2.1 Materials
Picatinny Arsenal provided the NTO. ATO (> 95% purity) was purchased from Princeton Biomolecular Research (Monmouth Junction, NJ, USA). All other chemicals used were of ACS standard. Luria-Bertani (LB) broth and Tryptic soy broth (TSB) were purchased pre-mixed from the manufacturer (Fisher Scientific, Waltham, MA, USA) and used at diluted concentrations indicated below. Sodium nitrate, and sodium sulfate were purchased from Fisher Scientific. Yeast extract, peptone, and glucose used to make YPG broth were purchased from Sigma Aldrich (St. Louis, MO, USA). YPG broth consisted of 5, 10, 10 g of yeast extract, peptone and glucose, respectively in 500 mL of sterile tap water.
Synthetic IX brine was made by combining Great Salt Lake (GSL) water with various electron donors, electron acceptors and NTO. Water was collected from the GSL Marina (Magna, UT, USA) every three months was used as the base of the reactor media into which the electron donors, electron acceptors and NTO were diluted. The water was stored at room temperature for up to three months until it was filtered through a 1.5 µm filter (Glass, Whatman), followed by a 0.45 µm sterile filter (Nylon, Millipore Sigma, Burlington, MA, USA) into a sterilized vacuum filtration apparatus. After filtering, the water was stored at 4oC to be used as feed for the SBR.
2.2 Great Salt Lake Culture Enrichment
Halophilic cultures were enriched from unfiltered GSL water using three separate electron acceptors and three carbon media as electron donors for a total of nine bioassays. Ferric chloride, sodium sulfate, and sodium nitrate were the electron acceptors. Carbon sources were 8.6% (w:v) strength each of LB, TSB, and YPG broths. The bioassays were conducted in 25 mL glass tubes with 20 mL of unfiltered GSL water spiked with carbon media and either sulfate (4,800 mg L− 1), iron (III) (2,790 mg L− 1), or nitrate (3,100 mg L− 1). All nine tubes were wrapped in aluminum foil and placed on a shaker table at 120 rpm at room temperature. The cultures were maintained for a month with weekly feedings. During feedings the cultures in the tubes were allowed to settle, supernatant was decanted and additional media was added to the same volume decanted. At the beginning of the second month, the cultures were supplemented with 50 mg L− 1 of NTO and biotransformation of NTO was observed over 28 days and 4 cycles. The enrichments showing the most NTO transformation were used to seed the 300 mL enrichment reactor described below. To ensure NTO and ATO were not degraded by abiotic reactions with sulfate or iron, aseptic control studies were conducted with the same media (i.e., LB, TSB and YPG), amended with sulfate or ferric chloride and spiked with 10 mg/L of either NTO and ATO. The control studies were incubated for one week and sampled periodically.
2.3 Sequencing Batch Reactor Operation and Sampling
Two different reactors were used in this study, an enrichment reactor and an SBR. The enrichment reactor operated from cycle 0 to 45 and was maintained at a working volume of 300 mL. It was made of a glass cylinder, 10.2 cm diameter, that was mixed continuously with an overhead mixer (Laboratory Stirrer, Yamato Scientific America) at 150 rpm. The reactor had an average hydraulic retention time (HRT) of \(4.2\)days. The GSL culture enrichment supplied with excess sulfate, and assumed to be sulfate reducing, was used to inoculate the first enrichment reactor. At the end of each cycle the overhead mixer was turned off and the reactor contents were allowed to settle for 10 min. After wasting the supernatant above the settled enrichment culture (typically 250 mL), approximately 50 mL of mixed liquor remained in the reactor. The mixed liquor was then fed with 250 mL of filtered GSL water, carbon media (5% strength), sulfate (4,900 mg L− 1), and NTO (50 mg L− 1). After 45 cycles, representing approximately 190 days, this enrichment culture was transferred to the larger SBR described below.
