Nickel Complexes with Soil Microbial MetabolitesMobility and

additional tests were conducted on 24-48 hr cultures (standard mineral base agar). These included acetoin production by the. Voges-Proskauer test; aci...
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9 N i c k e l Complexes w i t h Soil Microbial Metabolites-

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M o b i l i t y and Speciation in Soils R. E. WILDUNG, T. R. GARLAND, and H. DRUCKER Battelle, Pacific Northwest Laboratories, Richland, WA

99352

I t i s w e l l - e s t a b l i s h e d that i n o r g a n i c physicochemical mech­ anisms play a predominant r o l e i n c o n t r o l l i n g trace element s o l ­ u b i l i t y i n s o i l s and sediments. However, s o l u b l e species of trace elements which hydrolyze i n the n e u t r a l pH range, or tend to form c a t i o n i c i n o r g a n i c species w i t h intermediate to high i o n i c p o t e n t i a l s are often present i n n a t u r a l waters as organic complexes. Less i s known of the form of trace elements i n s o i l and sediment s o l u t i o n s , but on the b a s i s of d e t a i l e d reviews of i n o r g a n i c and organic processes i n f l u e n c i n g trace element c y c l i n g , i t r e c e n t l y has been concluded that d i s s o l v e d organic matter at the sediment water i n t e r f a c e serves to increase the c o n c e n t r a t i o n of trace elements i n waters by decreasing s o r p t i o n r a t e on the s o l i d phase Q, 2). Trace element organic complexes i n s o i l s may be g e n e r a l l y categorized on the b a s i s of t h e i r s o l u b i l i t y Ο), although con­ s i d e r a b l e overlap l i k e l y occurs. The major c l a s s e s of complexes are 1) r e l a t i v e l y high molecular weight humic substances that have a high a f f i n i t y for metals but are l a r g e l y i n s o l u b l e i n s o i l s , and 2) r e l a t i v e l y low molecular weight nonhumic substances derived l a r g e l y from m i c r o b i a l c e l l s and metabolism that e x h i b i t a range i n s o l u b i l i t i e s i n a s s o c i a t i o n w i t h metals. The o r i g i n and p r o p e r t i e s of organic m a t e r i a l s i n both c a t e g o r i e s i n r e l a ­ t i o n to metal complexation i n s o i l s and sediments were r e c e n t l y overviewed (_1, 4 ) . Of the humic substances, the humâtes ( a l k a l i s o l u b l e , a c i d i n s o l u b l e ) and f u l v a t e s ( a l k a l i and a c i d s o l u b l e ) c o n s t i t u t e up to 90% of the s o i l organic matter (.5)· Both f r a c t i o n s e x h i b i t high charge d e n s i t y due p r i n c i p a l l y to a c i d i c f u n c t i o n a l groups that lead to a strong pH dependent a f f i n i t y f o r cations i n s o l u t i o n and strong a s s o c i a t i o n with s o i l minerals and other organic c o n s t i t u e n t s i n s o i l s (6). Although l a r g e l y i n s o l uble i n s o i l s , the f u l v a t e s , thought to be of lower molecular weight than the humâtes, may, i n p a r t i c u l a r , have p o t e n t i a l f o r formation of s o l u b l e complexes w i t h metals ( 7^ . Nonhumate mater i a l s are a l s o l i k e l y to be of importance i n metal s o l u b i l i z a t i o n i n s o i l . These c o n s i s t of components of l i v i n g c e l l s , t h e i r 0-8412-0479-9/79/47-093-181$05.00/0 © 1979 American Chemical Society In Chemical Modeling in Aqueous Systems; Jenne, Everett A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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CHEMICAL MODELING IN AQUEOUS SYSTEMS

