WERF Phase 2: The Impact of Digestion and Dewatering on Reactivation and Regrowth of Viable but Non-Culturable Indicator Bacteria

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ABSTRACT
Recently, a number of reports have found much higher densities of the indicator bacteria, either fecal coliform or E. coli, in dewatered cakes compared to the samples just prior to the dewatering process for anaerobically digested solids. This phenomenon has been attributed to either reactivation and/or regrowth. The objectives of this study were to examine the impact of digestion and dewatering processes on reactivation and/or regrowth of FC and E. coli. Samples were collected from several anaerobic digestion processes, two thermophilic and five mesophilic, with either centrifuges or belt filter presses used for dewatering. Samples were enumerated using standard culturing methods for FC and E. coli as well as quantitative polymerase chain reaction (qPCR) to enumerate E. coli. The results demonstrated that for the single stage thermophilic processes sampled, a large proportion of FC or E. coli remain viable but nonculturable (therefore, they are not measured by culturing methods), and these bacteria can be reactivated during centrifuge dewatering. After reactivation, the bacteria can grow quickly and reach peak concentrations of greater than 107 per gram of dry solids within a few days of storage, followed by die-off with further storage. When thermophilic digestion utilized a reactors in series configuration, the E. coli were destroyed and no reactivation or growth was observed after
centrifuge dewatering. Samples after mesophilic digestion did not have significant quantities of viable but non-culturable E. coli. However, after centrifuge dewatering, growth of E. coli and FC occurred rapidly, reaching densities of greater than 107 per gram dry solids within a few days of storage, followed by a decrease in densities during continued storage. Cakes dewatered using a belt filter press did not show significant increases in the E. coli or FC density. The results suggest that centrifuge dewatering can reactivate viable but nonculturable bacteria, and the cake that is produced provides good growing conditions to support growth of FC and E. coli.

INTRODUCTION
Researchers and utilities have recently reported high concentrations of indicator organisms such as fecal coliform immediately after high solids centrifuge dewatering of mesophilic and thermophilic anaerobically digestion when just previous to dewatering the counts were significantly lower (Cheung et al., 2003, Iranpour et al., 2003, Erdal et al., 2003 and 2004, Hendrickson et al., 2004, Qi et al., 2004, Monteleone et al. 2004, Higgins et al., 2006a). For example, at one plant the FC densities were measured as <100 colony forming units (cfu) per gram of dry solids (DS) after thermophilic digestion; however, immediately after dewatering of the digested solids in a high solids centrifuge the measured counts were greater than 105 cfu/g DS. Different theories were promulgated to explain this phenomenon. For example, researchers suggested that the dewatering process caused floc breakup which led to increased enumeration efficiency during culturing methods used to enumerate FCs, other research suggested that the high counts were due to regrowth of bacteria present in the samples. Higgins et al. (2006a) suggested that during digestion, bacteria entered a viable but non-culturable (VBNC) state which means the bacteria were still present and viable, but they did not grow on standard culturing media (SCM), and were therefore not enumerated. Dewatering in high solids centrifuge resulted in ‘reactivation’ or ‘resuscitation’ of the bacteria. The term reactivation or resuscitation is defined here as the transition where bacteria become culturable when previously they were nonculturable. The researchers used molecular techniques to enumerate E. coli after digestion/before dewatering and also immediately after dewatering. The results showed that the counts based on copies of E. coli DNA were not significantly different before and after dewatering, despite the large difference in counts measured by the SCMs. These results supported the VBNC concept. This research leads to many important questions regarding the extent and nature of VBNC indicator organisms in digestion as well as their reactivation during dewatering.

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