13

Stability, persistence and resilience in anaerobic reactors" a c o m m u n i t y

unveiled
J. Tiedje a'c, A. Fernandez a*, S. Hashsham a'b, S. Dollhopf 'c, F. Dazzo a'~, R. Hickeya'd, and
C. Criddle at
aCenter for Microbial Ecology, bDepartment of Civil and Environmental Engineering,
CDepartment of Microbiology, all at Michigan State University, East Lansing, MI 48824,
dEFX Systems, Lansing, MI 48910.
The stability of function and its relationship to microbial community structure was studied
in anaerobic methanogenic reactors fed glucose over a 600 day period, and in reactors that
experienced a shock-load of glucose. In the first case, the Bacteria populations as measured
by 16S rDNA analysis varied in a chaotic manner throughout the period while function as
measured by pH and effluent COD remain stable. Hence, many combinations of populations
produced common function. Two different communities, one of which was high in
Spirochetes, but both functioning equally well in digesting glucose were fed a pulse of
glucose 10-fold higher than the normal feed. The high spirochete community showed more
resistance and resilience of function, had less diversity, and its community returned to its
original composition. Its superior performance was attributed to community flexibility, i.e.
minor populations which accommodate the shock-loading, while the more stable low
spirochete community did not tolerate the shock-loading as well.

1. INTRODUCTION
Reliable, efficient and rapid processing of organic matter is the goal of transferring natural
microbial communities to reactors for processing higher amounts of carbon. The environment
can be better controlled in reactors and the assumption is that this control also helps maintain
and stabilize microbial populations important to efficient function. We have studied two
questions concerning community stability with anaerobic reactors fed one carbon substrate,
glucose. The first question is how constant is the microbial community in a reactor
maintained under constant conditions for over 1500 days, and the second question is how fast
and reliably do communities return to their normal function and composition after they are
subject to perturbation. In this case the perturbation was a shock-load of glucose.
Anaerobic communities are particularly interesting for microbial stability studies because
substrate processing requires a food chain, a series of at least three guilds of microbes
specialized in the steps converting carbohydrate to methane and carbon dioxide. They must
operate in coordination for the community to prosper and the function to be efficient.
Current address: *Catedra de Microbiologica, Universidad de la Republica, Montevideo,
Uruguay, *Department of Civil & Environmental Engineering, Stanford University, CA
94305

14
Secondly, this ecosystem operates near the thermodynamic limits of stability and hence is
particularly sensitive to perturbations. Furthermore, the members of these communities are
thought to be well understood, particularly with regard to their metabolic pathways,
requirements, and identity.
This paper summarizes highlights of work presented in more detail elsewhere [ 1-3].

2. MATERIALS AND METHODS

Studies on Stability and Persistence. A 1.5-liter continuously stirred tank reactor was
operated anaerobically at 35~ for 1505 days [2,4]. Glucose was supplied in a periodic cycle:
16g/liter on one day followed by no glucose feeding the second day. A constant dilution rate
of 0.1 day-1 was maintained. Steady-state function as measured by effluent COD, volatile
fatty acids concentration, methane production, and pH. Steady-state was achieved after 400
days.
The microbial community structure was analyzed by amplified _ribosomal DNA restriction
analysis (ARDRA) [5]. Genomic DNA was extracted at seven sampling periods between day
900 and 1500. 16S rDNA was amplified from the extracted DNA using both Bacteria and
Archaea primers. Amplicons were cloned with a TA cloning kit, transformed into E. coli,
reamplified, and the appropriate sized amplicons were digested simultaneously with two
restriction enzymes, HaelII, and HhaI. The restriction fragments from each clone were
separated by electrophoresis on a 3.5% MetaPhor agarose gel. Restriction patterns were
normalized and compared with Gel Compare software. Pattern clustering was done by the
unweighted pair group method with averages with application of the Dice coefficient. Thirtysix ARDRA patterns were analyzed for each sample and frequencies were calculated as
percentages of the clones showing the same operational taxonomic unit (OTU). OTUs found
most frequently were sequenced, aligned and their nearest neighbors were identified using the
ribosomal database (RDP).
Studies on Response to Perturbations. In these experiments, we studied the response of
two very different anaerobic, glucose-fed communities to perturbation. One community was
comprised of 40% to 50% spirochetes and the other was comprised of more diverse organisms,
but typical of those more commonly found in anaerobic digesters. In this case, the reactor
volumes were smaller, 250-360 ml, so that we could manage 4 replicate reactors of each
community. Both reactor sets were subjected to an instantaneous 20-fold increase in glucose
concentration. The response to this shock-loading of substrate was followed by fatty acid
analysis, ARDRA, morphological analysis (image analysis) of the populations, T-RFLP, and
rRNA quantitative hybridization of certain populations. More detail on the reactor operation
and analyses is described in a companion manuscript in this volume [6].

