South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange
Electronic Theses and Dissertations
1986
Enrichment, Isolation and Characterization of Alachlor, Carbofuran, and Dicamba-degrading Bacteria Obtained from Soil
Neal R. Adrian
Follow this and additional works at: https://openprairie.sdstate.edu/etd
Recommended Citation Adrian, Neal R., "Enrichment, Isolation and Characterization of Alachlor, Carbofuran, and Dicamba- degrading Bacteria Obtained from Soil" (1986). Electronic Theses and Dissertations. 4342. https://openprairie.sdstate.edu/etd/4342
This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. EN RICHMENT , ISOLATION AND CHARACTERIZATION OF ALACHLOR,
CARBOFURAN, AND DICAMBA -DEGRAD ING BACTER IA
OBTAINED FROM SOIL
BY
NEAL R. ADRIAN
A thesis submitted in partial fulfillment of the requirement for the degree Mas ter of Science Maj or in Microbiology South Dakota State Univers ity 1986 ENRICHMENT, ISOLATION AND CHARACTERI ZATION OF ALACHLOR,
CARBOFURAN, AND DICAMBA-DEGRAD ING BACTERIA
OB TAINED FROM SOIL
Th is thesis is approved as a creditable and independent inv est igation by a candidate for the degree, Master of Sc i- ence, and is acceptable for meeting the thesis requi rements for this deg_ree . Acceptance of this thesis does not imply that the conclusions reached by the candidate are neces- sarily the · conclus ions of the maj or department.
Dr. W. Kennedy Gauger Date Thesis Advisor
.VI. • L\.UUt:::L" l.. .1.000 Date Hea d, Microbiology Dept. ACKNOWLEDGEMENTS
The author wishes to express thanks and appreciation to
Dr. W. Ken nedy Gauge r for his guidance, support and con struc tive crit icism in the preparation and editing of this manuscript .
A special thanks to Dr. Duane Matthees for his support , expert ise, and laboratory facilities throughout this stud y.
My time spent at South Dako ta St ate University's Mic ro biology Departme nt was an enj oyable and enlightening period that clarified my career go�ls .
i TABLE OF CONTENTS
Page
ACKNOWLEDGEMEN TS ...... i
TABLE OF CONTENTS ...... ; . . . ii
LIST OF FIGURES ...... 1v
LIST OF TAB LES ...... v i
INTRODUCTION ...... 1
Chapter 1 ...... • ...... • • . . . . . 3
LITERATURE REV IEW ...... 3
CHAPTER 2 ...... · ...... 34
SCREENI NG FIELD ISO LATES FOR INCIDENTAL AND CATABOLIC
PESTICIDE METABOLISM ...... 34
MATERIALS AND METHODS ...... 34
RES ULTS AND DI SCUSSION ...... 53
CHAPTER 3 ....· ...... 85 PESTIC IDE DEG RADATION BY BACTERIA ISOLATED FROM AN AGRICULTURE CHEMICAL SPILL 85
MATERIALS AND METHODS ...... 85
RES ULTS AND DI SCUSS ION ...... 93
ii CHAPTER 4 ...... •....•...... •...... •...... 101
ENRICHMENTS FOR MIC ROORG ANIS MS UTILIZI NG PES TICIDE S FOR CARBON,
NITROGEN , OR PHOSP HORU S SOURC ES ...... 101
MATERIALS AND METHODS ...... 101
RE S ULTS AND DI SCUSS ION ...... 10�
CONCLUSIONS ...... 111
LITERATURE CITED ...... 114
iii LIST OF FIGURES
Figure Page
1 a. Screening for pesticide degradat ion by bac- teria after four weeks incubat ion ..• ....•...... 59
lb . Screening for pest icide degradation by bac- teria after four weeks incubation ...... 60
1 c. Screening for pest icide degrada tion by bac- teria after four weeks incubat ion ...... 61
2 . Deg radat ion of alachlor in complex (tryp tone yeast extract) , defined + yeast extract (Hutner's basal + 0.05% yeast ext ract), and
def ined· (Hutne r's basal ) media ...... 64
3 • Degradat ion of ala chlor in complex (tryp tone yeast �xt rac t ), defined + yeast extract (Hutner's basal + 0.05% ye ast ext r act), and defined (Hutner's basal) media by
St rept omyces pilosu s ...... 65
Deg radat ion of ·al a chlo r in complex (tryp tone yeast extract) , defined + YE (Hutner's basal + 0.05% yeast ext ract) , and def ined (Hutner's
basal) media by St reptomycete · Isolate 5 ...... 67
s: Degradat ion of ala chlo r in comple x (tryp tone yeast extract), defined + YE (Hutne r's basal + 0.05% yeast extract), and def ined (Hutner's basal ) media by St rept omycete Isolate 26 ...... 68
6 . Rap id plasm id DNA sc reening of Isolates using either an alkaline or polye t hylene
glycol (PEG) procedure ...... 70
7a. Alachlor degradat ion by Isolate 5 in a soil
·slu rry supplemented with ala chlor ...... 75
7b . Alachlor degradati�n by Isolat� 5 in a soil slu rry supplemented with ala chlor 76
iv 8a . Alachlor degradation by Isola te 5 in a soil slurry from an agricultural chemical spill 77
8b . Alachlor degradat ion by Isolate 5 in a soil slurry from an agricultural chemical spill 78
9a. Alachlor degradat ion by Isolate 5 in a soil slurry supplemented with alachlor 7 9
9b. Alachlor degradat ion by Isolate 5 in a soil slurry supplemented with alachlor 80
