Alkylcyclohexanes in Environmental Geochemistry

Environmental Forensics (2002) 3, 293±301 doi:10.1006/enfo.2002.0100, available online at http://www.idealibrary.com on

Alkylcyclohexanes in Environmental Geochemistry Frances D. Hostettler* U.S. Geological Survey, 345 Middle®eld Rd, MS 409, Menlo Park, CA 94025, U.S.A.

Keith A. Kvenvolden U.S. Geological Survey, 345 Middle®eld Rd, MS 999, Menlo Park, CA 94025, U.S.A. (Received May 2002, Revised manuscript accepted July 2002) The n-alkylated cyclohexanes (CHs) are a homologous series of hydrocarbon compounds that are commonly present in crude oil and re®nery products such as diesel fuel. These compounds exhibit speci®c distribution patterns for di€erent fuel types, providing useful ®ngerprints for characterizing petroleum products, especially after degradation of n-alkanes has occurred. However, there are no published data to show how these compounds are altered in the environment after long-term spillage of petroleum products. This paper presents two case studies of oil spills that demonstrate the changing distribution patterns resulting from long-term anaerobic microbial degradation. These spills are the 1979 crude-oil spill in Bemidji, Minnesota, and a chronic diesel-fuel spillage from 1953±1991 at Mandan, North Dakota. The alkyl CHs in both spilled oil products are a€ected by similar biodegradative processes in which the compounds undergo a consistent pattern of loss from the high molecular weight end of the homolog distribution. Degradation results in a measurable increase in the concentrations of the homologs in the lower molecular weight range, a gradual lowering in carbon number of the homolog maximum, and a gradual decrease of the total homolog range from the high molecular weight end. This pattern is the opposite of low-end loss expected with weathering and aerobic biodegradation. The enhancement of the low molecular mass alkyl CH homologs, if not recognized as a degradative pathway of diesel fuel in an anaerobic environment, can potentially be misinterpreted in fuel-oil ®ngerprinting as deriving from lower distillation-range fuels or admixture of diesel with other fuels. Published by Elsevier Science Ltd on behalf of AEHS. Keywords: alkylcyclohexanes; diesel; anaerobic; biodegradation; Bemidji; Mandan.

Introduction

years before all the n-alkanes are completely degraded. Since many environmental diesel spills are weathered and have partial n-alkane loss, the more refractory alkyl CH distribution commonly has been used to de®ne diesel fuel contamination and to distinguish it from other mid-range re®nery products (Kaplan and Galperin, 1996). Alkyl CHs from approximately C7 to C24 with a Cmax at about C15 is a common distribution range for diesel fuel; indeed, it is characteristic of the railroad diesel standard from the Mandan re®nery (Hostettler et al., 2001) which is part of this study. The alkyl CHs, however, are only midway along the degradation continuum described by Kaplan et al. (1997) and there is no published documentation as to how their distribution pattern changes with long-term microbial degradation. Because of their utility in forensic geochemistry in de®ning di€erent re®nery distillates, this information is essential. Two hydrocarbon spills that have been in the environment for over 20 years are used in this study to investigate this degradation regime. The ®rst is a 1979 crude oil spill from a ruptured pipeline in Bemidji, Minnesota, and the second, a hydrocarbon accumulation, presumed to be primarily diesel fuel, in Mandan, North Dakota. The crude oil spill in Bemidji has been extensively studied (Cozzarelli et al., 2001; Bekins et al., 2001a; and references therein.). The prespill area was pristine, and there are no confounding

Fugitive oil spills are identi®ed and monitored according to their distinctive chemical ®ngerprints (Wang et al., 1999; Stout et al., 2002). Contributing to this signature are speci®c chemical constituents and chemical families, often in homologous series, relative distribution patterns of these constituents, and the geologic, thermal, depositional, and anthropogenic history of the oil. Once a hydrocarbon-containing mixture has been spilled and thus released from its source or containment, it carries its distinct chemical signature into the environment. However, after release and subsequent exposure to the environment, the oil is immediately subject to various forms of weathering. Any evaluation of spilled oil must consider the impact of this weathering on its chemical ®ngerprint. Historically, diesel fuel spills have been monitored according to a well-de®ned progression of weathering (Kaplan et al., 1997). Of the aliphatic constituents, the ®rst components to be removed are the n-alkanes, followed by the n-alkyl cyclohexanes (CHs), then branched-chain hydrocarbons, especially isoprenoids dominated by the ubiquitous pristane and phytane. Microbial degradation can be very slow, and, according to Christensen and Larsen (1993), it can take about 20 *Author for correspondence: E-mail: [email protected]

293 1527-5922/02/030293+09 $35.00/00

Published by Elsevier Science Ltd on behalf of AEHS.