3. RESULTS AND DISCUSSION

Stability and Persistence. The methanogenic reactor exhibited constant performance as
measured by effluent pH and COD removal from day 400 through day 1490 (Figure 1). The
pH ranged from 6.85 to 7.07 and the COD removal was between 89% and 99% until the
reactor began to fail on day 1491.

15
Functional efficiencv

loo.o
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90.0 % ....................................................................................................!
80.0 %

. . . . . . . . . . . . . . . . . . . . . . . . . .

1
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C o m m u n i t v structure

,~

Mb. formicicum
Ms. mazeii
Mb. bryantii

,,

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Spirochetes Gr. 1

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Eubacterium
P ropion ibacteri u m
Unaffiliated OTU

EO-VII

D-I

II

800 900 1000 1100 1200 1300 1400 1500 1600

Age of the reactor (days)
Figure 1. Summary of the performance (function) and dominant populations in an anaerobic
reactor maintained under constant conditions for over 1500 days. Redrawn from Fernandez et
al.[2].
Changes in Archaea populations were observed by comparing ARDRA pattern
frequencies over the 600 day time period shown in Figure 1. The dominant Archaea
population was rather constant from day 900 through day 1325, but then abruptly shifted to a
completely different community at day 1437 and maintained this community through the last
days of stable performance and even for the 15 days during the reactor failure. Organisms
closely related to Methanobacterium formicicum dominated the first period and
Methanosarcina mazei and Methanobacterium bryantii dominated the later period.
The Bacteria community was much more variable than the Archaea community (Figure 2).
Rapid population shifts occurred and no long periods of particular OTU persistence could be
distinguished. Furthermore, almost no trends in population succession could be detected,
even for an interval as short as 33 days. The diversity as measured by ARDRA profile
richness was also chaotic. The total number of OTUs at each sampling point range between 8
and 24. The OTUs that were sequenced were not closely related to other known organisms.
They represented the four clusters shown in Figure 1. The most dominant group of organisms
recovered by the rDNA method were members of the Spirochetes. The other dominant
organisms are affiliated with Eubacterium, Propionibacterium and one unusual OTU that had
less than 80% similarity to any known organism. While the successional pattem of Bacteria

16

Figure 2. Variation in dominant Bacteria over time in a stable anaerobic reactor. Each
pattern represents a different OTU and the bar size represents its frequency in the 16S rDNA
clone library. Redrawn from Fernandez et al.[2].
was chaotic, the first period was dominated by different groups of Spirochetes. Hence, there
was cycling of dominance within the Spirochetes.
Ecosystem stability often means different things to different disciplines. To some,
stability refers to function and to others, it implies constant composition of the community. In
this paper we are referring to the later as persistence. A difficulty in limiting the concept of
stability to function is that information about the populations is ot~en ignored, however, it is
the properties of the populations, particularly their fitness parameters, that are the heart of the
information needed to more intelligently manage communities. The results from our work
indicate that an extremely dynamic community sustains a functionally stable system.
Dominant members change dramatically, even over periods as short of 3.3 retention times.
These results also indicate that functional parameters, like pH and COD, are inadequate to
reveal community structure variation.
Phylogenetic analysis revealed that the genetic changes detected by ARDRA suggested
metabolic variation in the community. However, if similar physiology is assumed for
phylogenetically related taxa, the metabolic variation was less dramatic than were the genetic
changes. Since the anaerobic food chains involve close interactions between different guilds,
we expected that there would be a correlation between any changes in the Bacteria and
Archaea communities. This was only moderately apparent in our study. The most obvious
was that the Spirochetes occurred during the period when the Archaea were dominated by
Methanobacterium formicicum but disappeared in the later period when the methanogen
community shifted. We do not know if this relationship is causal and if so which component
would have initiated the change (Figure 1).
Response to Perturbations.
Ecologists quantify several parameters of stability as
described in Figure 3. The two main parameters commonly measured in this amplification

17

Amplification envelope
C
O

=m,

E
o
o

=o

Reactivity

Q,
O
m

====

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o

E
i_

Perturbation

! Resistance
i/

~,~_~./.!.m.a_,_._>:,max',

Resilience"
"

............. .v.......... /................

