et al. 2015,
Wainwright et al. 2018
Acosta et al 2015, Albright et
al. 2020, De Roy et al. 2013,
Fodelianakis et al 2020, Jones
et al. 2017, Ketola et al.
2017,Mallon et al. 2015,
Rivett et al. 2018, van Elsas et
al. 2012, Vila et al. 2019,
Yang et al. 2017, Zhou and
Ning 2017
Khan and Chousalkar 2020,
Nguyen et al 2018, Petschow
et al. 2005, Xing et al. 2017
Bai et al. 2002
Comeau et al. 1993, Nam et
al. 2005
Choudhary and
Schmidt-Dannert
2010
Kreitschitz et al. 2021
Gornish et al. 2019
Howard et al. 2017, Kaminsky
et al. 2019, O'Callaghan 2016
Ali et al. 2015

Environmental Filtering (EF)
Niche availability
Disturbance
Bioremediation
Microbiome engineering
(other)
Invasion biology
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Invasion biology
Restoration ecology
Power et al. 2011
Du et al. 2019
Cadotte et al. 2018, Gallien et
al. 2015, Maitner et al. 2012
Fick et al. 2016, Fick et al.
2020, James et al. 2019,
Nuttle et al 2007, Sheley et
al. 2009, Vasquez et al. 2011,
Wainwright et al. 2018
Bell and Bell 2021,
Fahimipour and Gross 2020,
Muthukrishnan et al. 2018,
Sriswadsi et al. 2017,
Zampieri et al. 2019, Zhao et
al. 2019
Campieri et al. 2000,
Costabile et al. 2017, Enam
and Mansell 2019, Freter et
al. 1983, Frese et al. 2017,
Markowiak and Slizewska
2017, McNally and Brown
2015, Pereira and Berry
2017, Perraudeau et al. 2020,
Pokusaeva et al. 2011,
Shepard et al. 2018, Umu et
al. 2017
Arif et al. 2020, Albareda et
al. 2008, Haskett et al. 2020,
Lennon et al. 2012, Remans
et al. 2007, Sasse et al. 2018
Loffler and Edwards 2006,
Otte et al. 1994, Thompson
et al. 2005
Shahab et al. 2020, Shaw et
al. 2016
Lembrechts et al. 2016,
Miller et al. 2021
Corbin and Holl 2012, Hulvey
et al. 2017
 Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Biotic interactions (BI)
Antagonism Via Simple Invasion biology
Competition
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Antagonism Via Antibiotics Invasion biology
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Gibbons et al. 2016, Shade et
al. 2012
Ali et al. 2020, Bezkorovainy
2001, Lewis et al. 2018,
Praveschotinunt et al 2019,
Sharma and Kanwar 2017,
Zhao et al. 2020
Bach and Hofmockel 2016,
Beauregard et al. 2013, de
Souza et al. 2015, Maltz and
Treseder 2015, Tripathi et al.
2020, Upton et al. 2019
Mansfeldt et al. 2019, Zhang
et al. 2018, Wang et al. 2020
Baltzis and Frederickson
1983, Dykhuizen and Hartl
1983, Lewis et al. 2018
Beaury et al. 2020, Corbin
and D'Antonio et al. 2004
Herron et al. 2013
Bauer et al. 2018, Eisenhauer
et al. 2013, Fuschlin et al.
2012, Hibbing et al. 2010
Panigrahi et al. 2018,
Perraudeau et al. 2020, Suez
et al. 2018
Capdevilla et al. 2004
Long et al. 2005, Perrez-
Gutierrez et al. 2013, Russel
et al. 2017, Safferman and
Morris 1962
Hecht et al. 2016
 Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Antagonism Via Predation Invasion biology
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Facilitation Invasion biology
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Deoliveira et al. 1995
Dwidar et al. 2015
Blumenthal 2005, Keane and
Crawley 2002, Kinnear et al.
2002, Mitchell and Power
2003
Bahr 1954, Barker and Brown
1994, Greub and Raoult
2004, Koskella 2019, Matz
and Kjelleberg 2005,
Pernthaler et al. 2005,
Shapiro et al. 2010,
Soundararajan et al. 2019,
Thingstad 2000
Das et al. 2015, Dedrick et al.