294 F. D. Hostettler and K. A. Kvenvolden

Figure 1. Subsurface accumulations of oil.

hydrocarbons to interfere with the oil signature. The original crude oil has been well characterized and shown to be a paranic-naphthenic crude oil (Eganhouse et al., 1993). The main body of the spilled oil

lies on top of the water table in a shallow sand and gravel aquifer (Figure 1(A), adapted from Bekins et al., 2001a). Although aerobic oxidation occurs in the contaminant plume, away from the main body, the

Alkylcyclohexanes in Environmental Geochemistry 295

Figure 2. Map of downtown Mandan, ND-LNAPL accumulation and sample sites.

main hydrocarbon accumulation is known to be in an anoxic environment, as shown in Figure 1(A), and is undergoing anaerobic biodegradation. The great preponderance of microorganisms are methanogens or other anaerobes (Bekins et al., 2001a). The rate of degradation of the aliphatic hydrocarbons at di€erent sites within the oil body is variable, most likely related to the availability of water and oil saturation (Bekins et al., 2001b). The spill at Mandan is, in part, older than the Bemidji spill. The large hydrocarbon accumulation of 5.7±11  106 L ¯oats on the water table about 7 m below ground surface in downtown Mandan (Figure 1(B), adapted from Hostettler et al., 2001). The only documented spill component is diesel fuel from the railway yard in the downtown area (Roberts, 2001). All locally supplied diesel fuel is produced at nearby re®neries, including the BP Amoco Mandan Re®nery, that utilize only crude oil from the nearby Williston Basin. Chronic, substantial spillage at the rail yard is known to have occurred since 1951, when the railroad started using diesel to fuel its engines, and continued until 1991, when over¯ow containment trays were installed to prevent further spillage. A map of this study site showing the downtown spill area with the sampled wells is shown in Figure 2. In addition to the main hydrocarbon accumulation there are secondary spill accumulations within the railroad yard at the east and west fueling areas (EF1 and WF11), at well PL4, where there was a spill from a leak at the railroad yard end of a pipeline from the Amoco re®nery, and at well RH1, where a small roundhouse once stood. The spills at PL4, EF1, and WF11 are the most recent, with the fueling area accumulations occurring between 1979, when the fueling depots were moved out of the downtown area to the EF and WF areas, and 1991, when the spillage trays were installed; the pipeline spill occurred about 1990 (Roberts, 2001). All the other sampled wells are in the main light nonaqueous phase liquid (LNAPL) accumulation beneath several blocks of the downtown

area, including part of the railroad yard. The samples from the downtown area would have been in the subsurface longest, from 1951 at the earliest to 1979 when downtown fueling was moved to EF and WF. Some samples collected for this study have aliphatic pro®les indicating diesel fuel at more than one degradation stage due to the chronic spillage (Hostettler et al., 2001). A sample of the re®nery diesel (RR40) currently in use by the railroad is also included in this sample set. The purpose of this work is to compare the degradation patterns of these two long-term hydrocarbon spills, focusing on the early aliphatic degradates, the n-alkanes and especially the n-alkyl CHs. The crude oil has a greater diversity and range of components than the diesel fuel, and so the n-alkanes and alkyl CHs in crude oil have a broader distribution range. However, these components are prominent in both types of oil, and their degradation patterns can be tracked over time. In similar environments, the progression of these degradation patterns can be compared in both hydrocarbon systems. The Bemidji samples are known to be undergoing anaerobic biodegradation; in the Mandan samples the similarities of the physical environment, the long-term nature of the spill, and the degradation progression make it likely that they are undergoing anaerobic biodegradation as well.

Methods Sampling and analyses Procedures used for the collection of residual oil in sediment cores from the Bemidji site are similar to those given in Eganhouse et al. (1993). New core samples were collected in 2001, and some archived samples from collections in 1996 and 1997, along with an archived sample of the original crude oil, were also analyzed. Locations of the samples included in this work are shown in Figure 1(A) and listed in Table 1.