exudates and the e n t i r e spectrum of degradation products which u l t i m a t e l y serve as b u i l d i n g u n i t s f o r the s o i l humic f r a c t i o n . Because of the g e n e r a l l y high turnover r a t e of microorganisms and r e a d i l y decomposable organic matter i n s o i l s , nonhumate m a t e r i a l s , i n c o n t r a s t to humic substances, are i n h e r e n t l y t r a n s i t o r y and the r e l a t i v e q u a n t i t i e s and composition i n s o i l may be expected to vary w i t h carbon sources and environmental c o n d i t i o n s (8, 9 ) . L i m i t e d i n v e s t i g a t i o n s (_7) have a t t r i b u t e d most of the t i t r a t able a c i d i t y i n s o i l s o l u t i o n to the a l i p h a t i c a c i d s (^0) and amino a c i d s ( 1 1 ) . However, a wide range of organic a c i d s and bases of m i c r o b i a l o r i g i n i n c l u d i n g simple a l i p h a t i c a c i d s , c a r b o x y l i c a c i d s d e r i v e d from monosaccharides, products o f the c i t r i c a c i d c y c l e , and aromatic a c i d s are l i k e l y present i n s o i l s o l u t i o n ( J J ) , 12^). Recent evidence i n d i c a t e s that s o i l microorganisms produce water s o l u b l e ligands w i t h a h i g h a f f i n i t y f o r a range of metals (L3, J A ) and m i c r o b i a l a c t i v i t y i n f l u e n c e s Pu s o l u b i l i t y i n s o i l (15). U n f o r t u n a t e l y , although the presence o f organic complexes of Cu, Zn, and Mn i n s o i l s o l u t i o n has been reported (JL6, 17) few i n v e s t i g a t i o n s , summarized by Mortensen ( 18) and Stevenson and Ardakani (12), have i d e n t i f i e d s p e c i f i c water-soluble ligands capable o f metal complexation i n s o i l , and i n t a c t organometal complexes have not been i s o l a t e d and i d e n t i f i e d . Thus, evidence i n support of the presence of s o l u b l e metal complexes i n s o i l i s l a r g e l y c i r c u m s t a n t i a l and there i s a p a u c i t y of knowledge r e g a r d i n g the nature, behavior, and r o l e of organic ligands i n geochemical c y c l i n g . The development of an understanding of the r o l e of microorganisms i n metal complexation has been l i m i t e d by the complexity of s o i l , sediment and m i c r o b i a l systems and d i f f i c u l t i e s i n the experimental s e p a r a t i o n of the e f f e c t s of m i c r o b i a l processes from physicochemical processes i n s o i l s and sediments. To a i d i n understanding the mechanisms of t r a c e metal comp l e x a t i o n by s o i l microorganisms, an experimental approach was developed which e n t a i l e d 1) i s o l a t i o n of organisms from s o i l on the b a s i s of trace metal t o l e r a n c e and C (carbon) requirements, 2) examination of metal transformations and form on growth of i s o l a t e d organisms i n v i t r o , 3) d e t e r m i n a t i o n of the m o b i l i t y and form of s t a b l e metal complexes i n s o i l , and 4) i d e n t i f i c a t i o n and d e t a i l e d study of metal complexes e x h i b i t i n g h i g h metal a f f i n i t i e s and s o i l s o l u b i l i t y as determined from steps 1) through 3 ) . This p r o t o c o l was a p p l i e d to the i s o l a t i o n and examination of metal complexes formed on growth of s o i l b a c t e r i a and f u n g i exposed to metals (Cd, Cr, N i , Pu, T l ) w i t h a range of p r o p e r t i e s and the chemistry of important N i complexes was examined i n detail.

In Chemical Modeling in Aqueous Systems; Jenne, Everett A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

WILDUNG E T A L .

Nickel

Complexes

with

Soil

Microbial

Metabolites

183

M a t e r i a l s and Methods

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B a c t e r i a and fungi were i s o l a t e d from s o i l on the b a s i s o f metal tolerance and C source, grouped on the b a s i s of morpho­ l o g i c a l , growth and p h y s i o l o g i c a l parameters and examined f o r a b i l i t y to a l t e r the s o l u b i l i t y and form of metals i n e x o c e l l u l a r s o l u t i o n s and a l t e r metal s o l u b i l i t y i n s o i l . S o l u b l e N i com­ plexes were subsequently c h a r a c t e r i z e d i n d e t a i l . I s o l a t i o n of B a c t e r i a and Fungi from S o i l Using Enrichment Techniques. Two enrichment procedures were employed to i s o l a t e microorganisms from s o i l : a t e r t i a r y enrichment, i n which organ­ isms were i s o l a t e d from s o i l a f t e r i n c u b a t i o n to l o g phase i n the presence of added metal, and a two-phase enrichment, i n which organisms were i s o l a t e d from the unincubated s o i l and grown on a number of d i f f e r e n t C sources i n the presence of metals. In the t e r t i a r y enrichment procedure, s o i l ( R i t z v i l l e s i l t loam) was amended w i t h s t a r c h (1.0%), NH4NO3 (0.5%), and Cd,

