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Microbiomes in bioenergy production: From analysis to
management
Christin Koch, Susann Müller, Hauke Harms and Falk Harnisch
Currently, bioreactors exploiting natural microbial
communities, that is, microbiomes, for bioenergy production
are almost exclusively operated based on bulk parameters and
empirical expert knowledge. The microbiome of these
bioreactors often remains a ‘‘black box’’, that is, its
composition and function are only analyzed retrospectively
(mostly in a case of failure). Here, on-time microbiome analysis
can allow a proactive process management. However, today’s
sophisticated molecular ecology methods appear inaccessible
for the routine analysis of reactor microbiomes in bioenergy
production. This review analyzes the requirements of methods
for routine microbiome diagnostics. Especially the ability of
current molecular and cell based methods to derive structure-
function-relationships, that is, correlations between the
microbial community structure and dynamics and the reactor
performance, are emphasized and key-criteria for routine on-
site monitoring are defined. Finally, a critical assessment of
selected methods for microbiome monitoring is performed
focusing on (i) the production of biogas in anaerobic digesters
and (ii) the production of the biofuel precursor n-butyrate.
Addresses
UFZ – Helmholtz-Centre for Environmental Research, Department of
Environmental Microbiology, Permoserstrasse 15, 04318 Leipzig,
Germany
Corresponding author: Harnisch, Falk (falk.harnisch@ufz.de)
Current Opinion in Biotechnology 2014, 27:65–72
This review comes from a themed issue on Energy biotechnology
Edited by Arthur J Ragauskas and Korneel Rabaey
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights
reserved.
http://dx.doi.org/10.1016/j.copbio.2013.11.006
Introduction
Microbial resource management (MRM) [1] respectively
mixed culture biotechnology (MCB) [2] is a key challenge
for future bioenergy production. MRM exploits natural
microbial communities, so called microbiomes (e.g. [3],
see also Box 1), for bioenergy production ranging from
biogas in anaerobic digesters (AD) via electric current
generation to the production of biobased chemicals and
sustainable recovery of resources [2,4,5]. The reactor
microbiome composition, structure, and activity, often
involving food-webs, strongly depend on its environment.
Thousands of different microbial species can contribute
to a microbiome and the contributions of each cell to its
functionality can, so far, not be determined.
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Owing to their complexity, microbiome based biopro-
cesses are often operated based on bulk parameters,
resulting from classical biotechnological methods cover-
ing physical, chemical, and biological parameters, such
as temperature, substrate and product concentrations,
turbidity, or enzyme activities [6,7]. When applied to
natural microbial communities, these methods are
representative for total system performance, and thus
the entire reactor microbiome, but do not allow a seg-
regated analysis of individual sub-functions. Recent
studies, however, have demonstrated that this segre-
gated information is needed to assess the organisms’
individual performances and thus to predict the per-
formance of an entire microbiome — for bioenergy
production, see, for example [8,9]. Consequently, a
proactive microbiome based management of bioreactors
seems only feasible, when including detailed, segre-
gated information on the biological key component, that
is, the reactor microbiome. For this purpose an adequate
monitoring is indispensable, but universal protocols do
not exist. Furthermore, because of the diversity of
bioenergy production systems standard operating pro-
cedures (SOPs) cannot be easily established. Therefore,
the establishment of reactor microbiome based routine
analyses — in addition to the established biotechnolo-
gical methods — is highly desired in order to enable the
steering of bioenergy production processes in future. So
far, many techniques used for microbiome analysis in
research are very sophisticated, requiring expensive
specialized equipment and specialized personnel. In
addition, signal acquisition and signal interpretation
are often time-consuming. These properties are contrary
to the required for routine analyses, as here the perfect
method would be as simple as a temperature sensor and
the final results as intuitive as traffic lights.
Present monitoring techniques for functional
microbiome diagnostics
The microbial ecology toolbox offers a wide variety of
molecular and cell based analysis techniques which can
be applied for microbiome diagnostics (for extensive
methods review see [10–12]). Fig. 1 illustrates the differ-
ent targets and levels of microbiome analysis. Classical
community analyses start with extraction of molecules,
for example, DNA, RNA, proteins, or phospholipid fatty
acids (PLFA). DNA and RNA can be analyzed using
DNA-based techniques like DGGE (denaturing gradient
gel electrophoresis, [13,14]), SSCP (single-strand confor-
mation polymorphism, [15]), and T-RFLP (terminal
restriction fragment length polymorphism, [16,17,18])
as well as high-throughput sequencing of DNA and
Current Opinion in Biotechnology 2014, 27:65–72
66 Energy biotechnology
Box 1 The reactor microbiome
Reactor microbiomes can be defined as natural microbial commu-
nities shaped for a certain purpose, here bioenergy production.
These are comprised of manifold different species, possessing
individual metabolic potentials, being metabolically interconnected
and representing a high level of functional organization [3]. To
