UCRL-JC-118024 PREPRINT &%J=---?m~r- -7

Laboratory Treatability Studies Preparatory to Field Testing a Resting-Cell In Situ Microbial Filter

Bioremediation Strategy

RECEIVED R. T. Taylor M. L. Hanna

N T 0 6 \ggg 0 sT:1

This paper was prepared for submittal to In Situ and On-Site Bioreclamafion

Third International Symposium San Diego, CA April 19,1995

April 1995

This is a preprint of a paper intended for publication in a journal or proceedings. Since changes may be made before publication, this preprint is made available with the understanding that it will not be cited or reproduced without the permission of the author.

7

r .* . r k . . ---

DISCLAIMER

This document was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor the University of California nor any of their employees, makes any warranty, express or implied, or assume any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Referene herein to any specific commercial product, process, or service by trade name, trademark, manufactum, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or the University of California The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or the U N v d t y of California, and shall not be used for advertising or product endorsement purposes.

*". , I '

*

DISCLAIMER

Portions of this document may be illegible in electronic image products. Images are produced from the best available original document.

.- i ..

Laboratory Treatability Studies Preparatory to Field Testing a Resting-Ceil I n SirU Microbial Filter Bioremedlation Strategy

Robcrt T. Taylor and M. Leslie Hanna Earth Sciences Division,

Lawreace Livermare National Laboratory

ABSTRACT

Prior to a down-holecolumn treatability test of a Mefhyfosiw friciwsporium OB3b attached- restingell in situ biofilw strategy, a set of three sequential laboratory experiments w e n carried out to d e f m several key operational parameters and to evaluate the likely degree of success at a NASA Kennedy Space Center site. They involved the cell attachment to site-specific sediments, the intrinsic restingccll biotransformation capacities for the contaminants of interest plus their time-dependent extents of biodegradative removal at the concentrations of concern, and a scaled ex situ mini-flow-through-column system that closely mimics the subsurface conditions during a field-treatability or pilot test of an emplaced restingcell filter. These experiments established the conditions rquired for the complete metabolic removal of a vinyl chJoride (VC), cisdichloroethylene (cis-DE), and trichloroethylene (TCE) mixture. However, the gas chromatographic (GC) procedures that we utilized and the mini-flow-through column data demonstrated that, at most, only about 50-70% of the site-water VC, cis-DCE, and TCE would be biodegraded. This occurred because of a limiting level of dissolved oxygea, which was exacerbated by the simultaneous presence of several additional previously unncognized groundwater compontnts, esptcially methane, that are also competing substrates for thc wholecell soluble methane monooxygenase (sh4h40) enzyme complex. Iriespective, collectively the simplicity of the methods that we have developed and the results obtainable with them appear to provide relevant laboratory-based test-criteria before taking our microbial filter strategy to an in situ field treatability or pilot demonstration stage at orkt sites in the future.

XNTRODUCTION

A common approach to the in sifu bioremediation of groundwaters contaminated with chlorinated solvents has been to pump a suite of nutrients or a combination of gases into &e subsurface to stimulate the growth of useful indigenous microorganisms and to induct their cometabolic degradative activities (Hazen et d. 1995, Lee et al. 1988, MahaEey et al. 1992). However. such nutrient Stirnutation packages generally cause displacement of the soluble contaminants,.do not sekdvely d c h the subsurface with the most efficient degraders (for example even when cH4 in injected), and they contain comp&ng substrates for the desired Come-tabolic contaminant degradations. Moreover, since this injection process is 3dimensional in nature, it attempts to biotreat the subsurface volumetrically. Quantifmtion of the actual mounts of specific subsurface microbial remediation is often difficult or impossible to assess on a volumetric basis (Taylor et d., references within, 1993, Knapp tt al. 1995a. National Restarch council 1993). To adhere to a strictly in situ microbial treatment but circumvextt most of these difficulties and simplify the rcquircd cngineering field practices, we have continued to investigate as an alternative a Zdimensimal in situ micfobial filter strategy

’

1

(Knapp ct a1 1995a. Taylor ct al 1993). It is basad on che use of nondividing (resting-state) cells that me produccd in bioreaccors, hmestcd, resuspended in a bufftr or clean groundwater. and then injected io form an attached biocatalytic filter. lnespcctivc of how it is anplaced to intercept groundwattx flow - thin wall or annular cylinda - thc effectiveness of such a rcsting-cell filter will degend on several key parameters: the filter's required residence time (dcpcnds on the attached cell population [biocatalyst density] and its degradative complettntss over the, the filtcr ~clcncss, and the contaminant flow-through rate); &e filter's contaminant biotransformation capacity; and tk filter's catalytic longevity.

