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Integrated Chemical/Bfological Treatment of a Paintstripper Mixed Waste: Metals Toxicity and Separation

Laura Vanderberg-Twary, CST-18 Rose K. Grumbine, CST-18 Trudi M. Foreman, CST-18 John L. Hanners, CST-18 James R. Brainard, CST-18 Nancy N. Sauer, CST-18 Pat J. Unkefer, CST-18

ASME Third Biennial Mixed Waste Symposium, Baltimore, MD, August 7-1 1, 1995

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Integrated chemicalhiological treatment of a paint stripper mixed waste: Metals toxicity and separation

Laura Vanderberg-Twary*, Rose K. Grumbine, Trudi M. Foreman, John L. Hanners, James R. Brainard, Nancy N. Sauer, Pat J. Unkefer

Chemical Science and Technology Division, CST-18 Mailstop C-346, Los Alamos National Laboratory

Los Alamos, NM 87545

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Integrated chemicalhiological treatment of paint stripper mixed waste: Metals toxicity and separation Laura Vanderberg Twary*, Rose K. Grumbine, Trudi Foreman, John L. Hanners, James R. Brainard, Nancy N. Sauer, and Pat J. Unkefer. Chemical Science and Technology Division, CST- 18, Los Alamos National Laboratory, Los Alamos, NM 87545. *Presenting author: phone 505- 665-6493; fax 505-665-3688

The DOE complex has generated vast quantities of complex

heterogeneous mixed wastes. Paint stripper waste (PSW) is a complex waste that arose from decontamination and decommissioning activities. It

contains paint stripper, cheesecloth, cellulose-based paints with Pb and Cr,

and suspect PU. Los Alamos National Laboratory has 150-200 barrels of

PSW and other national laboratories such as Rocky Flats Plant have many

more barrels of heterogeneous waste. Few technologies exist that can treat this complex waste. Our approach to solving this problem is the

integration of two established technologies: biodegradation and metals

chelation. Bioremediation of hazardous organics found in paint stripper has

been demonstrated in our laboratory. A defined consortium (1 g / L dry

weight cells) in minimal salts medium mineralized 50 ppm each of

dichloromethane (DCM), toluene, and acetone simultaneously in 24 h. At higher concentrations of solvents, this mixture was degraded within 48 h.

Analytical techniques included head space analysis by gas chromatography-

mass spectrometry and spectrophotometry. Actively growing cultures of

individual consortium members were exposed to Cr(VI) (1-100 ppm) as

K2Cr04, Cr(III) (5-500 ppm) as CrN309.9H20, or Pb (50-1000 ppm) as

Pb(N03)2for 24 h. Expected concentrations of metals in PSW are: Cr (combined)<l 00 ppm, Pb 200- 1000 ppm, Pu<<l00 ppm. Concentrations

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of 1 ppm Cr(VI) and 500 ppm Cr(III) resulted in 95% toxicity to the

bacterial cultures as determined by dilution plating. Pb (1000 ppm) caused

a 50% reduction in growth. Various types and combinations of metal

chelators were ineffective at sequestering metals from microbes.

Combining bioremediation and metals chelation results in an

environmentally benign, cost effective technology to treat PSW. This technology may also prove applicable to a variety of other complex waste

streams.

Introduction

The DOE complex has, during past practices, generated large

quantities of complex heterogeneous mixed wastes. We define

heterogeneous mixed waste ( H M W ) as waste that contains RCRA organics,

solid bulk materials, rad metals and RCRA metals. Development of

treatment methods for mixed wastes generated by DOE facilities is

mandated by law (Los Alamos National Laboratory agreements with the

EPA under the Federal Facilities Compliance Agreement).

HMW was, and still is, generated through Laboratory operations such as decontamination and decommissioning of former actinide processing facilities. We are examining a particular target class of H M W that was generated by stripping the paint from surfaces contaminated with

Pu (further referred to as paint stripping waste, or PSW). PSW contains

dichloromethane (DCM) paint stripper with other solvents, Pb and cellulose-based paint, Pu, cheesecloth rags, and other cellulosic material

like cardboard and cotton lab coats. Chromates from paint pigments may

also be present. There are approximately 150-200 barrels of this PSW

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stored on site at Los Alamos National Laboratory (LANL), and other sites

in the DOE complex have been identified with many more barrels of this

heterogeneous waste.

