Effects of Chitin Purity and Proppant Loading on the Bioremediation of Chloroethenes Jennifer A. McElhoe1 and Rachel A. Brennan2 Abstract: Hydraulic fracturing (or fracking) of substrates and proppants into contaminated soils is a developing, but understudied, practice of stimulating in situ bioremediation. In this work, three different purities of the substrate crab shell chitin (SC-20, SC-40, and SC-80), two proppant loadings (sand:chitin mass ratios of 5∶1 and 15∶1), and three chloroethene concentrations (1 and 10 mg∕L trichloroethene, and 1:5mg∕L cis-1,2-dichloroethene) were experimentally and statistically examined to determine their effects on halorespiration. The least refined crab shell, SC-20, produced the greatest variety of electron donors, converted the highest percentage of contaminant mass to ethene, and supported a significantly greater Dehalococcoides population than the other substrates. Although influent chloroethene concentration and proppant loading did not significantly affect halorespiration (p-values > 0:079), decreasing the proppant loading from 15∶1to5∶1 increased the longevity of electron-donor production. These results indicate that funds need not be expended for purification of crab shell substrates, and that SC-20 should be used with proppant loadings of 5∶1 or lower to maximize the duration of electron-donor production at sites with potential biodegradation rate limitations. DOI: 10.1061/(ASCE)EE.1943-7870.0000541. © 2012 American Society of Civil Engineers. CE Database subject headings: Remediation; Biological processes; Groundwater pollution; Soil pollution; Solvents; TCE. Author keywords: Halorespiration; Trichloroethene; TCE; Crab shell; Fracking. Introduction electron donors generally limits natural attenuation in subsurface environments (He et al. 2002). Soluble electron donors such as mo- Trichloroethene (TCE), a persistent groundwater pollutant, is cur- lasses (Kao et al. 2003), glucose (He et al. 2002), ethanol (Fennell rently one of the most prevalent contaminants found in industrial- et al. 1997), and lactate (de Bruin et al. 1992) have been applied to ized countries (He et al. 2003a; Stroo et al. 2003; Guilbeault et al. contaminated aquifers to overcome electron-donor limitations and 2005; McKnight et al. 2010). According to the Agency for Toxic effectively enhance anaerobic reductive dechlorination (ARD), but Substances and Disease Registry (ATSDR), TCE and its chlori- these substances degrade quickly and require continuous or semi- nated ethene daughter products, cis-1,2-dichloroethene (cDCE) and continuous addition to the subsurface, increasing operation and vinyl chloride (VC), have been detected at 60, 10, and 37% of the maintenance expenditures, and material costs. As a result, interest National Priorities List (NPL) sites, respectively (Griffin et al. has increased in the application of slow-release electron donors 2004). Traditional remediation strategies, such as pump and treat, such as tetrabutoxysilane (TBOS) (Seungho and Semprini 2009; are not only costly and generally inefficient at removing dense non- Yu and Semprini 2002), emulsified soybean oil (Long and Borden aqueous phase liquids (DNAPLs) like TCE (He et al. 2003a; Quinn 2006), olive oil (Yang and McCarty 2002), vegetable oil (Bell et al. et al. 2005), but also tend to transfer the contaminant from one 2001; Wiedemeier et al. 2001), oleate, and cell biomass (Yang and phase to another versus complete destruction (Freedman and McCarty 2000). Slow-release substrates provide a sustained, con- Gossett 1989). tinuous source of electron donors [like volatile fatty acids (VFAs)] One potential alternative to the conventional treatment of chlori- and hydrogen (H2) to the contaminated zone without active main- nated ethenes is in situ bioremediation using halorespiring bacteria, tenance, which can increase the effectiveness and reduce the overall like Dehalococcoides ethenogenes (Fennell et al. 2004), which cost of remediation. couple the energy released during reductive dehalogenation with Another slow-release electron donor that has recently been ap- growth (Löffler et al. 1999) by using a chlorinated ethene as a ter- plied for passive bioremediation is chitin (Harkness et al. 2003). minal electron acceptor (Major et al. 2002). Although the potential Chitin, derived from the shell material of crustaceans such as for complete destruction of the contaminant makes in situ bioreme- crab and shrimp, is the second most abundant biopolymer on earth diation an attractive alternative, the unavailability of appropriate after cellulose (Beaney et al. 2005). With a chemical formula of – C8H13NO5 (6 7% nitrogen), chitin has a nearly ideal carbon-to- Downloaded from ascelibrary.org by Pennsylvania State University on 07/14/16. Copyright ASCE. For personal use only; all rights reserved. 