Our current focus is on the usc of a methanotroph. M. rrictrosporium 083b, as a rcsting-ccfl biodegradative filter fot certain chtorinatcd ethents. It is wcll documentGd that cells of this strain containing the s M M 0 enzyme complex can catalyze the cpxidation of a variety of ethene compounds that have at least one C-H bond and we have demonstrated the potential feasibility and effwtiveness of an attached restingGcl1 filter strategy over extended periods of time for degrading a single contaminant, TCE, Rowing through a saturated homogeneous quartz sand, Oklahoma (OK) No. 1 (Hanna and Taylor 1995, Knapp et al. 1995a, Shonnard et al. 1994, Taylor et al. 1W3). In preparation for a field-treatability study at theWilson Comers (WC) site, NASA K e ~ e d y Space Center, Florida (Jackson et al. 1995). we carried out a sef of simple laboratory experiments directed at the restingcell biodegradation of a mixture of chlorinated ethenes. Previous site characterization work carried out by others indicated that the predominant contaminants in the shallow (e15 m). slow-moving (4 cmlday). U)-2l0C plume at WC were VC (-0.2-1.0 pprn), cis-- (-1.4-3.0 ppm), and TCE (-0.2-1.0 ppm) and it established that the WC subsurface was composed of shell fragments (80%) plus quartz sand (20%) and a carbonate-based groundwater (PH 7.0; ionic sltength 0.01 molal) witb -0.5-2 ppm of dissolved oxygen (Jackson et al. 1995).

I ROUTINE MATERIALS AM) METHODS UTILIZED

2

M. trichosporium OB3b cells were batch grown at 3OOC in a 5-L Moreactor to an early stationary phase in a modified Higgins' minimal salts medium lacking Cu (Park et al. 1991. Taylor et aI. 1993). The bacteria were harvested by ctnuihgation, and washed and resuspended in 10 mM Higgins' medium phosphate buffer (XPB), pH 7.0. Thtst Suspensions wtft adjusted to either -1.0 mg of dry cell wt/mL (-2 x IO9 ceIls/mL) for attachment assays and the mini-flow-through column loading or to -5 mghnL (-1 x 1010 cellsiml) for use as concentrated st& in the frceu3l biotransformation measurements (Hanna ct at. 1994, Park et al. 1991, Shonnard et aL 1994. Taylor et al. 1993).

The concentrations of methane. VC, cis-DCE, and TCE, as well as the GC peak areas given by a group of additional highly-volatile, undifferentiated compounds in the WC groundwater were determined with a Hewlett-Packard (HP) Modd 5890A instrument It was equipped with a 6 ft stainless-steel column (outside diameter of 1/8 in) packed with a 0.1% AT-lo00 on Graphac 80/100 mesh (Alltech) and a flame ionization detector (FID). Nitrogen was the carrier gas (30 mumin) and samples were run at column temperatures of 35OC and 100°C. The presence of methane also was confirmed and quantified by GC analysis with a Hach-Carle series 100 instrument that contained 6-7 ft stainless-steel, packed-bed mokcdar sieve 13x (80/100 mesh) and Haysep A (60/80 mesh) coIumns in series and was quipped with a thermal

conductivity detector. Putative mefhanc GC peaks fmm this hstrumcnt w m cofkcccd and found to give the cracking pattern expected for CH4, when they werc subjcctad to mass spcctromeay.

