The Effect of Klozur® Activated Persulfate ISCO on Microbial Populations

The impact of chemical on the native microbial population during in situ chemical oxidation (ISCO) is not well understood. As a result, some concerns exist within the remediation community as to the potential of negative impacts ISCO may have on bioremediation processes, especially its impact on down-gradient plume control via bioremediation and its impact on subsequent natural attenuation processes. While there have been several studies in the literature looking at the impact of various oxidants on microbial viability, there have been few published investigations involving activated persulfate. In this edition of Peroxygen Talk, we will take a look at persulfate chemical oxidation and its effect on microbial populations.

Population Impacts

Several years ago it could be heard at conferences that chemical oxidation events “sterilize” the soil, completely wiping out the natural microbial population, rendering the soil lifeless. This belief is no longer pervasive due and oxidation-induced sterilization has not been observed in the field. It is difficult in actuality to sterilize dirt, unless you have an autoclave. In addition, within the soil column, there are many “hiding places” or pockets for microbes to survive during a chemical oxidation event. Coupled with the diversity and pervasiveness of microbial life and natural replenishment from incoming groundwater into the treatment zone, re-population of the microbial population post an oxidation event is generally anticipated.

In fact, to some extent the conceptual image of ISCO’s impact has shifted, and there is now a generalized hypothesis that injection of oxidants can ultimately enhance long term bioremediation and natural attenuation capability. By this theory, oxidants may actually improve microbial function by: reducing the level of contaminants to less than toxic concentrations; breaking down contaminants to more useable fragment sizes; and increasing the levels of dissolved naturally occurring organics, which can be utilized as a food source. However, while ISCO is recognized as having potential benefit to subsequent aerobic bioremediation processes, such as the biodegradation of petroleum hydrocarbons, there is the thought that chemical oxidation must be harmful to any subsequent anaerobic bio-processes, such as the anaerobic bioremediation of chlorinated ethenes, as anaerobes loathe oxic conditions. Interestingly, Droste et al¹ reported that data from an application of persulfate and permanganate (sequentially injected) in a pilot field test to treat chlorinated solvents supports evidence of ongoing sulfate-reducing bacterial activity post-injection. In fact these results indicate that the reductive dechlorination of TCE may actually have been enhanced by the oxidant application, based on their assessment of TCE to vinyl chloride ratios. This conclusion may indicate that even for subsequent anaerobic bio-remediation process, the benefits of reduced contaminant loading and increased natural dissolved organics more than offset the impacts of increased oxygen content on the anaerobic population.

Tsitonaki et al² published one of the few peer-reviewed studies on the impact of persulfate, in this case activated by heat (40 C), on soil microorganisms. They investigated the effects on indigenous microorganisms as well as soils spiked with P. putida, at persulfate concentrations ranging from 0.1 to 10 g / L over a period of fourteen days. Their work indicated that the microbial populations of spiked samples were reduced greatly by the application of activated persulfate, which is consistent with other works referenced in their paper showing that “spiked” microbes are very susceptible to chemical oxidation. However, the indigenous microbial populations in their soil samples showed a high degree of resistance in terms of cellular integrity. (Discussion on the impact of substrate utilization will follow below). Bou-Nasr et al³ also reported little impact on indigenous cell concentrations when exposed to iron activated persulfate. Crimi et al⁴ reported that the biomass was not altered significantly (the population was not eradicated) and the sulfate-reducing bacteria were present and remained active when the their soil samples were treated with persulfate, activated by either Fe-EDTA, Fe-citrate or high pH.

Cassidy⁵ recently performed a laboratory study looking at the effect of Klozur CR (a formulation of calcium peroxide and persulfate designed to provide both ISCO and long-term oxygen release benefits) on sulfate-reducing bacteria (SRB) in sediments contaminated with polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons. The impact on SRBs may be significant, as they are strict anaerobes. Previous studies identified Desulfovibrio, Desulfobacteriaceae and Desulfobulbus as the predominant SRB species present in these soils. In the study, Klozur CR was dosed at three concentrations: 4 g / kg sediment (Dose 1), 50 (Dose 2) and 100 g / kg (Dose 3). Relative abundance data (displayed as Most Probable Number [MPN]) for the microbes is shown in the figure below.