Time

Figure 3. Ecological parameters used to describe components of stability.

envelope are resistance, which is the degree of deviation from the steady state, and the
resilience, which is the time required for the community to return to its normal state[7,8]. We
measured the fatty acids produced in response to the shock-loading for the two communities
and evaluated their resistance and resilience. Another component that can be useful in
characterizing stability is the reactivity which is defined by the maximum slope of the rising
limb of the envelope.
All reactors performed uniformly for the 2 weeks before the shock-loading indicating
replication of function without replication of community structure, consistent with the finding
in the previous long-term study.
The low spirochete reactors (LS) had less resistance and resilience to perturbation since
the fatty acid accumulation was higher and took longer for the fatty acids to return to steadystate levels than for the high spirochete reactors (HS) (Figure 4). The low spirochete reactor,
however, showed more diversity both by image analysis and ARDRA showing that diversity
does not correlate with stability in this case, at least by these measures. The recovery of the
community as measured by morphotype similarity analysis showed that the high spirochete
reactor returned to its original community composition, while the low spirochete reactor did
not (Figure 5). The ARDRA analysis confirmed the morphotype analysis, both showing that
Spirochetes, which initially dominated the high spirochete reactors, were diluted by

18

1~1
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r
9I ~

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Cj r
40

(u

O
I-

HS set

,--

LS set

,w

16

24

32

Time from perturbation

(days)
Figure 4. Recovery of community function measured as % of electron flow in fatty
acids following the glucose shock-loading at day 0.

100
HS set
A

80
LS set

t__

60

.=,,

r
o

4O

.=,,

o
Q.
o
L
a.

20

'

'

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16

24

32

Time from perturbation (days)

Figure 5. Recovery of the community structure as measured by image analysis following
the glucose shock-loading at day 0.

19
organisms that grew faster on the glucose, but then returned to dominance at day 16 and
thereafter following the glucose perturbation.
The high spirochete reactors appeared to accommodate the high glucose loading because
minor members of the population could rapidly ferment the extra glucose producing butyrate.
ARDRA and 16S rDNA sequence analysis showed that this fast responding population was
related to Eubacterium hadrum.The large population of Spirochetes in a high spirochete
community is thought to ferment glucose to acetate, but when a large amount of glucose was
fed, a fast growing Eubacterium that was initially present in very low numbers emerged, out
competing the slower growing Spirochete populations, eventually shifting 30% of the electron
and carbon flow through butyrate (Figure 6). This was accompanied by a high proportion of
fast-growing, acetoclastic Methanosarcina species which rapidly metabolized the acetate
generated after the perturbation, resulting in little accumulation of acetate.
In the low spirochete reactor, Streptococcus-like organisms responded to the glucose
addition and produced lactate which appeared to be followed by clostridia converting the
lactate to butyrate. Acetate subsequently accumulated because the methanogenic population
was dominated by the slow growing Methanosaeta species. Organisms like Eubacterium and
Clostridium probably produce large amounts of H2 shifting the reducing equivalents directly
to a neutral methanogenic substrate that is rapidly utilized and possibly conferring functional
stability to the system.
Our studies show that functional stability could not be attributed to higher species
diversity, rather, a flexible community structure allowed minor members to rapidly
accommodate the high flux of glucose. Consequently our conclusion is that a stable
community structure may be inflexible and does not allow populations to quickly respond to
new conditions. Hence, flexibility rather than diversity or persistence may be important traits
for stably functioning reactor ecosystems.

100

IIIIII11
IIIIIIII

80 84

8
n

...... e
IIil111
II II111
r!!!!!!!

I--I Unique: unidentified

Ill
II]
III

II
11
II

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HS (IV-XXV): unidentified

I1|
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III
.!!!.!!

II
II

I~1

HS-III: Eubacterium hadrum

HS-II: Spirochaeta caldaria (98.7%)

I~

HS-I: Spirochaeta caldaria

60

t4o
?
% 20

(94.7%)

(98.7%)

16
0
8
Time from perturbation (days)

Figure 6. Succession of OTU's in the high spirochete reactor following the glucose shockloading at day 0.

20
ACKNOWLEDGMENTS
This research was supported by NSF grant DEB9120006 to the Center for Microbial Ecology.

REFERENCES

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