reveal this microbiome complexity and identify process relevant
interconnections that in turn may allow a prospective management of
the reactor, insights on the level of the smallest biological entity, the
microbial cell, have to be gained in the time-frame of the resulting
process dynamics. As the microbiomes functionality depends on a
highly complex interplay rather than the potentials of individual
species, the search for the species marking a healthy, high-
performance process or indicating imbalances cannot be performed.
Hence, the microbiome as a whole has to be in the focus of interest
(see also [55] in this issue). A change of the microbiome structure
itself is indicative of a changing environment and thus can potentially
be used as a early indicator of process (in)stability in bioenergy
production.
RNA [8,19–22], and microarray technologies [23–27].
Fewer applications in the bioenergy sector are reported
for proteomic approaches [28,29] or PLFA analysis [30].
In general, DNA-based methods only provide infor-
mation on the presence of organisms and their metabolic
capacities but lack information on actual activities.
Obviously, this activity information will be indispensible
for a future management of microbiomes. It could partly
be provided by RNA analysis. Yet, the additional efforts
as well as the challenges of RNA extraction [31] are
probably the reasons for its rare application to reactor
microbiomes, so far.
In contrast, single cell based techniques assign infor-
mation to an individual cell and thus generally require
specific markers, for example, FISH probes, lectines, or
antibodies, limiting their applicability to complex reactor
microbiomes comprising a priori known key-players [32–
34] (see Box 1).
Better suited are single cell techniques using general
marker types [9,35–37]. Commonly, all techniques for
microbiome diagnostics require protocol optimization for
the respective samples and here, even ‘‘standard’’ tech-
niques like DNA extraction or cell fixation can become
challenging for microbiomes of different habitats [38,39].
Case studies
Obviously, the routine monitoring of reactor microbiomes
has different requirements and constraints in comparison
to its scientific study, especially in terms of operator
knowledge, costs, and acquisition time. In both cases,
however, the choice of method determines the level on
which process relevant changes in the microbiome can be
detected, for example, as already mentioned DNA is only
a marker for presence but not for activity. Therefore, the
selected method has to be adequate for assessing the
relevant structure-function relationships (see also Box 1)
Current Opinion in Biotechnology 2014, 27:65–72
of the microbiome. In the following, two representative
case studies are evaluated, based on proposed key-criteria
(Box 2), in order to illustrate the potential of a molecular
and a cell based method for routine monitoring in bioe-
nergy production. Finally, an evaluation for the case
studies as well as the general potential of the respective
techniques for microbiome monitoring is performed.
Case study 1: Anaerobic digestion monitored by
community flow cytometry
The microbiome of a mesophilic anaerobic digester for
biogas production was regularly analyzed for nine months
using community flow cytometry based on DAPI-staining
in combination with the routine assessment of process
parameters (e.g. total gas and methane production, pH,
volatile fatty acids (VFA) accumulation) during phases of
stability and perturbation [40]. The microbiome com-
prised twenty sub-communities displaying distinct cell
abundance changes, clearly related to process parameter
changes. Thus, it was demonstrated that the changes in
the microbiome are of functional diagnostic process value.
For example, twofold and fourfold substrate overloads led
to significantly increased cell numbers in two sub-com-
munities (Fig. 2a). These changes were clearly an effect
of the disturbance. For instance, the increase of the cell
abundance in both sub-communities was more intense for
the four- than for the twofold substrate overload. In both
cases, the cell numbers returned to their original values
when the standard process parameters were applied. In
addition, strong positive correlations between VFA con-
centrations and cell abundance in these sub-communities
were found, based on Spearman’s rank order correlation
coefficient, while they were negative for methane pro-
duction. Therewith, the study demonstrated that flow
cytometry provides an excellent tool to monitor the
response of the reactor microbiome of an anaerobic
digester towards process variations. In combination with
the accordant functional knowledge there is a high poten-
tial for microbiome management, that is, an immediate
associated counteraction based on microbiome changes
for the process stabilization and optimization seem
possible.
Signal acquisition: The sample preparation follows a
simple protocol [35] being suitable for on-site conduction.
Including incubation times, measurements are performed
within few hours. Dynamic variation can be analyzed
down to bacterial generation times of relevant organisms
and thus being below the typical feeding intervals and
hydraulic retention times of agricultural biogas plants.
Measurements were performed using a high-end instru-
ment obviously inadequate for routine application. Here,
we and others are developing benchtop instruments
tailored for on-site application.
Costs: Benchtop instruments suitable for the approach
described in this case study will be available for about
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 Microbiomes in bioenergy production Koch et al. 67