The biotransformations of CK4, VC, cis-DCE, TCE, and the volatile undiffercntiated compounds by both frecccll suspensions and by attached cells within mini-flow-through columns was gmexally monitored by &e disappearance of the parent compound(s), using the fIP5890A-FID GC combination (Park et d. 1991, Shah et al. 1995, Taylor e( al. 1993). However, the biotransformation of TCE also was corroborattd quantitatively by the conversion of [ 12-1eJTCE to radiolabeled water-soluble mctabotitcs (Taylor et al. 1993).

It is important to note that for all of the foregoing GC analyses 30 pL to 500 p.L gas-phase (head- space) samples were removed with gas-tight syringes (Hamilton) from sealed vials and then were manually injected into the HP5890A or the Hach Series lo0 instnunent. To create such samples, precisely 1.0 mL aliquots of WC welt water were first transfmed with a syringe into 5.0 mL glass vials that were sealed with open-topclosure screw caps and PTFEfaced red rubber septa (the PTFE facings were placed downward towards the gas phase) and the vial contents were allowed to equilibrate at room temperature. The vial head- space GC peak areas for C&, VC, cis-DCE, and TCE were directly compared to those generated with known amounts of these compounds (standard curves) that were processed and head-space sampled from 5.0 mL vials in an identical manner. Similarly, for the cell suspension biotransformation capacity determinations (0.25 mL reaction volumes), the steady-state rate mcasurments (05 mL reaction votumcs), and the low-level, chlorinatedcrhene-mixture biodegradation time curves (1 .O mL reaction volumes), bead- space samples were removed form sealed 5.0 mL vials and analyzed by their GC peak areas against standards (Park et al. 1991, Shah et al. 1995, Taylor et al. 1993). Likewise, in the I cm x 10 cm horimtal column flow-through expeximent, 1.0 mL effluent &actions wecc collcct& directly into seaIed 5.0. mL glass vials via a WO-bent stainless-steel cannula and the vial head-spaces were sampled for GC analyses. '

RESULTS AND DISCUSSION

Site-specific well sediments were cotlected by Jackson et al. (1995) and thtn lyophilized and utilized by us to estimate &e maximal cell attachment that would be attainable by injection-pumping. For this measurement a disposabfe glass, Pastewpipetcolumn assay procedure was developed (Figure 1). AI1 Pipet- column attachment assays were run in duplicates or triplicates. Oklahoma No. 1 sand-ccdumns were always run in parallel as a historical refcrence material for M. trichosporiwn OB3b attachment. Negative ConttoIS for wn-bacterial particle subtraction consisted of packed columns through which HPB or site groundwtm was pumped. but no cells were loaded (Hanna et al. 1993, Shonnard et al. 1994). Table 2 shows that comparable attachments in the range of 4.0 to 10.0 x 10s CelWg of column material were obtained for the WC sediments and the OK No. 1 reference sand. Like tbe OK No. 1 sand (Hanna ct al. 1993, Shonnard et al. 1994). attachment of M. trkhuspotim OB3b to the WC sediments was marlcdly dependent on the ioaic strength of the aqueous medium. An ionic strength of approximately 0.01 molal is needed to promote maximal attachment to sand (Hanna et al. 1993, Shonnard et al. 1994) or the WC sediments (data not shown). But the process is not elcctrolyte-specific, since fhc 10 mM HPB (0.01 molal) and the carbo~tc- based JLNL and WC groundwaters (both 0.01 molal) promote similar cell attachment densities (Table I).

Atso, M. trichosporiUm OB3b attachment to &e OK No. 1 sand @&ma ct al. 1993, Knapp ct d. 199%) as we11 as the WC sdimcnts (data not shown) exhibits a s a d o n kinetics (Km-like) tyj~ of behavior. ThC OK No. 1 sand and the WC sediments in Table 1 have porosities of 0.32 and 0.40, rcspcctively, and permeabilities -9 Darcys (Jackson ct al. 1995, Taylor ct al 1993).