The second SBR was a 500 mL glass graduated cylinder, with a 4.8 cm diameter and a working volume of 400 mL. The reactor had ports at 5 cm from the base to withdraw effluent and sample the reactor, and a port at 15.2 cm from the base for feeding the reactor. When air was supplied by an air stone, it was supplied at a third port that was 3 cm from the reactor base. Stainless steel hose barbs (3/8 x 3/8 IN 316 SS, Grainger Choice) were fitted into the holes and held in place with stainless steel washers, and marine-grade silicone sealant was applied to the outside to prevent leaking. The reactor was mixed with an overhead mixer at 150 rpm. The mixer and influent and effluent pumps (VWR® Variable-Speed Peristaltic Pump, VWR) were controlled by timer (Chrontol XT4, Fisher Scientific). The average HRT was 3.4 ± 1.0 days over all cycles monitored. The average volume exchange ratio was 77%. Sludge was not wasted at any point during the operation of the reactor, but the effluent did contain approximately 1 to 2 g/L volatile suspended solids (VSS, Table 1). After wasting the effluent at the end of the draw cycle, approximately100 mL of mixed liquor remained in the reactor. It was fed with 300 mL of filtered GSL water, carbon media (YPG (2.7%), TSB (2.7%), LB (2.7%), strength based on the manufacturer’s preparation recommendation of 8% total strength), sulfate (10,000 mg L− 1), and NTO (36 ± 34 mg L− 1). No evaporative losses occurred in the reactor since the final cycle volume plus the volume taken for samples was equal to the initial cycle volume. The temperature averaged 21.6oC. In phases I, III and IV the SBR cycle consisted of 3 min feed with no mixing, 47.85 hr of reaction with mixing, 3 min settling, and 3 min decant. During phase III an additional 3 hr aeration phase was added immediately after the 47.85 hr of reaction prior to settling. The different operating phases are summarized in Table 1.
The SBR initial and final samples were taken directly from the reactor and were collected for most cycles. Influent sampling occurring within 10 min of adding new media to the SBR. Periodically, a complete cycle analysis was conducted and samples were collected at: 0, 1, 2, 4, 8, 12, 24, 36, and 48 hr. For sampling the reactor, a 10 mL sample of either the influent (from within the tank after mixing), mid cycle or end of cycle (i.e., effluent) was collected and filtered through a 0.45 µm non-sterile syringe filter (PTFE, Phenomenex, Torrance, CA, USA). Oxidation reduction potential (ORP) and pH were measured from the first 5 mL. The remaining 5 mL were diluted to 15 mL with deionized (DI) water, spilt for analysis of NTO, ATO, perchlorate, nitrogen species (i.e., nitrate, nitrite, ammonium and TKN), and sulfate. After dilution of the 5 ml with 15 ml DI water, 0.5 mL was transferred into an amber high-pressure liquid chromatography (HPLC) vial (Phenomenex) and diluted to 1.5 mL with DI water to quantitate NTO and ATO. Dilution was required to reduce the salt concentration prior to liquid chromatography. The remining 14.5 mL was kept for analysis of the other parameters. All samples were stored at \(4℃\) until analysis. Samples analyzed with HACH vial test kits were processed within 24 hr of the sample collection.
Table 1
Operational phases and parameters monitored
|
Feed conditions and phase of study
|
|
Anaerobic
|
Anaerobic
|
Anaerobic-aerobic
|
Anaerobic
|
Stress test
|
Parameters
|
Enrichment reactor
|
SBR
Phase I
|
SBR
Phase II
|
SBR
Phase III
|
SBR
Phase IV
|
Working volume, mL
|
300
|
400
|
400
|
400
|
400
|
Cycles
|
45
|
0 to 41
|
42 to 81
|
82 to 177
|
178 to 210
|
Sulfate, mg L-1
|
4,800
|
4,480 ± 350
|
1,445 ± 46
|
2,990 ± 640
|
2,651 ± 25
|
Salinity, %
|
NR b
|
NR
|
NR
|
4 ± 1
|
4 ± 1
|
Influent NTO, mg L-1
|
69 ± 64
|
69 ± 57
|
19 ± 11
|
28 ± 16
|
47 ± 6.7
|
Influent NO3-, mg-N L-1
|
NA a
|
NA
|
9.5 ± 2.7
|
5.7 ± 0.1
|
13 ± 1.1
|
Influent ClO4-, mg L-1
|
NA
|
NA
|
NA
|
NA
|
25 ± 6.2
|
HRT, day d
|
7.0 ± 1.0
|
4.3 ± 0.8
|
3.2 ± 0.7
|
3.0 ± 0.8
|
3.0 ± 0.8
|
VSS in effluent, g/L e
|
NA
|
NA
|
1.0 ± 0.5
|
1.4 ± 0.9
|
1.4 ± 0.5
|
NTO removal, %
|
88 ± 7.6 c
|
77 ± 17
|
44 ± 25
|
68 ± 19
|
91 ± 9.7
|
pH
|
NA
|
7.5 ± 0.2
|
7.3 ± 0.3
|
6.9 ± 0.2
|
6.7 ± 0.1
|
ORP, mV f
|
NA
|
NA
|
NA
|
-263 ± 56
|
-290 ± 20
|
a NA = not applicable, b NR = not recorded, c average and standard deviation of all three enrichment cultures, d HRT = hydraulic retention time, e VSS = volatile suspended solids, f ORP = oxidation reduction potential |
