Age of the reactor days

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 often 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

TO 3

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 steady-state 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

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

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 Q

HS set

LS set

HS set

LS set

8 16 24

Time from perturbation (days)

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

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 accompa nied 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.

100i 80

S 60

□ Unique: unidentified 0 HS (IV-XXV): unidentified

HS-III: Eubacterium hadrum (94.7%) ■ HS-II: Spirochaeta caldaria (98.7%) gg HS-I: Spirochaeta caldaria (98.7%)

00 8 16 Time from perturbation (days)

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

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