Treatment and disposal of PSW has been at best, difficult.

Treatments such as incineration, advanced oxidation technology, and acid

digestion all present problems for the treatment of these wastes be it in

permitting, safety issues, cost, or secondary waste minimization.

Consequently, this type of waste is generally stored. Current storage

practices are a great expense for DOE and are often in conflict with laws governing the storage and handling of mixed wastes. An environmentally

benign, publicly acceptable solution needs to be identified which results in

destruction of the hazardous organic components, removal of the toxic (Pb,

Cr) and rad (Pu) metals and minimization of the final waste volume. We are developing a technology that uses two established technologies to accomplish these treatment objectives. Bioremediation with chelation of metals is a novel combination of proven technologies. Each of these

technologies are, in themselves, efficient, cost effective and publicly

approved. Bioremediation technology has been utilized effectively in the

degradation of toxic compounds. Cobb et al. (5) and Arquiaga and Canter (2) have investigated microbial destruction of phenolic and aircraft paint

stripping waste waters, but the presence and effect of metals was not

addressed. DCM and hydrocarbons are also common components of

paintstrippers. Organisms that utilize these substrates can be readily

isolated from the environment (for example 1,6,8). The effects of toxic

metals on microbes has been documented (for example 3,4,10), but not with respect to microbes employed in the remediation of mixed wastes.

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The overall objective of this project was to determine the toxicity of

metals in PSW to a test consortium and to evaluate the potential of water

soluble chelators to separate metals from the PSW and/or mitigate metal

toxicity. These preliminary findings are part of the research and

development of a modular treatment train for volume reduction and waste minimization of PSW.

Materials and Methods

Consortium and growth conditions. The 2-membered consortium (Rhodococcus sp. and Hyphomicrobiurn sp.) was maintained on basal salts

medium (9) agar plates or in liquid culture at 4" C. Cultures were grown

in Microflex teflon screw cap flasks filled to 1/5 volume with sterile medium. Cultures were supplemented with 13.6 mM acetone, 10 mM

dichloromethane (DCM) or 5 mM toluene (expressed as if all of the

volatile substrate was in solution). The consortium was routinely grown at 30" C on a rotary shaker (140 rpm).

Biodegradation experiments. Cell suspensions (1 -1 2 mg/mL dry weight) were amended with toluene, acetone, and/or DCM at the

appropriate concentrations in a volumetric flask. Aliquots (3 ml) were

dispensed into 22 ml head space vials that were sealed with teflon lined

septa and crimped. The vials were incubated as described above and at selected time points vials were removed and maintained at 0" C until

analysis. Experiments were run a minimum of 3 times in duplicate.

Controls employing killed cells and no cells were also included to account

for absorption of solvents to cells and abiotic transformations.

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Metals toxicity determinations. The consortium was amended with

the appropriate growth substrate and various concentrations of Pb, Cr(III)

and Cr(VI) from stock solutions in triple distilled water: Pb as Pb(NO3)2, 10 g L-1; Cr(III) as Cr(N03)3*9 H20, 1.0 g L-1; Cr(VI) as K2Cr04, 1.0 g L-1. Total reaction volume was 5 ml. Following 24 h incubation, a sample

was removed for dilution plating. Dilutions were done in triplicate. Plates were incubated with volatile carbon sources present in the vapor phase of a

dessicator. Colonies were counted and the colony forming units mL-1 was

determined. Experiments were performed a 'minimum of two times.

Results are expressed as a percent reduction in growth with respect to

control cultures that did not receive any metals.

Metals in paint. Concentrations of Pb and Cr(combined) in two representative paints were determined by digestion of solid samples with

1M HNO3 followed by ICP-AES.

Metals chelation. Water soluble polymers (10 mg) were exposed to

Pb and Cr(V1) solutions (100 mg L-1) in 20 mL at pHs between 1 and 11

for 30 min to 24 h. Following exposure, solutions were filtered and metal

concentrations in the retentate and permeate were quantified by inductively

coupled plasma - atomic emission spectrometry (ICP-AES). Analytical methods. Biodegradation was monitored by gas

chromatography-mass spectrometry (GC-MS) using head space analysis.

Analysis was performed on an HP 5890 Series 11 chromatograph equipped

with a mass selective detector and interfaced to a Telunar 7300 head space

analyzer. The column employed was an HP-1 capillary column using split

injection. The split was altered depending upon the initial concentrations

of substrates. A 3 point external standard curve was run along with each

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experiment,

coupled plasma spectrometer.