Fig. 1

Microbiome Complex microbial community

composed of microbial cells with
different phylogenetic back-
ground, cellular properties and
Cell wall
physiological potentials
DNA
Cytoplasm

Signal aquisition
Measurement of extracted molecules after cell lysis Single cell based techniques

Proteomics Microarray DNA based FISH Flow cytometry

techniques
Protein separation, Hybridization of Sequence analysis Microscopy of cells Measuring optical
e.g. using SDS gel, nucleid acids with usually after DNA labeled with characteristics, e.g.
with subsequent the complementary amplification sequence specific cell size and DNA
analysis sequences on a slide fluorescence probes content, in high-
and spot-specific throughput
detection

DAPI-DNA fluorescence

FSC

Interpretation + Implementation

Operator guideline Microbiome management

Current Opinion in Biotechnology

Overview on conceptual steps and potential methods for diagnostic microbiome monitoring in bioenergy production processes. Complex reactor
microbiomes can be characterized by a variety of techniques based on either extracted molecules or intact cells. The interpretation depth and
implementation feasibility define the methods’ potential for deriving operator guidelines and, as most desired aim, perform proactive microbiome
management.

www.sciencedirect.com Current Opinion in Biotechnology 2014, 27:65–72

68 Energy biotechnology
Box 2 Proposed criteria for monitoring techniques
What are the desired properties of the analysis methods applied
for microbiome based reactor management? A general answer
cannot be provided, but from our perspective the following has to be
considered. One main prerequisite is that the methods are not or only
minimal invasive, that is, sampling and signal acquisition have no
impact. We further suggest four key-criteria and their benchmarks for
method evaluation. As routine microbiome monitoring is still in its
infancy a rating of the adequacy of a given method for following a
certain microbiome cannot be provided. The benchmarks have to be
tailor-made and can only be related to the respective process
properties as illustrated.
(i) Signal acquisition:In optimum, an on-line signal is continuously
provided. The further evaluation of this criterion is strongly
depending on the process dynamics and we propose that the
information should best be available within the generation time
of the respective organisms. Often acceptable, but not sufficient
for all processes, is a signal acquisition within one hydraulic
retention time or feeding interval of the reactor. Further delay will
lead to decreased scoring and only retrospective methods are
not suitable.
(ii) Costs:Here the capital costs (including depreciation) and
operating costs (including consumables as well as personnel)
have to be defined, thereby the sum of both in a given time has
to be related to the respective product or valorization step and
the productivity increase by proactive management.
(iii) Automatization:In optimum, the measurements are performed
automatically without operator impact. Nearly optimal are
methods executed on-site and requiring no or little training, even
for unskilled personnel. Acceptable is the need for simple wet-
laboratory equipment and operation by a laboratory technician
or an intensively trained person. Mostly unsuited are techniques
requiring demanding manual handling.
(iv) Interpretation & Implementation:In best case, an implementation
in routine process control can be realized. Nearly optimal are
methods producing results leading to straightforward operator
guidelines. Acceptable are methods requiring little additional
data analysis for interpretation, whereas the need of expert
knowledge or intensive computation is not acceptable.
80.000 s (including a UV laser) and the operating costs
are about 10 s per sample (with a single-digit cent
amount for consumables), which appears reasonable con-
sidering that every day of shut-down for a typical German
agricultural biogas plant can cost about 1000 s [41].
Automatization: Wet-laboratory equipment and operation
by a laboratory technician or trained person are necessary
with the current equipment, but may become obsolete for
future instruments.
Interpretation & Implementation: Person assisted automated
data analysis is already possible [35,42] and the potential
to derive operator guidelines is shown in the case
study.
Outlook: The current developments of on-line flow cyt-