296 F. D. Hostettler and K. A. Kvenvolden Table 1. Geochemical parameters of Bemidji and Mandan samples Bemidji Samples BE-5 BE-6 BE-7 BE-8 BE-9 BE-10 BE-11 BE-12 BE-13 BE-20 BE-22 BE-23 BE-24 BE-25 BE-26 BE-27 BE-28 BE-31

Deg'n State*

Year Collected

Pr/Ph

C C C C C B A A A A C C C B C C B A

2001 2001 2001 2001 2001 2001 2001 2001 2001 1996 1997 1997 1997 1997 1997 1997 1997 1979

1.1 1.2 1.2 1.2 1.1 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.2 1.3

n-C17/Pr 0 0 0 0 0 0.68 1.0 1.0 1.1 1.3 0 0 0 0.64 0 0 0.33 1.3

Mandan Samples

Location

d13C-

Pr/Ph

MW2R MW17 PZ2 P5 P1R EF1 PL4 RH1 WF11 EW2 FR2 PZ6 MW44 MW43 MW45 MW7R RR40

RR yard RR yard RR yard RR yard RR yard E Fueling RR yard RR yard W Fueling RR yard RR yard N Main St N Main St N Main St N Main St N Main St Re®nery

28.6 28.7 28.6 28.8 29.1 29.2 29.6 28.5 29.0 28.7 28.7 28.6 28.7 28.4 28.4 28.8 29.5

1.2 1.3 1.2 1.2 1.2 1.2 1.3 1.3 1.3 1.3 1.3 1.2 1.2 1.2 1.3 1.2 1.5

n-C17/Pr 0 0 0 1.5 0.17 0.91 4.5 0 3.0 0.83 0.83 3.6 3.3 0 0 0 3.7

*Degradation state, as de®ned in Figure 3.

Sediment samples were kept frozen until analysis, at which time they were thawed in their glass jars. Approximately 10 g of thawed sample was placed in a pear ¯ask, and extracted with successive volumes of dichloromethane (DCM). Anhydrous sodium sulfate (to remove water) and freshly activated copper powder (to remove any traces of sulfur) were added to the ¯ask and the mixture refrigerated overnight. The sample extract was ®ltered, and the DCM ®ltrate evaporated under a stream of nitrogen to a ®nal volume of 5.0 mL. The approximate concentration of oil was ascertained by evaporating a 50 mL aliquot onto a tared aluminum boat and weighing the residue. A volume containing about 20±40 mg of oil was removed, and the DCM evaporated. The residue, or, in the case of the pure crude oil, about 25 mg, was taken up in 5.0 mL of hexane and the samples fractionated on a mixed silica and alumina column into two fractions, aliphatic and aromatic. The fractions were analyzed by gas chromatography/ mass spectrometry (GC/MS) and, for aliphatic sterane and hopane biomarkers, by GC/MS in the selected ion monitoring mode (SIM), monitoring m/z 191 (terpanes) and m/z 217 (steranes). Compound identi®cations were based on comparison with known compounds or with previously characterized reference oils. Members of homologous series were identi®ed by extracted ion chromatograms of characteristic fragment ions. These included n-alkanes (m/z 57) and n-alkylated CHs (m/z 83). Ratios of selected components were calculated using peak heights. Only the aliphatic fraction will be discussed here. Mandan samples and locations are also included in Table 1. Sampling methods and analytical techniques for the Mandan samples are detailed in Hostettler et al. (2001). Brie¯y, samples were analyzed as whole oils using 10±20 mg of sample diluted to 5.0 mL in hexane. GC/MS analysis utilized a lower range temperature program than was used for the crude oil samples, because of the lower boiling range of diesel fuel relative to crude oil. No hopane and sterane biomarker analysis was done because these compounds are not present in

diesel fuel. However, alternate biomarkers, bicyclic sesquiterpanes of molecular weight 208 and 222, monitored by m/z 123, were found to remain at constant levels over the sample set, and thus were used as conserved internal standards. Stable carbon isotope analysis for the whole oil samples was performed by Zymax Corp, San Luis Obispo, CA. As with the Bemidji oil, focus for this study was on the n-alkanes and the n-alkyl CHs.