Cr, N i , T l (1, 10, and 100 μg/g) or Pu (0.05, 5 and 10

μα/g).

The s o i l , w i t h amendments, was brought to 22% moisture and i n c u ­ bated (2£PC) under aerobic c o n d i t i o n s (continuous flow of CO2 free a i r ) . At mid-log growth phase, as determined by CO2 e v o l u ­ t i o n r a t e , an a l i q u o t (1 ml) of a 1:10 incubated s o i l : w a t e r s l u r r y was i n o c u l a t e d i n t o an enriched s o i l e x t r a c t medium c o n t a i n i n g glucose (1%), NH4NO3 (0.5%), K HP0 (0.05%), s o i l e x t r a c t (30%) and Cd, Cr, N i , Pu, or T l added i n s o l u b l e form, to achieve Cd, Cr, Ni and T l c o n c e n t r a t i o n s o f 1, 10, and 100 μg/ml and Pu c o n c e n t r a t i o n s of 0.05, 5 and 10 μ0ΐ/πι1. Con­ t r o l s were i d e n t i c a l except metals were not added. The pH was adjusted to 7.0 f o r aerobic b a c t e r i a and 6.0 f o r f u n g i . S t r e p t o ­ mycin s u l f a t e (0.4%) was added to the fungal enrichment to p r e ­ vent growth of b a c t e r i a . The i n o c u l a t e d c u l t u r e s were incubated (28°C)with shaking (150 rpm) u n t i l maximum c e l l d e n s i t y was obtained and secondary enrichments were i n i t i a t e d by t r a n s f e r r i n g an inoculum of each o f the primary enrichments to f r e s h , enriched s o i l e x t r a c t medium c o n t a i n i n g the metals at the same concentra­ t i o n s . The t e r t i a r y enrichment was then conducted i n a manner s i m i l a r to the secondary enrichment. Pure c u l t u r e s of organisms were i s o l a t e d at the end of each enrichment u s i n g successive pour p l a t e and s t r e a k i n g u n t i l c u l t u r e s d i f f e r i n g i n colony morphology were r e s o l v e d . The pure c u l t u r e s were maintained i n stock on agar s l a n t s i n the presence and absence o f the metals a t the concentra­ t i o n s used i n i s o l a t i o n . The stock c u l t u r e s were passed to f r e s h s l a n t s monthly i n order to assure v i a b i l i t y . A two-phase enrichment procedure was u t i l i z e d to s e l e c t s o i l organisms on the b a s i s of a b i l i t y to metabolize c l a s s e s o f organic compounds i n the f i r s t phase; and on a b i l i t y to grow i n the presence of metals i n the second phase. In the f i r s t phase, a l i q u o t s of a standard m i n e r a l base medium (Table I , glucose used only i n c o n t r o l ) were s e p a r a t e l y amended w i t h 1) aromatic a c i d s , 2

4

In Chemical Modeling in Aqueous Systems; Jenne, Everett A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

184

C H E M I C A L MODELING IN AQUEOUS SYSTEMS

Table I .

Solution Designation A

Composition o f standard m i n e r a l base medium employed i n m i c r o b i a l s t u d i e s . Volume per l i t e r o f Medium (ml) 100

Composition 685 ml o f KH PO (0.2M), 2

315

2

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diluted 10

4

ml o f Na HPO (0.2M)

20.0 g MgS0

4

t o 2000 ml · 7H 0,

4

1.325 g C a C l

2

' 2H 0

2

2

d i s s o l v e d i n 1000 ml C

1

50 mg ZnSO^ ' 7H 0, 2

50 mg MnSO^ ' H 0, 2

12 mg CuSO^ ' 5H 0, 2

15.9

mg C o ( N 0 ) 3

19.0 mg N a B 0 2

235

4

mg Na Mo0 2

4

?