2.4 Perchlorate, Nitrate and ATO Inhibition of NTO Transformation and Degradation
Batch studies were conducted to determine if the presence of perchlorate and nitrate would inhibit the halophilic NTO degrading cultures since perchlorate and nitrate were previously reported to prevent souring in oil and gas reservoirs (Okpala and Voordouw 2018; Wang and Coates 2017; Wu et al. 2018). Souring occurs in oil and gas reservoirs when sulfate (i.e., the electron acceptor in excess in the SBR) is reduced into hydrogen sulfide gas. The batch studies were conducted in 50 mL amber glass vials and contained the same electron donors and acceptors as in the SBR. The first study included 50 mg L− 1 NTO and 20 mg L− 1 of perchlorate and/or nitrate. The second study included 50 mg L− 1 of NTO and 100 mg L− 1 of perchlorate and/or nitrate. Controls for the batch studies included a cell free mixture of NTO, perchlorate, and nitrate and a second set of controls where only NTO, perchlorate, or nitrate were present with microorganisms. The first and second batch studies ran for seven or ten days, respectively with samples collected on days 0, 1, 3, 5, 7, and 10 days (second batch study only). At each sampling point, 5 ml of sample was collected, filtered first through a 0.45 µm non-sterile syringe filter (PTFE, Phenomenex). One mL was stored for NTO and ATO analysis, then the remainder was filtered through a 0.22 µm non-sterile syringe filter (Nylon, Thermo Fisher Scientific, Waltham, MA, USA) and stored at 4oC until analysis by methods described in Section 2.7.
Duplicate batch studies were performed with cells from the SBR to assess the effect of increasing concentrations of ATO (0, 5, 50 and 100 mg L− 1) on NTO transformation. Here, 50 mg/L of NTO was included in the batch studies. Controls were constructed similarly to the biotic treatments but without the addition of cells. Cells were taken from the decanted waste from cycle 131. The waste was separated into two 50 mL centrifuge tubes and centrifuged at 500 x g for 5 min at 4oC. Repeated centrifuging in same tubes until all decanted waste was used. Tubes were shaken in-between centrifuging. A total of 50 mL of centrifuged cells were resuspended in GSL feed amended with the same media as noted in Table 1 for Phase III and monitored for 7 days.
2.5 Enzyme Extraction
To evaluate if halophilic cultures degrading NTO were expressing enzymes specific to degrading this compound, soluble cell enzyme extracts were collected and exposed to NTO in the absence of other carbon and nitrogen sources. Absence of NTO transformation by soluble cell enzymes without the addition of alternative nitrogen and carbon sources is an indicator of non-specific biodegradation or cometabolism in enrichment cultures (Suttinun et al. 2010). For the soluble protein extraction, cells were harvested from the SBR 4 hr after the SBR reaction phase started. A total of 150 mL was collected and spilt by weight into three 50 mL centrifuge tubes. The tubes were centrifuged at 5,000 x g for 10 min at 4oC. The supernatant was removed, retaining the cell pellet in the tube. Using the weighed amounts of the tube, the mass of the pellet was calculated. Bacterial Protein Extraction Reagent (B-PER) in phosphate buffer (Thermo Fisher Scientific) was added to the centrifuge tube immediately after decanting the supernatant at a volume of 4 mL of B-PER per 1 g of cell weight. The tube was incubated at room temperature for 15 min then centrifuged at 15,000 x g for 5 min at 4\(℃\). The supernatant was then immediately added to HPLC water spiked with 50 mg L− 1 NTO. Initial and final samples (t = 15 min) were taken, syringe filtered to remove excess bacterial enzymes and analyzed on the HPLC for NTO degradation.
2.6 NTO sorption to cells
To assess the potential for loss of NTO from the reactor due to sorption of the munition to cells that are periodically removed with the effluent, rather than biodegradation, a batch cell dissolution study was conducted. Cells from the reactor effluent were harvested and immediately concentrated by centrifugation (5,000 x g for 10 min at 4oC). After removal of the supernatant the cells were resuspended in either water, acetonitrile or phosphorus buffered saline solution. The cells were then sonicated for 24 hr and the concentration of NTO in the supernatant was determined. Total suspended and dissolved solids in the resuspended solutions were determined via gravimetric methods.