ICP-AED was performed on a Varian 2000 inductively

Figure 1. Degradation of low levels of solvents by a test consortium

80

60 .-. A 80 E * 40 B e

3 Y

s 20

Acetone

__t_ DCM

Toluene

0 0 5 10 15

Time, hrs 20 25

Results Organics destruction. The test consortium degraded 80 mg L-1 each

acetone and DCM in 18 h and the same concentration of toluene in 24 h (Fig. 1). When levels of these substrates were increased to those normally

employed in growth of the organisms, all substrates were degraded within

45 h (Fig. 2).

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Figure 2. Degradation of high levels of solvents by a test consortium*.

E

* Acetone + D c M * Toluene

0 10 20 30 40 50 Time, hrs

*Biodegradation assays were performed as described in Materials and methods.

Metals Toxicity. The expected concentration range of toxic metals in

PSW are: Cr(III and VI) 2 100 mg L" and Pb 0.2 - 1.0 g L-l. When the consortium was grown on DCM in the presence of metals typically found

in PSW, the most toxic effects were observed at low concentrations of

Cr(V1). However, when acetone served as growth substrate, the effect of Cr(VI) was not as obvious (Table 1). Higher concentrations of Cr(III) and

Pb were also toxic when DCM was growth substrate, but toxicity was not as great when acetone was the growth substrate (Table 2). Addition of Pb to the basal salts medium resulted in a milky white precipitate.

Table 1. Effect of substrate.

on growth of consortium with DCM or acetone as growth

Cr(VI), % of control Concentration growth

of metal, Acetone grown DCM mg L-1 grown

1 ND 76 5 80 5

25 ND 2

50 80 1

100 80 1

250 ND ND

500 ND ND

lo00 ND ND

Consortium was grown on basal salts medium with 13.6 mM acetone or 10 mM DCM as carbon and energy source. Cells were washed in 50 mM phosphate buffer, pH 7.2 and suspended in 5 mL basal salts medium (1 mg dry wt. cells mL-l) containing the metal and acetone or DCM. After 24 h incubation at 300 C colony-forming units (CFU)mL-I were determined as described in Materials and methods. *ND, not determined.

Concentration of metals in paint. Two representative paints were

digested to estimate the total concentrations of Pb and Cr that would be

expected in PSW. A polyurethane-based paint contained 238 mg Pb g-1

paint and 37 mg Cr g-1 paint. A cellulose-based paint contained 1.7 mg Pb g - l p a i n t and 0.36 mg Cr g - 1 p a i n t .

Table 2. Effect of C r O and Pb on growth of consortium with DCM or acetone as growth substrate.

Cr(III), % of control Concentration growth growth

Pb, 9% of control

of metal, Acetone DCM Acetone m mg L-1 grown grown grown grown

1

5 ND*

ND ND

100 ND

ND ND

100

25 ND 100 ND ND

50 ND 100

100 100 100

ND

ND

100

100

250 ND ND ND 100

500 54 0 ND 58

lo00 ND ND 100 51 Consortium was grown on basal salts medium with 13.6 mM acetone or 10 mM DCM as carbon and energy source. Cells were washed in 50 mM phosphate buffer, pH 7.2 and suspended in 5 mL basal salts medium (1 mg dry wt. cells mL-l) containing the metal and acetone or DCM. After 24 h incubation at 300 C colony-forming units (CFU)mL-1 were determined as described in Materials and methods. *ND, not determined.

Metals CheEation. Two different water soluble polymer chelators of

10,000 m.wt., one anionic and one zwitterionic, were studied for their

ability to bind Pb and Cr(VI). The binding capacities for Pb and Cr(V1)

were determined under optimum conditions at a range of pHs. Both polymers had pH optima of 643 and similar binding capacities of the metals

after 1 h (Fig. 3).

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- - . .-

Figure 3. Binding Capacities of Pb and C r o on Anionic and Zwitterionic Polymers.*

800 I

600

00 1 400 - z

200

0

* + **

- m vi .-i - PH

As described in Materials and Methods Anionic Water Soluble Polymer Zwitterionic Water Soluble Polymer

Mitigation of MetaZs Toxicity. Both water soluble polymers were

investigated for their ability to mitigate Pb and Cr(V1) toxicity. The

zwitterionic polymer did not affect the growth of the consortium on DCM.