2019, Nale et al. 2018, Ott et
al. 2017
Balogh et al. 2010, Marsh
and Wellington 1994, Postma
et al. 1990, Wang et al.
2019, Wright et al. 1995
Feitchmayer et al. 2017,
Manefield et al 2007
Bouchez et al. 2000
Barrangou et al. 2007, Lopez-
Igual et al. 2019, Nakai and
Park 2002
Byers et al 2006, Gagnon et
al. 2020, Valdez et al. 2020
Foster and Bell 2012,
Pascual-Garcia et al. 2020
Anandham et al. 2007
 Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Not Categorized Invasion biology
Restoration ecology
Microbial ecology
Human/animal microbiome
engineering
Agriculture/soil microbiome
engineering
Wastewater / Drinking water
treatment
Bioremediation
Microbiome engineering
(other)
Piccardi et al. 2019
Li et al. 2017, Zhang et al.
2020
Berner and Bruckhart 2005,
Corbin and D'Antonio et al.
2004, Gallien and Carboni
2017, Kinnear et al. 2002,
Mitchell et al. 2006,
Northfield et al. 2018,
Seastedt and Pysek 2011,
Tillman et al. 2004, White et
al. 2006
Carroll 2017, Estes et al.
2011, Funk et al. 2008, Paine
et al. 1966, Palmer et al.
1997, Ripple and Beschta
2012, Ripple et al. 2014, Shea
and Chesson 2002, Sinclair et
al. 2007
Albright et al. 2020, Berry
and Widder 2014, Brunel et
al. 2020, Garcia-Bayona and
Comstock 2018, Herren and
McMahon 2018, Maynard et
al. 2017, Zapien-Campos et
al. 2015
Baktash et al. 2018, Frese et
al. 2017, Panigrahi et al.
2018, Seekatz et al. 2014,
Suez et al. 2018, Trosvik and
de Muinck 2015
Kong et al. 2018
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Table S2. Terminology across disciplines related to key concepts in microbiome engineering

Alternative Terms/Related
Term Field Concepts
probiotics, microbiome transfers,
live bio-therapeutic products, fecal
transplants, colonizers, donor
Inoculant Human/animal microbiome microbiome/strains
inoculants, biocontrol agents, plant
growth promoting microbes
(PGPMs), probiotics, biofertilizer,
biostimulant. bioprotectant,
Agriculture/soil microbiome propagules, Immigrants
Wastewater / Drinking water inoculants, bioaugmentation,
treatment biostimulant
bioaugmentation, biostimulant,
Bioremediation propagule, target species
invaders, aliens, exotic species,
non-native species, biocontrol
Invasion Biology agents
colonists, focal species, recruits,
priority species, desirable species,
Restoration Ecology target species, seed mix
initial microbiome, recipient
Resident Human/animal microbiome microbiome
Agriculture/soil microbiome initial microbiome
native community, strict colonizer,
Wastewater / Drinking water leaky colonizer, transcient
treatment colonizer, biofilm community
Bioremediation native microbiome
Invasion Biology native species
endogenous species, seed
community, native species, local
Restoration Ecology species
healthy microbiome,
reprogrammed community, final
End Community Human/animal microbiome microbiome, altered microbiome
Agriculture/soil microbiome final microbiome
Wastewater / Drinking water biofilm community, planktonic
treatment community
Bioremediation bioaugmented community
invaded communiy, endpoint,
Invasion Biology altered community
historical state, native state,
Restoration Ecology endpoint, target community
Propagule
Pressure Human/animal microbiome
Agriculture/soil microbiome
Wastewater / Drinking water
treatment
Bioremediation
Invasion Biology
Restoration Ecology
dose, frequency, delivery mode
application amount, number of
propagules, inoculant dose,
frequency of application, delivery
method
concentration, relative abundance,
symbiotic pressures, addition
method, environmental factors,
engineering parameters (e.g., flow
rate, surface area to volume ratio
etc)
propagule density, enrichment
methodology,
dispersal rate, regional factors,
species transport
dispersal rate, regional factors,
species transport, propagule
delivery