Rcsting-state M. trichosporh OB3b biovansfonn~on capaci~es for thc chlorinated ethtncs found at WC were examined by h e experimental protocol in Figure 2. It was empIoycd previously for TCE (Taylor et al. 1993) and was designed to measure the intrinsic biotransfonaation capacity by using conditioos that resemble a flow-through biofiltcr. An e m p l d microbial filter will operate in a dynamic, not a static, manna with respect to thc contaminant biodegradation products. For &is reason, in Figure 2 the cells arc spun down at 2 hour intervals and resuspended in fresh HPB (or clean site water) for tach rc- exposure to the chlorinated VOC. Any convenient short-time interval can be Sclectcd, but the purpose of these repetitive centrifugations is to separate frequently the cells from any rckased metabolic products - analogous to a flow-through biofilter. Enough replicate vials are set up to permit successive chlorinated ethene reexposures until no restingcell biodegradation is detectable.

With this protocol, we determined the biotransformation capacities of five chlorinated ethenes without the potentially confounding influence of a continual static build up of toxic metabolites (Table 2). The f ~ t c transformation capacity for VC is 'much lower than the values for TCE and cis--, however, VC was not the predominant contaminant at WC. If 1 , I - m was the predominant contaminant, a resting- cell strategy probabIy would not be feasibte with our chosen bacterium because filter replacement would be necessary too frequently. Our data further demonstrate clearly that the biotransformation capacity of trans- DCE is much higher than that of cis-DCE. It probably arises from the stereochemical sMMOcacalyzcd epoxidation of these geometrical 1,2-DCE isomers and differences in their resulting sttrtocpoxide reactivity or toxicity. Table 2 also shows that sodium formate consistently enhanctd the biotransfmation capacity for each compound teste& The mechanism for this is unknown, but it is most likely rclattd to the increased intracellular NADH regeneration that results from formate oxidation to by formate dehydrogenase (Shah et al. 1995 and references thaein). Higher inracellular NADH concentrations may protect the sMMO enzyme complex from the transient, but highly reactive and toxic, initial products of aerobic chlorinated &em biodegradation, namely their tpoxidts.

Steady-state kinetic constants were determined for the three major WC contaminants of concern, along with those for uans-DCE and 1.1-DCE for comparison (TabIc 3). Thert is very limited kinetic data in the literature for these chlorinated ethenes and none for M. trichosporim OB3b cells that have been deliberately cultured to a nitmedepletion condition in a modified Higgins' medium formulation (Park et at. 1991, Taylor ct al. 1993). From plots of the initial rate (obtained from early, closely-spaced time points) of chlorinated ethene disappcaranct versus the starting chlorinattd &me concentration, typical Michaclis- Menton, enzyme-saturation data were collected. Accurate initial aqueous-phase concentrations of the chlorinated ethenes were readily calculated from the amounts of each compound that wccc injected into tbe sealed 5.0 mL glass vials by utilizing experi~~entatly determined aqutous-phase/gas-phast distribution coefficients that had been obtained previously with 0.5 mL of HPB in the vials. Apparent Kms and maximal specific degradation velocities (Vmaxs) were then read direclly off such curves and similar vaiues were also derived from a linear Lineweaver and Burk type of plot in the fm of (S)h versus (S). An example of the

4

linwity and the fit of such I plot to the data is illustrattd for ciS-DcE b t b w Table 3. The Vmax and Km values in Tablc 3 werc generated in HPB @H 7.0) in the presence of sodium formate to optimize the intracellulw NADH substrate concentration and at the optimal growth temperature of 3OOC to approxirnatc the maximal theoretical wholeGetl specific bioconversion veIocity. Overall, Table 3 shows that tbc maximat degradation ratcs per mg of dry cell wt are similar (-60-80 nmolcs/min), except for l.l-IX3E which is somewhat lower. In addition, among thc three major WC contaminants of interest TCE and VC have similar whole41 Kms, while that of &-D& is decidedry smdler. Since the Km is a crude indicator of the enzyme-substrate dissocialion constant, the kinetic values in Table 3 predict that at very low concentrations (far below their Kms) of thcse three compounds in a mixture thc degradation of cis-DCl3 should reach cumpletion the earliest, followed by VC and TCE later, but appr~ximately together. it is noteworthy that differences between the cis-DCE and trans-DCE structuraI isomers are a h reflected in their widely differing Km values. In other words, these two isomers are either docking into the intracellular sMM0 active site differently or else they are yielding stereospecific intermediate transition states within the sMh40 complex.