ometers, including automated sampling and sample prep-
aration, for monitoring of drinking water and pure culture
bioprocesses are highly promising regarding optimum
Signal acquisition and Automatization [43,44,45]. Owing
Current Opinion in Biotechnology 2014, 27:65–72
to their heterogeneity reactor microbiomes in bioenergy
production are more difficult to sample than, for example,
drinking water. Thus, the development of tailor-made
machines will be necessary for full automatization, which
might be achievable within the next decade for individual
setups.
Case study 2: Production of a biofuel precursor monitored
with high-throughput sequencing
The microbiome of thermophilic bioreactors for the pro-
duction of the biofuel precursor n-butyrate from cellulosic
biomass waste was studied in terms of biomass pretreat-
ment, operating conditions and reactor perturbations
[8]. The microbiome was monitored using high-
throughput sequencing (16S rRNA gene as target).
The impact of changed operating conditions on micro-
biome composition and function was evaluated. Key-
interconnections were determined for the most abundant
species and a functional microbial correlation network,
dominated by Thermoanaerobacterium spp. was derived
(Fig. 2b). The relative abundance of Thermoanaerobacter-
ium spp. corresponded to the total fermentation pro-
duction rate, although principle coordinate analysis
revealed that unique microbiomes developed depending
on biomass pretreatment. Thus, functional redundancy,
that is, parallel pathways for substrate conversions, was
identified as one guarantor for overall functional stability.
Key control parameters for maximum production rates as
well as process stability were further derived, for
example, in situ product extraction. This illustrates the
potential of high-throughput sequencing for microbiome
management.
Signal acquisition: Sample preparation includes DNA
extraction, amplification, and sequencing, for example,
using a 454 pyrosequencing platform. Under standard
laboratory conditions the procedure is performed within
weeks which might be acceptable for some slow processes
with long hydraulic retention times but not for fast
processes which need immediate response. Recent
advances suggest signal acquisition within less than
24 h, for example [46].
Costs: Capital costs include a benchtop sequencer
(75,000 s), an extensive lab-infrastructure and trained
personal. The costs per run, covering in maximum about
2000 samples, do vary between 170 and 830 s, [46,47].
Therefore, outsourcing to a sequencing service is an
option that does currently cost 500–800 s per sample.
Automatization: On-site automatization seems currently
not feasible as laboratory equipment and extensive train-
ing are required and outsourcing is more likely.
Interpretation & Implementation: Data analysis is extensive
and can hardly be performed without specific training, but
will enable deriving operator guidelines.
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 Microbiomes in bioenergy production Koch et al. 69

Fig. 2

Case study 1 Case study 2

Anaerobic digestion monitored by community Production of a biofuel precursor monitored with
flow cytometry high-throughput sequencing
(a) flow cytometric microbial cell number variation (b) Thermodesulfobiaceae Clostridiaceae
community pattern Bacteria (k)

normal Firmicutes (p)

reactor
regime Planococcaceae Thermoanaerobacteraceae
Firmicutes (p)
Planococcaceae

2fold cell Bacteria (k)

substrate number
Rhodocyclaceae
overload increase
Sphingobacteriaceae Firmicutes (p)
primary fermentation
Bacillaceae as central pathway
Porphyromonadaceae Bacteria (k)
normal
reactor Clostridia (c) Family
regime Thermoanaerobacterales
Clostridiales (o)
4fold cell
substrate number Ruminococcaceae
Firmicutes (p)
overload increase
Lachnospiraceae
G8 G11 Lactobacillaceae
index gates Veillonellaceae

Current Opinion in Biotechnology

The possible results of microbiome monitoring demonstrating structure–function relationships for the discussed case studies: (a) Correlation of
cell abundance of microbial sub-communities, measured by flow cytometry, in anaerobic digester for varying substrate load. (b) Food-web for an
n-butyrate producing microbiome, resulting from high-throughput sequencing. The key-nodes for central fermentation pathways, based on
Thermoanaerobacterium spp., are indicated.
Figures modified from [8,40] (copyrights 2013 American Chemical Society).