1 0 • Growth of two isolates obtained from Brook ings chemical spill in a basal med ium with alachlor as sole source of carbon and energy ...... 94
1 1 . Growth of two isola t es obtained from Brookings chemical spill in bas al medium with tri flu ralin as sole source of carbon and ene rgy ...... 95
1 2 • Growth-of two isola tes obtained from Brookings chemical spill in basal medium not containing an addit ional source of carb�n and ene rgy ...... 96
1 3 . Ala chlor degradat ion by two isola tes obtained from Brookings chemical sp ill ...... 97
v LIST OF TABLES
Tab le Page
1 . Growth of isolates on 2% unwashed and washed agar in HB broth supplement ed with various carbon and ene rgy sources ...... •.. 54
2. Growth of isola tes in broth media containing various carbon and ene rgy sources 57
3 . Milligrams of mycelial biomass harvested from each culture flask following degradat ion of alachlor in three types of culture media ...... 63
4. Summary of taxonomic characteristics for alachlor-degrading bacteria ...... 84
5 . Ala chlor degradation after four weeks by eight bacteria isolated from an agricul- tural chemical spill ...... 99
6 . Enrichments performed with various pesticides added as either carbon and ene rgy, nitrogen, or phosphorus sources ...... 105
vi INTRODUCTION
The prod uction and use o � synthetic chemicals fpr
pest icide control increase d dram atically after World Wa r
II. Th ey were found to be highly effective , cheap to pro-
duce and easy to apply. Development of these chemic als has
been one of the major developments for agricul ture .
In the 1970's public health risks and environme ntal
damage caused by agricultural chemic als was recog nized .
Problems of chronic exposure� improper waste disposal and
environmental dam age developed wh ich led to the federal gov-
ernment regul ating their use and manufacture .
Pest icide manu facturers have a variety of physical
/ · and chemical disposal methods for pest icide wast es. Dis-
posal or detoxification me thods for pesticides that have
become enviro nmental pollutants through sp ills or accumu-
la tion in · high use areas are limited or nonexistent . Th ere
1s a �otential for biodegradation to help decontaminate
these environmental pollutants, since the degradation of
organic compounds in the environment is predominantly micro-
biologic ally mediated . Further research on microbial
metabolism of xenobioti�s and their ability to degrade
these comp ound s under different growth condit ions is
needed. 2
Four objectives for this thesis research were to:
1. Screen lab oratory field isolates for their· capability to degrade the pesticides alachl or, carbof uran, and dicamba.
2a . Take key isolates obtained from objective 1 and determine in vitro broth degradat ion in three types of culture media.
2b . With these same isolates determine thei� in vitro soil slurry degradation capability .
3. Determine alachlor degradation by eight isolates obt ained from an agricultural chemical spill.
4. Determine if any observed pest icide degradation was mediated . by plasmid or chromoso mal DNA . 3
CHAPTER 1
LITERATURE REV IEW
Pesticide degradat ion occurs thr ough chemical and _ biological metabolic processes wh ich are char acteri zed as either abiotic or biotic, respectively (8, 48). Abiotic degradat ion is not dependent on specific soil enzymes, and involves chemical reactions with other organic mole cule s.
A me tabolic reaction is one wh ere alterat ions in the pesti cide are mediated by an enzyme .
ABIOTIC DEGRADATION
EFFECTS MED IATED BY HUMUS FORMA TION: Ab iotic degradation of pesticides may occur · during humus form ation in the soil. The organic fraction of soil may contain carboxyl, phenolic, aliphatic , hydroxyl, carbon yl, as well as le ss frequently found functional groups such as amino, imino , and sulfhydryl groups (1, 8). These highly react ive functional groups link together chemically with polysacchar ides , am ino acids , peptides and proteins , and other organic matter. This results iri a complex �ixture of polymerized material called humus and pesticid� molecule s or their me t abol ites may become incorporated during " its form ation
( 8 ) . 4
Mic roorganisms may produce enzymes that gen erate
highly reactive pesticid e molecule s that are otherwis e
unreactive . Th ese then combine with functional groups
present in the soil organic ma tter. For example phenoli c
pesticides can be transf ormed in to active phenolic radic als
by the action of phenoloxidases (8) . Th ese radicals then
stabil ize by linkin g together or by further oxid ation to
quinones.