The ability of cells in suspension, at the same approximate volumetric catalyst density as the WC attachment data {Table l), to effect thc oxidative biotransformation of a VC (1.0 pg) + cis-DCE (3.0 pg) + TCE (1.0 pg) mixture was then monitored at thc WC plume temperame versus time in oxygen-rich HPB. These contaminant levels were the maximal anticipated amounts of each chlorinated ethene per mL and the approximate ratios in which they were expected to exist in WC groundwater. Egure 3 illustrates that the biodegradation of cis-DCE occurred more rapidly and was complete within 5 h, while VC and TCE were - 9 5 8 consumed in -12 h.

when cell-suspension time curves were generated as above with WC groundwater. comptete biodegradations of the small amounts of VC, cis-WE, and TCE occurred in -10 h (Figure 4). However. comparatively much larger mole amounts of several other highly volatile components were also metabolized by M. trichusputium OB3b. Further GC evidence and mass sptctrometry established that thc major compound in this highly volatile group was methane Figure 4). In a striking way, this group of highly volatile components yielded prominent peak areas in our HP5890A RD-GC profiles. e.&: volatile undifferentiated cornmds 461,800 units (fiom Figure 4). methane 2373,979 units, VC 768,270 units, cis- DCE 593.540 units, and TCE 65,963 units. Methane and the volatile undifferentiated compounds were not detected in the earlier site characterization work by others that relied solely on an automated standard purge- and-trap GC procedure (EPA m h o d 601) for all of the well water analyses (Jackson et d. 1995). The undifferentiated volatile mix- litstad in figure 4 may consist of several fluorinated ethenes (Jackson et al. 1995). Methane and UIC undifferedated volatile mixture were r@ly dctccted during all of our rnctabo1ic experiments with WC groundwater because manual gas-phase sample injections were perfomed directly into packed-bed GC columns that were Iinkcd to FIQ or TCD detectors (Material and Methods)

Because of the likely increased oxygen burden imposed by the additional unexpected mtrabolizablc components in the WC groundwater, sealed 1 cm x 10 cm horizontal, gIasscolumn, flow-through systems were prepared (Figure 5). Thest glass C-10 columns (Phannacia) were packed with WC well I7 sediment. loaded with bacteria to an attachment density of -10 x IO8 celldg of sediments, and then subjtcted to a continuous input flow of WC well 17 water from 50 mL gas-tight syringes (Hamilton). They served as

5

closed itstrvoirs that prevented the loss of a and other volatile compounds (e.g. methane) present in collected wet1 water samples. A controlled rate of well water deIivery to the test and control (minus cell) columns was achieved with a muiti-channel syringe pump (Harvard Apparatus 22) at a controlled rate of 4 - 8 5 linear column cm/h (I 1-12 h filter residence time). Emucnt fractions (1.0 mL each) collcctcd at 4 h intervals in 5.0 mL sealcd vials over 72 h nvcalcd that only about 50-709b of the WC well 17 water components VC, cis-DCE, and TCE were metaboliztd (r;igurc 6). along with simiiar puctntagcs of the CH4 and the volatile undifferentiated compounds (data not shown). Thus, this mini-column study predicted C O K C ~ Y that only a panid biotransformation of the chlorinated erhencs would be did in a down-hole column treatability experiment at WC welt 17.

In conclusion, we have devised a series of Lhrae simple laboratory tests that are appIicabIe to the design and treatability succcss of an in sifu resting cell filter: (1) Pastw-pipct-column attachment assays with sitc-specific sediments; (2) intrinsic biotransformation capacities and acfobic suspension metabolic time cwves for the contaminants of interest alone and within tht site groundwater matrix; and (3) GX situ scaled mini-flow-througb column systems that employ site-specific sediments and can mimic and maintain the temperature, pH, volatile compound composition etc. of site-specific groundwaters. These exsiru mini- columns can m e to develop better estimates of the required filter residence time, the filter thickness, and its replenishment intervals. But, in particular, they can dso provide an early-warning about unexpected metabolizable compounds or inhibitors in the site groundwater of interest.