The cationic polymer caused a 10 fold reduction in consortium growth.

Because Cr(V1) was the most toxic metal, preliminary studies were undertaken with this metal and the zwitterionic polymer. Initial studies

with this polymer did not mitigate the Cr(VI) toxicity. Discussion

A consortium was optimized for the rapid degradation of DCM, acetone and toluene. These substrates served as model compounds for RCRA organics expected in typical PSW. At concentrations of these

substrates that support growth, complete degradation of all substrates was accomplished in 2 d.

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When the consortium was amended with concentrations of metals

suspected in PSW, inhibition of microbial growth was observed. This was

not surprising as the toxicity of metals to microbes has been well

documented. It is likely that in the case of Pb, the precipitation product

was causing toxicity to the microbes. The concentration of Pb in solution

was always at a maximum, and toxicity was only observed in the flasks

containing larger concentrations of Pb as a precipitate. Further experiments to determine the mechanism of inhibition were not

undertaken.

Decreased growth indicated that biodegradation of substrates would be sub optimal so the use of water soluble metals chelators to mitigate

metals toxicity was investigated. It was necessary to determine whether the

polymer itself had an effect on the growth of the consortium. The cationic polymer proved to be inhibitory to the microbes without any metals

present. This negative effect may have been due to binding of the polymer to the anionic cell walls of the microorganisms. On the other hand, the

zwitterionic polymer was not toxic to the microbes, but preliminary

experiments indicated that this polymer did not mitigate the toxicity of Cr(V1) under the experimental conditions employed. The ability of this polymer to mitigate Pb toxicity has not yet been investigated. In later

experiments the amount of polymer required to bind Cr(VI) and Pb under

optimum conditions was determined (Fig. 3). These experiments suggested

that additional polymer is required to completely bind the Cr(V1).

Additional experiments are needed to determine if alleviation of toxicity is possible at higher concentrations of polymer.

The water soluble polymers have advantages over other chelators in

this system. They have higher capacities than other metal chelators and can

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* +

bind metals in homogeneous solutions. Recovery and regeneration of the

water soluble polymers is facile. The polymers may be employed for metals chelation prior to the introduction of microbes to the PSW or

following microbial introduction if further experimentation indicates that

higher concentrations of PSW can mitigate toxicity of Cr(VI) and Pb.

Mixed wastes containing a bulk component cannot be treated effectively by any one technology for solid or liquid waste treatment

technologies. Bioremediation and metals chelation, two effective and proven technologies, insure a cost-effective, environmentally benign

solution for PSW treatment. This novel technology may prove applicable

to an array of other complex waste streams.

References

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4.

5.

Alvarez, P. J. J.; Vogel, T. M. Substrate interactions of benzene, toluene and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries. Appl. Environ. Microbiol. 57:2981; 1991.

Arquiaga, M. C.; Canter, L. W. Microbiology associated with the biological treatment of aircraft paint stripping wastewater. Wat. Sci. Tech. 20525; 1988.

Babich, H.; Schiffenbauer, M.; Stotsky, G. Comparative toxicity of trivalent and hexavalent chromium to fungi. Bull. Environ. Toxicol. 28:452; 1982.

Barrow, W; Tournabene, T. G. Chemical and ultrastructural examination of lead induced morphological convertants of Bacillus subtilis. Chem.-Biol. Interactions 26:207; 1979.

Cobb, H. D.; Egan, J. W.; Olive, W. E.; Hansen, D.J. Biodegradation of phenolic paint-stripping waste: Laboratory evaluation of a fixed film batch reactor. Tyndall Air Force Base, FL: Air Force Engineering and Services Center, ESL-TR-79-11; 1979.

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Goulding, C; Gillen, C. J.; Bolton, E. Biodegradation of substituted benzenes. J. Appl. Bacteriol. 65:l-5; 1988.

Leisinger, T. General Aspects. Microorganisms and xenobiotic compounds. Experientia 39:1183; 1983.

Leisinger, T. ; Kohler-Staub, D. Dichloromethane dehalogenase from Hyphomicrobium DM2. Meth. Enzymol. 188:355; 1990.

Ross, D. S . ; Sjogren, R. E.; Bartlett, R. J. Behavior of chromium in soils: IV. Toxicity to microorganisms. J. Environ. Qual. 10: 145; 1981.

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