Results Biodegradation of Bemidji samples, although substantial, has not yet impacted the isoprenoids pristane and phytane. Thus these compounds can be used to normalize the n-alkanes and alkyl CHs to monitor their changing concentrations and distribution patterns and to compare samples directly with other samples. This observation concerning the unweathered nature of the isoprenoids is based on comparison with the hopanes present in the crude oil, which are unaltered at this stage of biodegradation, and, thus, can serve as conserved internal standards (Wang et al., 1998). Extracted ion chromatograms which highlight n-alkanes (m/z 57) and n-alkylated CHs (m/z 83) are shown for representative Bemidji samples in Figure 3. Although the Bemidji oil spill occurred in 1979, the oil at di€erent sites within the oil body varies considerably in degradation level due to physical factors and microbial accessibility. A rough degradation continuum can be inferred and used to divide the samples into three groups: A ˆ little degradation with n-alkanes showing a characteristic oil distribution, relatively unchanged; B ˆ partially degraded, low end n-alkanes and isoprenoids dominant; C ˆ more degraded, n-alkanes absent, isoprenoids dominant. Table 1 includes the degradation group attributed to each sample. To display further the approximate degradation continuum, pairs of n-alkanes (n-C13 plus n-C14 at the low molecular weight end of the n-alkane distribution, and n-C25 plus n-C26 at the high molecular weight end), and pairs of n-alkyl CHs (C13 plus C14, and C18 plus C19, on

Alkylcyclohexanes in Environmental Geochemistry 297

Figure 3. Constituent extracted ion pro®les of representative Bemidji samples.

either side of the original crude oil CH homolog maximum) were normalized to pristane and plotted (Figure 4(A) and (B), respectively). The samples in these plots were arranged along the x-axis in order of increasing biodegradation, according to decreasing values of the commonly used degradation ratio, n-C17/ pristane (Peters and Moldowan, 1993), until that parameter decreased to zero (Table 1), followed by an order determined by the gradual loss of alkyl CHs. Both Figures 3 and 4(A) show that the high mass n-alkanes are degraded preferentially to those of low mass. Figure 4(B) indicates that once the CHs begin to be degraded, there is an increase in both the relative and absolute concentrations of the low molecular mass CHs as the high molecular mass CHs diminish. The progression of diesel alteration at the Mandan site is shown in representative extracted ion chromatograms,

Figure 5. The order of alteration is similar to that at Bemidji. In this case the samples are divided into four groups indicating progressive degradation: fresh (nalkanes prominent), low weathering (n-alkanes slightly less prominent), moderate weathering (only low end nalkanes prominent), and most advanced weathering (isoprenoids dominant). Values for d13C ratios are included with the ®gures of the four samples and can be seen to increase with increasing biodegradation. In addition, representative samples from the suite of Mandan samples, in order of increasing biodegradation beginning with the reference diesel, are listed in Table 2. Relative concentrations of a typical low molecular mass n-alkane, n-C11, and a low, middle, and high molecular mass n-alkyl CH, C10, C15, and C17, respectively, are listed. Normalization to the conserved internal standard allows comparison within the entire

298 F. D. Hostettler and K. A. Kvenvolden

Figure 4. Graph of low and high molecular weight homologs in the biodegradation continuum, Bemidji samples.

Table 2. Relative concentrations of n-alkanes and n-alkyl CHs in Mandan samples (normalized to conserved internal standard) Sample

n-C11

CH±C10

CH±C15

CH±C17

RR40 (reference diesel) WF11 P5 MW43 PZ2 MW45

14 25 31 24 12 7.4

0.79 2.0 2.6 2.4 2.4 2.4

4.1 4.0 2.7 2.3 1.7 1.7

2.8 2.4 1.4 1.4 1.2 1.1

sample suite. The data in Table 2 show that the low molecular mass homologs from both the n-alkane and n-alkyl CH suites increase in both relative and absolute concentrations compared to the undergraded RR-40.