This work was funded by the National Aeronautics and Space Administration and the Office of Technology Development within the Department of Energy's Office of Environmental Management, under its In Situ Remediation Integrated Program. All work was performed under the auspices of the Department of Energy by Lawrence Livermore National Laboratory under Contract W-7405-Eng-48.

REFERENCES

Hanna, M. L., D. R. Shonnard, and R. T. Taylor. 1993. "Measurements of Methybsinus trichosporium OB3b Attachment to Sand with the Use of Disposable Mini-Cotumns." Abstracts of the

Hanna, M. L. and R. T. Taylor. 1995. "Auachment/Detachment and Trichloroethylene Degradation by Resting Cells of Methylosinus zrichosporium OB3b." Abstracts of the of %

Y for Mt-, Atlanta, GA, May 16-20: p. 417.

Washington, DC May 21-25: submitted.

Hazen, T. C., B. B. Looney, M. Wen, J. M. Dougherty, J. Wear, C. B. Fliermans, and C. A. Eddy. 1995. "In Situ Bioremcdiation of CkIorinated-Solvents Via Horizontal Wells," Abstracts of the 94th General l, Las Vegas, NV, May 23-27: p. 329,

ins, G. D., J. Munakata, L. Sempdni, and P. L. McCarty. 1993. "TrichIoroetbyIene Concentration ts on PiIot Reld-Scale In Siru Groundwater Bioremcdiation by Phenol-Oxidizing Microorganisma"

Y U O ~ C I . TKhn& (12): 2542-2547.

Jackson, K. J., A. G. Duba, M. L, Hanna, h4. C Jovanovich, R B. Knapp, N. N. Shah, and R T. Taylor. 1995. "Field Treatability Experiment of In Situ Microbid Fdtu Bioremediation: Wilson Coma, Kennedy Space Center, Florida." and On S i t t Bioreclamat ion: The Th ird International svrnwsium. San Diego, aYpril24-27.

racts of In

6

Knapp, R, B., R. 7'. Taylor, A. G. Duba, K. J Jackson, M. C. Jovanovich, A. M. Wijesinghe, and M. L. Hanna 1995a. "EngincCring an In Situ Microbial Filter for Groundwater Biorunediation." submitted.

Knap , R. B., R. T. Taylor, M. L. Hanna, and D. R. Shonnafd. 1995b. "Mathematical Description for thc Attac ent of Resting-State cells to a Quartz Sand." & . . -, S a Diego, CAS Am 24-27.

Rn Lee, M. D., J. M. Thomas, R C. Borden, P. B. &dicot, C. H. Ward, and J. T. Wilson. 1988. "Biortstoration

18 of Aquifus Contaminated with Organic Compounds." -ws *- ..

(I): 29-89.

Mahaffey, W. R., G. Compcau, h4. Nelson, and I. K i d l a 1992. "TCE Bioremadiation." ~4(1):48-51.

National Research Council (Committee on In Situ Biormediation, Water and Science Technology Board, and Commission on Engineering and Technical Systems). 1993. Insiizr Biormcd &ion. When d a work? Park, S., M. L. Hanna, R. T. Taylor, and M. W. Droege. 1991. "Batch Cultivation of Methylosinus rrichosporim OB3b. I: Production of Soluble Methane Monooxygenase." j$&xhno 1 Bioeng, 3 (4): 423- 433.

Semprini, L. and P. L. McCarty. 1992. "Comparison Between Model Simulations and Field Results for In Situ Biorestoration of Chlorinated Aliphatics. Part 2 Cometablic Transformations." ground Water 3 (1): 3744.