Outlook: Current developments indicate further improve-
ments regarding Signal acquisition, Costs and Automatiza-
tion (e.g. [46,47], MinIONTM and GridIONTM device).
The main restrictions for routine application are data
analysis and interpretation [48]. Therefore, the devel-
opment of automated data analysis and interpretation
platforms will probably decide on the capability for
prospective routine monitoring. Considering the efforts
performed in this direction also in medical research, these
developments are optimistically likely within the next
five years. Despite developmental potentials high-
throughput sequencing remains a time-demanding
method, thus being so far inadequate for monitoring of
microbial dynamics in the range of hours or days. Never-
theless, the unprecedented information depth justifies its
application, especially for research and development in
bioenergy production.
Towards microbiome-based reactor
management
The management of reactor microbiomes requires appro-
priate monitoring of complex natural communities. The
case studies have shown two promising examples how
functional relevant changes in the microbiome structure
can be determined. Thereby we illustrated some of the
basic concepts on how operator guidelines can be derived
in future, but also highlighted current constraints for
routine microbiome analysis. So far it is possible to
reliably measure microbiome structures, including their
variation, using different levels of analysis. However,
despite technological developments like automated
sample preparation and data analysis that will certainly
improve within the next years, one fundamental chal-
lenge for routine microbiome analysis and management
remains: Linking a microbiome’s structure to its perform-
ance. To achieve this, detailed knowledge of structure-
function relationships within the microbiome is necess-
ary, thus requiring segregated analysis of individual
sub-functions. When applying marker-based methods
the existence and prior knowledge on key-processes or
microbial key-communities is required. However as
demonstrated in case study 2, functionally relevant
changes in the microbiome, for instance sub-functions
like metabolic pathways, can be redundant or bypassed.
Thus an analytical result may not be unambiguously
linked to the process performance. On the other hand,
cell based analyses performed regularly within generation
times of microorganisms allow monitoring microbiome
structure variations in process relevant time scales. Here,
however, the application of a general marker allowing to
monitor all cells requires further correlation analysis to
reveal the functional importance of sub-communities, as
for instance illustrated in case study 1. In summary, a
delicate balance between universality and specificity of
the analysis method is required. In all cases, however, a
comprehensive set of process relevant (abiotic)
parameters is indispensable for functional correlation
and thus prospective microbiome management.

www.sciencedirect.com Current Opinion in Biotechnology 2014, 27:65–72

70 Energy biotechnology
The prediction of microbiome performances is very com-
plex. The functional performance of cells as well as sub-
communities, including, for example, their metabolic
pathways and replication, their interrelation and response
to environmental parameters have to be considered and
mathematically represented. Deriving there from
straightforward operator guidelines like SOPs is even
more challenging. The mathematical description of
microbial structure-function relationships is already
demanding for pure culture systems, where several sig-
nificant advances were made in the past [49,50], but even
more complex for microbiome based processes like
anaerobic digestion. For AD the general principles, in-
cluding biochemical pathways representing the major
trophic levels present in bioreactors, are well understood
and described in the anaerobic digestion model ADM1
[51]. Nevertheless, because of the complexity SOPs are
not yet established in this field. Thus, the correlation of
results of different studies or the performance of different
AD plants are often impossible, which makes mathemat-
ical description even more difficult. Here the consider-
ation of microbiome data will be an interesting option.
These present constraints might be one reason why
more general concepts introducing ecological principles
for the description of bioprocesses have been discussed
[52,53,54].
In practice, however, the main goals are clear: successful
product formation (with high rate, titer etc.) and process
stability. The underlying principles for achieving these
goals are more in the focus of fundamental research as
well as for the development of SOPs for microbiome
management and respective analysis instruments and
protocols. One may doubt if it is feasible and affordable
for highly diverse, ever-changing microbiomes for bioe-
nergy production to gain sufficiently detailed knowledge
to mathematically describe all processes on cellular level.
Therefore, we speculate that for practice (i) deducing
general rules for the management of certain types of
microbiomes and (ii) monitoring reactors routinely on a
more global level will be a first big step, which will
provide already an excellent benefit.
Acknowledgements
F.H. acknowledges support by the BMBF (Research Award ‘‘Next
generation biotechnological processes — Biotechnology 2020+’’) and the
Helmholtz-Association (Young Investigators Group). S.M. acknowledges
support by the EFRE (European Fund for Regional Development).
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