2

* 6H 0, 2

" 10H 0, 2

* 2H 0, 2

10 ml Fe-EDTA s o l u t i o n (17.9 g Na EDTA-2H 0 and 3.23 g KOH i n 2

186

2

ml added t o 13.7 g F e S 0 « 7 H 0 4

i n 364 ml H o. 2

2

Bubbled f i l t e r e d

a i r through s o l u t i o n o v e r n i g h t and a d j u s t e d pH t o 5.0 w i t h 8M HN0 ) 3

d i s s o l v e d i n 100 ml D

10

10 g NH C1 d i s s o l v e d i n 100 ml 4

Ε ^

100

15 g glucose d i s s o l v e d i n 1000 ml

F

100

2.5 g yeast d i s s o l v e d i n 1000 ml

1/ Glucose was not used i n s t u d i e s o f the e f f e c t

of C sources.

i n c l u d i n g benzoate (0.5%), £ hydroxy benzoate (0.5%), m hydroxy benzoate (0.5%), and tryptophan (0.11%); 2) organic a c i d s , i n c l u d i n g succinate (0.5%), malate (0.5%), l a c t a t e (0.5%), and acetate (0.5%); 3) sugars, i n c l u d i n g glucose (0.5%), sucrose (0.5%), and f r u c t o s e ( 0 . 5 % ) ; and 4) an o r g a n i c a l l y r i c h medium c o n t a i n i n g yeast e x t r a c t and peptone. A l i q u o t s o f these media were i n o c u l a t e d w i t h a s o i l (1 g) and incubated w i t h shaking (200 rpm) f o r 120 hours. From each o f these primary enrichments, a secondary enrichment was performed by i n o c u l a t i n g a l i q u o t s o f s t e r i l e medium c o n t a i n i n g the same C sources and Cd, C r , * N i , T l ( 1 , 10, and 100 μg/ml) o r Pu (0.05, 5 and 10 μΟί/τηΙ). The inocu­ l a t e d m e t a l - c o n t a i n i n g medium was incubated (28°C) w i t h shaking

In Chemical Modeling in Aqueous Systems; Jenne, Everett A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

9.

wiLDUNG ET AL.