Shah, N. N., M. L. Hanna, K. J. Jackson, and R. T. Taylor. 1995. "Batch Cultivation of MerhyIosinus tricbsporium OB3b: IV. Production of Hydrogen-Driven Soluble or Particulate Methane Monooxygenase

Shonnard, D. R., R. T. Taylor, M. L. Hanna, C. 0. B w , and A. G. Duba. 1994. "Injection-Attachment of Methylosinus trichosponm OB3b in a Two-Dimensional Miniature Sand-Filled Aquifer Simulator." Water &sour. Res, 3 (1): 25-35.

Activity." Biottchnot Bioerg. : in press.

Taylor, R. T., M. L. Hanna, N. N. Shah, D. R. Shonnard, A. G. Dub, W. B. Durham, K. J. Jackson, R. B. Knapp, A. M. Wijesinghe, J. P. Knezovich, and M. C. Jovanovich. 1993. "In Situ Bioremediation of Trichlorocthylenc-Contaminated Water by a Rtsting-Cttl Mcthanotrophic Microbial Filter." &drolo- Sciences J. (IAHS) 3 (4): 323-342.

LIST OF FIGURE AND TABLE CAPTIONS

FIGURE 1.

Table 1.

FIGURE2

TABLE 2.

TABLE 3.

FIGURE 3.

FIGURE 4.

FIGURE 5.

FIGURE 6.

Pasteur-pipetcolumn assay procedure for bacterial attachment

Attachment of M. trichusporium OB3b to Okiaborna No. 1 sand versus Wilson Corners

sediments.

QlIorinated cthenc biotransfmationcapacity protocol with resting cell suspensions.

Biotransformation capacities of M. VichoSporiUm OB3b for S C Y ~ chlorinated ethenes.

Summary of the restingdl kinetic constants for several chlorinated ethenes.

Time course for the biodegradation of a VC, cis-DCE, TCE mixture by ceIl suspensions in

HPB under excess oxygen conditions.

Time course for the biodegradation of the chlorinated ethenes in WC well 17 groundwater by

cell suspensions under excess oxygen conditions.

Photograph of the 1 cm x 10 cm horizontal, glasscolumn, flow-through systems.

A representative scaled laboratoxycolumn metabolic experiment with WC sediments and

WC well 17 groundwater flow-through

Key word list: I n situ microbial filter, Methylosinus trichosporium OB3b. resting cells, laboratory

treatability experiments, attachment assays, biotransformation capacities. vinyl chloride

(VC), cisdichloroetbylene (cis-DCE), trichIorotthylem (TCE), scaled mini-flow-through

columns.

t

connectors

Columns (0.6 crn x 10 cm) are prepared in disposable glass Pasteur pipets with either Oklahoma No. I sand or site-specific sediment material saturated in either 10 mM HPB (pH 7.0) or site-specific groundwater.

Bacteria are then pulled onto the column at 0.1 mVmin (63 cm/h column linear flow rate) for 2 h with a peristaltic pump (8 channel, Minipuls 3, Gilson).

Unattached bacteria are washed out at 0.034 rnl/min (21 cm/h column linear flow rate) for 16 h.

Figure 1

5.

6.

The top 2 cm of the column are removed by scoring with a metal file and snapping the glass to eliminate any column-surface strained cells from the count data

The sand and bacteria in the remaining 8 cm of column are extruded into a 10 mi graduated cylinder and the volume is brought to exactly 10 mf with distilled water. Cells are removed by shaking for 10 seconds and allowing the sand or site-sediment material to settle.

Bacteria are enumerated with a Coulter electronic particle counter having a 30 pm aperture and equipped with a Coulter Channelyzer. Twice filtered (0.22 pn) 4% NaCl is used as the counting solution (Hanna et al. 1993, Shonnard et al. 1994)

TABLE 1. Attachment of M. trichuspdzim OB3b to Oklahoma No. 1 sand versus Wilson Corners sediments.