Figure 1 MPN of SRB in the Reactors (error bars show the std dev of the MPN)

In the control (no Klozur CR added), no significant change in the MPN for the SRB were observed. Addition of the Klozur CR did result in significant decreases in the relative abundance of SRBs, with an increasing loss of population as the dosage increased. However, within the timescale of the study (8 weeks), the microbial population rebounded in the reactors under Dose 1 and Dose 2 conditions. In fact, the population in the reactor with Dose 1 resulted in a greater MPN value after eight weeks than the control. These data are especially interesting given the fact that part of the Klozur CR product is a slowly releasing oxygen source. Cassidy attributes the rebound of the microbial population as probably due to an increase in sulfate concentration (a by-product of the persulfate reaction) and the accumulation of low molecular weight fatty acids and alcohols, such as acetate, oxalate, propionate and ethanol resulting from the oxidation of the PCBs and aromatic hydrocarbons. Analysis of the microbial distribution showed that the three SRBs mentioned above represented approximately 30% of the total bacterial populations, and that the SRB concentrations had a greater decrease as the oxidant dosage increased, commensurate with the relative abundance of all species data. Studer et al⁶ presented data from a field application of Klozur CR at a site contaminated with BTEX and fuel constituents. Bio-Trap® monitoring indicated the presence of both aerobic and anaerobic hydrocarbon degraders, and that significant total bacterial and Proteobacetrial populations exist three months post chemical oxidant application. Population levels did decrease, on the order of about 20%, but the microbial population remained viable.

Impacts on Substrate Utilization

The data discussed indicate that in the short-term, application of activated persuflate to the subsurface will impact microbial populations in the short-term, but that they will eventually recover, even the anaerobic species. Also important is the impact activated persulfate may have on the substrate (contaminant) utilization efficiency of the microbes, as this is key to bioremediation and natural attenuation processes. Tsitonaki² investigated the acetone consumption of microbes in the presence of heat activated persulfate. While their data shows that the indigenous microbe population showed little influence by persulfate in terms of cell membrane integrity, they were vulnerable to the highest level of persulfate concentration (10 g / L in their study) in terms of acetone consumption. One explanation offered was that at the highest persulfate concentrations, the pH of their lab samples were low (pH = 3), which may have affected the proton motive force and thereby influenced the uptake of acetone. In the field work of Studer et al⁶, the inclusion of C¹³-labelled benzene in the Bio-Traps allowed for an analysis of benzene utilization by the indigenous population. Three months post application of the Klozur CR product, first-order estimated benzene utilization rates ranged from 0.023 mg / day to 0.043 mg /day. This indicated that after the chemical oxidation event, benzene utilization still continued and that the oxidant did not destroy the population nor eliminate its ability to utilize and destroy the contaminant. Likewise, the work of Droste¹ demonstrated that application of persulfate did not severely impact the utilization of TCE by the native dechlorinators, and in fact may have enhanced the bioremediation of residual contaminant.

Conclusions

While further laboratory study and field assessment is needed, it is clear that activated persulfate application into the subsurface does not sterilize the soil matrix. Impacts to the microbial populations are observed for both aerobic and anaerobic species, and it could be anticipated that population densities may drop 20 – 30% post-application. However, in all cases rebound of the indigenous microbial species are observed, and in several examples, resultant densities are greater than prior the oxidation event. Cell utilization of the contaminant substrate will be impacted as well. However, field evidence suggests that these bio-processes are not halted, but continue apace. As a result, the use of activated persulfate chemical can be used successfully in concert with subsequent bioremediation events or natural attenuation.

¹ E.X. Droste, M.C. Marley, J.M. Parikh, A.M. Lee, P.M. Dinardo, B.A. Woody, G.E. Hoag and P.V. Chheda. “Observed Enhanced Reductive Dechlorination After Chemical Oxidation Pilot Test”. Paper 2C-01 in Gavaskar and Chen (Eds), Remediation of Chlorinated and Recalcitrant Compounds – 2002. Proceeding of the 3^rd International Conference on Remediation of Chlorinated and Recalcitrant Compounds (Monterey, CA), published by Battelle Press, Columbus, OH.

²A. Tsitonaki, B.F. Smets and P.L. Bjerg. “Effects of heat-activated persulfate oxidation on soil microorganisms”. Water Research 42, p 1013 – 1022 (2008).

³ J. Bou-Nasr, D. Cassidy and D. Hampton. “Comparative study of the effect of four ISCO oxidants on PCE oxidation and aerobic microbial activity”. Proceedings of the 5^th International Conference on Remediation of Chlorinated and Recalcitrant Compounds (Monterey, CA), 2006.

⁴ L. Gallagher and M. Crimi. “Coupling Persulfate ISCO with Bioprcesses: A Review”. 5^th International Conference on Oxidation and Reduction Technologies for the In Situ Treatment of Soil and Groundwater (Niagara Falls, NY), 2007.

⁵D. Cassidy, Western Michigan University, “The Effect of Klozur CR on Sulfate-Reducing Bacteria (SRB) in Sediments from the Kalamazoo River”, a report to FMC.

⁶J. Studer and P. Block. “Design Considerations for Application of Klozur CR to Promote Sequential Chemical Oxidation and Oxidative Bioremediation”. AEHS West Coast Soils Conference (San Diego, CA), 2008.