Discussion Patterns of compound degradation within the n-alkane and CH homologous series are clearly seen in the Bemidji samples (Figures 3 and 4). The n-alkanes are degraded from the high molecular weight end, with a relative enhancement of the level of low molecular weight n-alkanes (5n-C17) before all the n-alkanes are completely biodegraded. Stout and Lundegard (1998) made a similar observation in another study of spilled diesel fuel, attributing the n-alkane weathering to intrinsic biodegradation. Because the Bemidji samples are samples of opportunity, collected after degradation had been occurring for over 20 years, there are only a few samples available to show the very early n-alkane degradation with respect to the unaltered crude oil (BE-31). For this reason, the Bemidji data do not show whether there is an absolute increase in the lower molecular weight n-alkanes. In Figure 4(A) the level of the low molecular weight n-alkanes stay approximately constant while the higher molecular weight n-alkanes are just beginning to be degraded, but none of the few samples available show more than level abundances before the low molecular weight n-alkanes themselves begin to be degraded.

The n-alkyl CH degradation pattern in Figure 4(B), however, is more informative. This distribution remains relatively constant for both high and low molecular weight homologs throughout most of the n-alkane loss, and begins to show degradation when the n-alkanes are nearly depleted. At this point, just as with the n-alkanes, the alkyl CH distribution shows a clear increase of the lower molecular weight homologs with respect to the higher molecular weight homologs as biodegradation increases. This pattern is accompanied by a decrease in the overall distribution range of the alkyl CHs, thus diminishing the overall range from the high molecular weight end. In addition, Figure 4 shows that the n-alkyl CHs exhibit an increase in absolute abundance (since the graphed homologs in each sample are normalized to pristane) of the low molecular weight homologs compared to the original crude oil and to the less degraded samples. The Mandan samples exhibit stages of degradation similar to the Bemidji samples, although most of the Mandan samples contain at least some n-alkanes. Thus, the n-alkane degradation sequence is present in more samples and therefore a more speci®c chronology can be inferred. To arrange the Mandan samples in order of increased biodegradation, however, is not completely straightforward, in part because of the chronic nature of the spillage. Several samples have more than one Cmax for the n-alkanes (Hostettler et al., 2001), suggesting mixtures of older and fresher product. Nevertheless, the samples can be sorted in an approximate biodegradation order by their m/z 57 pro®les, with samples representative of four progressive levels shown in Figure 5. As observed in the Bemidji samples, the same degradation progression of loss of n-alkanes and alkyl CHs from the high molecular weight end and enhancement of levels at the low molecular weight end can be clearly seen in the Mandan samples. In order to ascertain whether the enhancement is relative or if it re¯ects absolute increases in concentrations, a conserved chemical constituent is needed for normalization of the data. Pristane and phytane, which were applicable in the

Alkylcyclohexanes in Environmental Geochemistry 299

Figure 5. Constituent extracted ion pro®les of representative Mandan samples.

Bemidji samples, cannot be used here because even though they appear to remain constant in the less degraded samples, Figure 5 shows that the isoprenoid grouping also degrades slowly from the high molecular

weight end, i.e., from pristane and phytane. In the absence of hopane and sterane biomarkers, Stout et al. (2002) suggest utilizing less common biomarkers such as the bicyclic sesquiterpanes which can be pro®led in

300 F. D. Hostettler and K. A. Kvenvolden

Figure 6. Correlation of two biodegradation parameters in Mandan sample set.

extracted ion chromatograms at m/z 123. Indeed, all the Mandan samples show consistent m/z 123 chromatograms, with two major peaks tentatively identi®ed by comparing literature spectra as 8b(H)-drimane and homodrimane, molecular weights 208 and 222, respectively (Alexander et al., 1984). The abundance of the drimane compound was compared to some recalcitrant alkylated polycyclic aromatic hydrocarbons (PAH) present in the hydrocarbon matrix to verify constancy throughout the sample set; the compound was then used as an internal standard to normalize the n-alkanes and the alkyl CHs. Table 2 shows the results for speci®c compounds in the reference diesel fuel and in ®ve representative spill samples. The samples are listed in order of increasing biodegradation, with the sample MW45 the most degraded. Comparison of the ®ve spill samples to the unaltered diesel reference fuel (RR40) shows that in the ®ve spill samples, both of the low molecular weight series homologs, the n-C11 n-alkane and C10 CH, increase in both relative and absolute concentrations. In contrast, the higher molecular weight CHs do not increase. The C15 CH is constant initially, then decreases, but the C17 CH decreases sooner, as evident even within sample WF11 (note, however, that ``sooner'' is very relative, since sample WF11 is only reaching this degradation state after 10±20 years in the subsurface). The CH biodegradation progression in Mandan and Bemidji samples agree, both showing an initial relative and absolute increase in low molecular weight homologs. The n-alkane biodegradation at Mandan, with a broader sample representation, shows that a relative and absolute initial enhancement of the low molecular weight n-alkanes is the operative process at Mandan, and probably has occurred at Bemidji also. Furthermore, it is suggested that the enhanced levels of low molecular weight n-alkane and n-alkyl CH homologs most likely derive from reactions impacting the higher molecular weight homologs, possibly microbial (enzymatic) cleavage of terminal