Pasteur-pipe tcolumn btdmateriat

Oklahoma No. 1 sand

Oklahoma No. 1 sand

Oklahoma No. 1 sand

Wilson Corners sediments

10 mM HE%, pH 7.0

Distilled water

Cart>onate-bascd LLNL groundwater

10 mM HPB, pH 7.0

Wilson Corners sediments Distilled water

Wilson Corners sediments Carbonate-based WC well 17 water

3.2 - 5.0

0.1

4.0 - 8.0

4.7- 10.0

0.2

4.8 - 8.7

Chlorinated VOC (725 nmofes) added to cells (0.1 mg) i 2 mM Na formate within 5.0 mL viafs

Unexposed cantrol cells (0.1 mg) within 5.0 mC vials

U After 2 hours spin down cells and remove supernatant spin down celk and

Remove 400 pL of gas-phase or 50 pC of aqueous-phase samples at 15,30,60 and 120 rnin for biotransformation analyses

Standard steady-state rate assay for remaining whofe-cell degradation

remove supernatant

Add fresh HPB Add fresh HPB and test compound

for remaining degradation specific activity After2hours u

spin down cells and remov8 supernatant

U \ /

Remove samples at 15,30,60 and 120 rnin for anafyses

Standard kite assay for re rnaini ng deg radat ion

Figure 2 specific activity

TABLE 2. Biotansformation capacities of M. hiclrosponitm OB3b for several chlorinated ethenes.

Test compound (125 nmole repeated additions to 0.1 mg of cells)

Biotransformation capacity (mg of compound degradaa/mg of dry cell wt)

Minus formate PIus formate (2 mM)

TCE

VC

trans-DCE

cis-DCE

1.1-DCE

0.25

0.09

0.26

0.16

0.020

0.36

0.10

0.47

0.19

0.0 15

B Biotransformation capacities were determined at 20°C (Figure 2 and Taylor et al. 1993).

TABLE 3. Summary of the resthg-celJ kinetic parameters for several chlorhated ethenes.

Test compound Vmax (nmoles of Estimated whoIeccll Km test compound frommofthcobsavcd

ofdry cell wt) bidegtaddWmg appamt Vmax

TCE

vc OanS-DCE

cis-DCE

82

77

68

61

104 p.M (13.6 ppm)

90 ClM (5.6 P P I

25 p.M (2.4 ppm) or 19 ph4 (1.9 ppm) from s/v vesus s plot)

20 I I I I i

y = 0.31 + 0.016~ 9 = 0.998 Km = 18.6 pM Vmax = 60.8 nmoles/min/mg of dry cell wt

1s - v = nmoles cis-DCE consumed/mirJmg of dry cell wt

10 -

1

0 200 400 600 800 lo0

20 I I I I i

y = 0.31 + 0.016~ 9 = 0.998 Km = 18.6 pM Vmax = 60.8 nmoles/min/mg of dry cell wt

1s - v = nmoles cis-DCE consumed/mirJmg of dry cell wt

10 -

1

0 200 400 600 800 lo0

Aqueous phase cis-DCE, pM

1oc

80

60

40

20

0

Aerobic Incubation mixtures (1 .O ml) contained HP8 @H 7.0) and cells (0.25 mg dry cell wt) within sealed 5.0 mL glass vials.

Compound Amount added to the vials - vc - &-NE - TCE

1 .o pg

3.0 p.g

1.0J.q

0 10 20 30 40

Hours, 21°C

Figure 3

100

80

20

O C

# i I

Aerobic incubatlon mixtures contained WC NPSH- welt wafer (-pH 7) and cells (0.25 mg dry cell wt) Ir total volume of 1 .O mL within sealed 5.0 mC glass vials.

- w = - cis-DCE - TCE .

InLialamourllinldwenwater Total HP589QA GC peak area,

461,800

0.82 pg (S2nmoles)

0.71 crg (11 nmobs)

1 . 4 ~ (14nmofes) ’

0.76 pg (12 nmdes)

0 10 20 30 40 Hours, 21°C

Figure 4

. . . . . - . :. . . .._ .'. . .. . . .. ._ -.

. -. ._ : > . .

?

.

- 0.8 L % CT, 0.6

0.4

0.2

0

2.0

1.5

1 .o

0.5

0.0

- C Q) 2 0.05 5

0-72 h 8odegradation = I t

I I 1 1

L i Control column

Attached cell d u m n B 4 1

0.00 0 20 40 60 80

Elapsed time, hours Figure 6