2.7 Analytical Methods
Concentrations of NTO and ATO were monitored on an HPLC (LC-2030, Shimadzu) equipped with a UV detector. The stationary phase was a Synergi C18-reversed-phase column (250 x 4.6 mm, pore size: 4 µm, Phenomenex, Torrance, CA, USA), preceded by a guard cartridge (Polar C18, 4 x 3.0 mm, Phenomenex, Torrance, CA, USA). The mobile phases were HPLC grade water and acetonitrile. The mobile phase elution gradients were 95:5 [percent water:percent acetonitrile] at 0 min, a ramp to 5:95 between 4 and 5 min, isocratic at 5:95 between 5 and 9 min, a ramp to 95:5between 9 and 10 min, and isocratic at 95:5 between 10 and 13 min to prepare for the next injection. The flow rate was 0.75 mL min− 1. NTO was monitored at a wavelength of 348 nm, had a retention time of 3.2 min and a detection limit of 0.1 mg L− 1 after sample dilution. ATO was monitored at a wavelength of 216 nm, had a retention time of 3.5 min and a detection limit of 1 mg L− 1 after sample dilution.
Confirmation of ATO production and degradation was determined by liquid chromatography mass spectrometry (LC-MS) analysis on a Waters ACQUITY H-class UPLC coupled to a Waters ACQUITY TQD mass spectrometer. The chromatograph column used was a Waters ACQUITY BEH amide UPLC column (2.1 x 100 mm, 1.7 µm particle size). The mobile phase gradient ramped from 10:90 [percent water with 10% formic acid: percent acetonitrile] at 0 min to 70:30 at five min. The flow rate is 0.45 uL min− 1. The mass spectrometer was set at positive Single-Ion-Recording (SIR) mode for monitoring protonated ATO ions ([M + 1]+ : 101 m/z). The capillary voltage was 3 kV. The sample cone voltage was 30 V. The source temperature was 120oC, the desolvation temperature was 350oC, and the nitrogen desolvation gas flow was 650 L hr− 1.
Perchlorate and chloride concentrations were measured by an 883 Basic IC Plus (Metrohm, Riverview, FL, USA) ion chromatography (IC). Using a Metrosep A Supp 4 Column (4 x 250 mm) with a Metrosep A Supp 5 Guard Colum (4 mm). The eluent was 159.6 mg L− 1 NaHCO3, 201.4 mg L− 1 Na2CO3, and 5% (v/v) acetonitrile. The elution flow rate was 1 mL min− 1 and the injection volume was 20 µl. Based on the chloride concentration, the sodium chloride concentration and salinity were calculated. It is assumed that every 10,000 mg L− 1 of sodium chloride is equivalent to one percent salinity by volume.
Due to the high salinity of the GSL basal media, which would interfere with IC analysis, HACH kits were chosen to monitor the concentrations of sulfate (TNT 865), nitrate (TNT835), nitrite (TNT839), ammonia (TNT831), and TKN (TNT880). The reactor samples diluted 20x achieves similar readings between samples prepared in DI water compared to GSL water. With the 20x dilution, the detection limits were as follows: sulfate (3,000 mg L− 1), nitrate (4.6 mg-N L− 1), nitrite (0.3 mg-N L− 1), ammonia (20 mg-N L− 1), and TKN (0.2 mg-N L− 1). Sulfate was always detectable in the reactors despite the relatively high detection limit due to the dilution required for salinity. A portable spectrophotometer (DR1900, HACH, CO, USA) was used to analyze the test vials with preloaded programs. The ORP and pH were monitored on a VWR® benchtop meter (B40PCID, VWR®).
2.8 DNA Extraction and Sequencing
Samples for DNA sequencing were collected at the end of every second week starting at cycle 108. A total of 5 mL was collected for extraction and vacuum filtered through a 0.45 µm sterile nitrocellulose filter (Millipore Sigma). Filters were stored at -80oC until extractions were performed. DNA was extracted following previously published methods (Griffiths et al. 2000). DNA concentration was measured on a Synergy HTX Multi-Mode Reader (BioTek, Winooski, VT) using a Take3 plate. Extracted DNA was stored at -20oC until being sent for sequencing by RTL Genomics (Lubbock, TX, USA). Each DNA extraction was amplified using two primer sets, one for Archaea [Arch517F (5'-GCYTAAAGSRNCCGTAGC-3’) to Arch909R (5'-TTTCAGYCTTGCGRCCGTAC-3’)] and one for Bacteria [515yF (5'-GTGYCAGCMGCCGCGGTAA-3’) to 926pfR (5'-CCGYCAATTYMTTTRAGTTT-3’)].
2.9 Statistical Analysis
Paired Student’s T test was used to test if there was a significant difference between initial and final concentrations of NTO, perchlorate, and sulfate. Statistical analysis was performed using SigmaPlot Systat (Version 14.0, Systat Software Inc, San Jose, CA, USA).