1Ph.D. Candidate, Dept. of Civil and Environmental Engineering, nitrogen ratio for bacterial growth (Harkness et al. 2003). Because 212 Sackett Building, The Pennsylvania State Univ., University Park, of the abundance of this material as a waste product of the shellfish PA 16802-1408. E-mail: [email protected] industry, availability is not limited and costs are low. Different 2Associate Professor, Dept. of Civil and Environmental Engineering, grades (i.e., purities) of chitin are available, and the associated cost 212 Sackett Building, The Pennsylvania State Univ., University Park, increases with the extent to which the raw shell material is refined. PA 16802-1408 (corresponding author). E-mail: [email protected] In its natural form, the crustacean shell is a porous solid consisting Note. This manuscript was submitted on August 4, 2010; approved on primarily of calcium carbonate (CaCO ), protein, and chitin. The January 27, 2012; published online on January 31, 2012. Discussion period 3 open until January 1, 2013; separate discussions must be submitted for least refined and least expensive grade of chitin is crushed and dried individual papers. This paper is part of the Journal of Environmental crustacean shell, and contains all three components (CaCO3, pro- Engineering, Vol. 138, No. 8, August 1, 2012. ©ASCE, ISSN 0733- tein, and approximately 20% chitin ¼ SC-20). The next refined 9372/2012/8-862–872/$25.00. grade of chitin is produced through a caustic wash to remove 862 / JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / AUGUST 2012 J. Environ. Eng., 2012, 138(8): 862-872 ∼ % ¼ the protein (leaving CaCO3 and 40 chitin SC-40), and process configurations including chitin purity, proppant loading, the most refined grade is produced by applying a strong acid to and type and concentration of contaminant. remove the CaCO3 (leaving > 80% chitin ¼ SC-80). With each re- The purpose of this work was to examine the effect of chitin finement, the cost of chitin increases from $1:00–1:25∕lb, to purity, proppant loading, and contaminant feed on the rate and ex- $2:50–3:00∕lb, and finally to $4:00–6:00∕lb for SC-20, SC-40, tent of halorespiration through chemical and molecular analyses. and SC-80, respectively (JRW Bioremediation, personal communi- The concentrations of chlorinated solvents and chitin fermentation cation, 2010). products were monitored over time, and Dehalococcoides sp. num- Successful results have been obtained using crab shell chitin for bers were quantified at the conclusion of the experiment to evaluate anaerobic dechlorination of TCE (Jacob et al. 2005), tetrachloroe- the effect of the treatment on bioremediation. Because of its more thene (PCE) (Brennan et al. 2006a), chlorinated volatile organic complex composition, it was hypothesized that the cheaper, less compounds (VOCs) (Price 2007), PCE and 1,1,1-trichloroethane refined SC-20 would provide a more diverse suite of electron do- (1,1,1-TCA) (Buser et al. 2010), and for denitrification (Robinson- nors and support an equivalent or higher number of Dehalococ- Lora and Brennan 2009a) and acid mine drainage remediation coides spp. with greater dechlorination activity than the more (Daubert and Brennan 2007; Venot et al. 2008; Robinson-Lora refined and costly SC-40 and SC-80. If true, then the use of and Brennan 2009b, 2010; Newcombe and Brennan 2010). In SC-20 would be justified, and electron-donor costs using chitinous all of these studies, SC-20, rather than SC-40 or SC-80, was used materials could be significantly decreased in future field-scale ap- to keep material costs low, but the potential benefit of using a plications. It was also hypothesized that decreasing the proppant higher-purity chitin was not examined. In addition, previous studies loading would extend the longevity of electron-donor production, mixed the crab shell particles with sand as a proppant to maintain and the use of crab shell substrate would effectively enhance dech- hydraulic conductivity following hydraulic fracturing (or fracking) lorination of both TCE and cDCE at various concentrations. of the mixture into contaminated soils, but an evaluation of the ef- fect of the proppant loading (sand:chitin ratio) on dechlorination Materials and Methods has yet to be performed. While the distribution of Dehalococcoides in laboratory columns receiving a continuous feed of chitin fermen- tation products and 3:7mg∕L (22 μM) PCE was evaluated Chemicals and Substrates (Brennan 2003; Brennan et al. 2006a), the response of the organ- Both TCE and cDCE were purchased from VWR International isms to chitin purity, proppant loading, and other chloroethene (West Chester, Pennsylvania). Both VC and ethene gases were pur- types and concentrations