carbons as suggested by Stout and Lundegard (1998). It is particularly likely that the alkyl CH molecules, consisting as they do of a CH end and an n-alkane end, would be degraded from the n-alkane end of the molecule, resulting in the observed increase in shorter chain, lower molecular weight homologs. Finally, the observed increase in concentration of lower molecular weight n-alkane and CH homologs and the decrease in high molecular weight homologs leading to a diminished distribution range, occurring in an anaerobic system, have not been documented previously. These new data run counter to the assumption that biodegradation progresses only with losses from low molecular weight end of homolog distributions, as happens in physically weathered and aerobic systems (Wang et al., 1998). The resultant distribution demonstrated in this paper, particularly of the alkyl CHs, therefore, could be erroneously interpreted in a forensic analysis as deriving from a lower re®nery cut, nondiesel fuel or from admixture with lower cut re®nery fuels. Examples of these fuels might be: diesel (No.1), mineral spirits, and kerosene. Compared to the railroad diesel, RR-40, with a CH homolog range noted earlier of C7 ±C24 and a Cmax of C15, these fuels have distribution ranges with lower molecular weight homologs and typical Cmax values of C11, C9 ±C10, and C12, respectively (Kaplan et al., 1997). As further evidence that the primary process impacting the Mandan samples is biodegradation, a parameter re¯ecting the changing CH distributions, a(CH)C10 ± C12/a(CH)C10 ±C24 was plotted against the bulk stable carbon isotope values for the whole oils (Figure 6). Stout and Lundegard (1998) showed that intrinsic degradation in the spilled diesel hydrocarbon system noted above caused a measurable change in bulk stable isotope compositions, d13C, from lighter to heavier, over time. Thus, in certain systems, this parameter can be used as a biodegradation parameter. Figure 6 demonstrates that there is a strong correlation, R ˆ 0.80 and P5 0.001, between these parameters within this sample set. The samples group very well according to their ages in the environment. This graph,

Alkylcyclohexanes in Environmental Geochemistry 301

therefore, strongly suggests that both parameters are changing due to biodegradation.

Conclusions This work shows that under anaerobic conditions, as at the Bemidji spill site, aliphatic hydrocarbon biodegradation progresses slowly and the general order of hydrocarbon-family loss is the same as that listed in Kaplan et al. (1997). However, the progression within speci®c families is unique to an anaerobic system. This progression occurs with initial loss of high molecular weight n-alkanes and n-alkyl CHs, and enhancement of lower molecular weight homologs. At Mandan, where the degradation progression is very similar, anaerobic biodegradation is also inferred. The Mandan diesel fuel spill shows that this progressive enhancement of the low molecular mass n-alkane and n-alkyl CH homologs re¯ects an absolute increase in their concentrations as the high molecular mass homologs are diminishing. These degradation patterns are quite di€erent from those observed in aerobic or physically weathered systems, where loss of n-alkanes and other aliphatic homologs occurs from the low molecular weight end of the distributions. This degradation progression is such that it can potentially confound interpretation of longterm fuel spills. Spilled diesel and other mid-cut re®nery fuels are de®ned by the range and distribution of the n-alkyl CHs. If the biodegradation has progressed well into or beyond n-alkane loss and to the stage of low molecular mass n-alkyl CH enhancement and high molecular mass loss, the hydrocarbon pattern can be erroneously attributed to other lowerrange middle distillate fuels or admixtures of fuels.

Acknowledgments The authors thank Barbara Bekins and Geo€ Delin, both of Water Resources Division, USGS, who provided the Bemidji samples for this project, and especially, thanks to Barbara for helpful discussions on the history of the Bemidji study and its microbiology. We also thank Jon Kolak for his help with the analysis and Jeanne Dileo for her work on the graphics.

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