Nickel Complexes with Soil Microbial Metabolites

185

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(200 rpm) f o r 48 h r . Pure c u l t u r e s were i s o l a t e d from the media e x h i b i t i n g m i c r o b i a l growth ( t u r b i d i t y ) at the h i g h e s t metal c o n c e n t r a t i o n using pour p l a t e and successive s t r e a k i n g t e c h ­ niques. M o r p h o l o g i c a l l y d i f f e r e n t b a c t e r i a l and fungal c u l t u r e s were placed i n t o stock and maintained as p r e v i o u s l y d e s c r i b e d . B a c t e r i a l and fungal enrichments d i f f e r e d only i n that b a c t e r i a l enrichments were conducted w i t h media adjusted to pH 7.0 whereas the fungal enrichments were conducted w i t h media adjusted to pH 6 and contained streptomycin s u l f a t e (0.4%). Morphological and P h y s i o l o g i c a l C h a r a c t e r i s t i c s of Microorganisms I s o l a t e d from S o i l . The 239 b a c t e r i a l i s o l a t e s obtained i n pure c u l t u r e from the two enrichment procedures were c l a s s i f i e d on the b a s i s of morphological, growth and p h y s i o l o g ­ i c a l parameters. These were measured using standard methods (19). A l i q u o t s (2 ml) of stock c u l t u r e s were t r a n s f e r r e d to a standard m i n e r a l base medium (Table I ) , incubated (28°C) on a r o t a r y shaker (150 rpm) f o r 24 hr. M o t i l i t y (wet mount) and gram r e a c t i o n were measured. The c u l t u r e was then used to i n o c u ­ l a t e an agar p l a t e of standard mineral base medium which was c u l t u r e d f o r an a d d i t i o n a l 24 hr and used f o r the c a t a l a s e and oxidase t e s t s . For the gram p o s i t i v e and spore-forming b a c i l l i , a d d i t i o n a l t e s t s were conducted on 24-48 hr c u l t u r e s (standard mineral base agar). These included a c e t o i n production by the Voges-Proskauer t e s t ; a c i d production from the mixed sugars, arabinose, mannose, and x y l o s e ; a c i d production from glucose; and s t a r c h h y d r o l y s i s . Spore l o c a t i o n and morphology were determined i n o l d e r c u l t u r e s . Pigment production on s t a r c h agar was a l s o used to subdivide c e r t a i n of the spore-forming b a c i l l i i n t o major biotypes of B a c i l l u s mycoides. Twenty s t r a i n s considered r e p r e ­ s e n t a t i v e of the c u l t u r e s i n the c o l l e c t i o n on the b a s i s of these t e s t s and chemical c h a r a c t e r i s t i c s of e x c e l l u l a r products (described below) were examined to determine i f e x o c e l l u l a r com­ p l e x a t i o n of N i a l t e r e d N i m o b i l i t y i n s o i l r e l a t i v e to i n o r g a n i c forms. A t o t a l of 250 fungal i s o l a t e s were obtained i n pure c u l t u r e . Colony morphologies of many of the i s o l a t e s were s i m i l a r . On the b a s i s of colony morphology on standard m i n e r a l base medium, organ­ isms were separated i n t o 59 types. These types were r e t a i n e d i n stock to examine metal r e s i s t a n c e c h a r a c t e r i s t i c s and t h e i r a b i l i t y to e x o c e l l u l a r l y modify N i form and s o l u b i l i t y i n s o i l . Further c l a s s i f i c a t i o n to the species l e v e l i s underway. M o d i f i c a t i o n of N i c k e l Form by S o i l M i c r o b i a l I s o l a t e s . To determine the a b i l i t y of the m i c r o b i a l i s o l a t e s to modify the chemical form of N i , standard m i n e r a l base medium (15 l i t e r s ) was prepared (Table I ) and separated i n t o a l i q u o t s (500 m l ) . The a l i q u o t s were frozen r a p i d l y on dry i c e and stored (-26 C) u n t i l used. Immediately p r i o r to use, the a l i q u o t s were r a p i d l y thawed at 37°C i n a water bath, f i l t e r s t e r i l i z e d (0.2 μ) and s e p a r a t e l y P

In Chemical Modeling in Aqueous Systems; Jenne, Everett A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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CHEMICAL MODELING IN AQUEOUS SYSTEMS

amended w i t h unlabeled N i (as N 1 C I 2 ) and i t s r a d i o i s o t o p e ( 6 3 . 0.3 μ(Σΐ/πι1). In order to develop a b a c t e r i a l inoculum for t h i s medium, stock c u l t u r e s were grown to l o g phase i n s t a n ­ dard m i n e r a l base medium (10 ml) without metal. An a l i q u o t (0.25 ml) of t h i s c u l t u r e was used as an inoculum f o r the growth medium (10 ml) c o n t a i n i n g metal, and the c u l t u r e s were incubated (28°C)with shaking (150 rpm) f o r 48 h r . The fungal inoculum was developed s i m i l a r l y , except f u n g i from stock were streaked on standard m i n e r a l base agar from the stock s o l u t i o n and incubated ( 2 8 C l u n t i l s p o r u l a t i o n was evident. An a l i q u o t (10 ml) of s t e r ­ i l e b u f f e r (Table I , S o l u t i o n A) was added to the surface and the spore suspension p i p e t t e d a s e p t i c a l l y i n t o tubes. An inoculum (0.5 ml) of t h i s spore suspension was added to the growth medium c o n t a i n i n g N i (10 μg/ml). The organisms were incubated (28 C), w i t h shaking (150 rpm) f o r 72 h r . At the end of growth i n the N i - c o n t a i n i n g medium, the c e l l s were separated by f i l t r a t i o n (0.4 μ Nuclepore) and dry weights were determined. An a l i q u o t of the f i l t r a t e (