EFFECTS MED I ATED BY. CHANGE OF pH : The hydrogen ion concentration has a maj or effect on the reactivit y of
the pest icid� mo lecule by ionizing func tional groups of
pesticid es or functio nal groups present in soil organic mat ter (8, 64 ). This is important with reactions involving
/ th e pesticide and functiona l groups present in soil organic mat ter. Microorganisms are capabl� of chang ing the pH to
either an alk alin e or acidic environment . Consequ ently microb i ally mediated pH differences may facili t ate abio tic degradation . pH is the dominating factor controlling abiotic degradation for some pesticides. An alk aline pH of
8.5 will facilitate abio tic degradation of parathion; and
(8). This phenom- amit roi e is optimally degraded at pH 6. 5 . enon may be more apparent in the mic roenvironment due to
_ the large buffering capacit y of soil with no apparent effect observed in the macroenvir onment (8)� 5
EFFECTS ME DIATED BY OXI DATI ON-REDUCTION
CONDITIONS: Chang ing the reduct ion- o xidatio n potenti al may als o affect the degradation of certain pest icides (8,
64 ). Microorganisms may alter the oxidat ion-reduction
condit ions by consuming avai lable oxygen thereby producing anaerobic condit ions . The prevailing reducing enviro nment can modify the '' ...ra tes and products of purely chemical reactions ..." fo r some pesticides (8). The anaerobic conditions can also produce a totall y differ ent flora of microorganisms that have '' ...specific abilities for metabolic transformation of substrates" (8).
EFFECTS MED I ATED BY PHYS I CAL PARAMETERS: Many physical factors influence the tra nsformation and movement
/ of pest icides . Rates of degradation of pest icides in so{l are a function of temperature , moisture , sorption coeffi- cients , extent of occlusion in the soil organic matrix, and the intrinsic capacity of the pesticide to resist degrada- tion (1, 8, 29, 48, 75). Of the physical factors known to affect the res idual fate of pesticides , sunlight is the most important (48) . Sunlight may activate compounds cal led photosens itizers tha t tra nsfer the capt ured light ene rgy to pesticides re�ulting in pho�olysis of pesticides .
Other photochemical reactio ns that occur are dehalog ena- tion, photochemical oxidation and photon uc1eoph ilic reactio ns. 6
Increasing the temperature increases the loss of
some pest icides by volat ilization and degradation ( 1, 33, 44). Volatilization is chiefly responsible for lo sses of
pest icide res idues from the substrate to wh ich they were-
applied. Degradation does not occur during volat ilization .
The degradation of the herbicide alachlor was sho wn to be
influenced by tem perature . An alachlor metabolite was ° formed -in soil when incubated at 46 C, but not at 22° C
(33).
DIFFERENTIATING BETWEEN BIOTIC AND ABIOTIC DEGRADATION
Oft en the result of these abiotic processes is the
erroneous presumption that the observed degradation is
attributable to microbial activity (8). Ideally one wants to isolate the enzyme that is responsible for the me tabolic
tra nsformation of the pesticide. Th is is seldom done due
to time constraints imposed, lack of availab le equipment ,
or lack of technical expertise.
Often initial comparisons are made between sterile
and nons terile soil to show that the degradation is mi cro-
bially med iated . But factors even such as the method of
soil sterilization may affect the outcome of an experiment .
· It was shown that autoclav ing soil '' ...des tcoys the free
radical me chanism involved in the nonbiological degradat ion 7
of amitrole , wh ile sterilizing soil with ethyle ne oxide . " had no effect on the degradation of ami tro le (8).
One must care fully evaluate the environmenta l parameters and circums tances under wh ich degradation takes place in order to class ify it as abiotic or biotic. Soil is a highly com plex environment and many physical and chem- ical factors influence pesticide degradation . Experiments performed in the lab orat ory are com ple ted under exact param eters which may alter phys io-chemical conditi�ns more rap- idly than under field condi�ions (8). Consequently in vitro pesticide transformation reactions are representative only for a specific experimen t.
BIOTIC DEGRADATION: Matsumura divides bioti c degradat ion, wh ich he ref e rs to as enzy m atic degradation, into three categories : 1) incidental me t abolism 2) catab- olism and 3 ) detoxificat ion metabolism (48). Incidental met abolism is some t imes referred to as cometabol ism, although Matsumura refers to com etaboli sm in more restric- tive terms . Incidental metab o lism occurs when the pesti- cide mo lecule is transformed to varying degrees by a microorganism; how ever the organism derives no carbon or energy ben efit as a result of the process. The metabolites formed may become substrates for other microorganisms . 8
Catabolism refers to the metabolic process wheie
the microorganism derives carbon or energy from the pesti-
cide molecule and th�re fore growth of the microorgans im
occurs (48). Only a portion of the mole cule may be cata -
bolic ally metabol ized by a given organism le avin� the res t
of the pest icide as a substrate for subsequent reactions .
The pesticide molec ule may als o serve as a nitrogen or phos-
phorus source (18, 60).
De toxification metabolism occurs through the action
of mutant str ains that have _developed res istance and are
una ffected by toxic effects of a pesticide . The mutant
strains metabolically , or otherwise, detoxify the pesti-
cide , or withstand any of its toxic effects . If the
pesticide is not toxic to the organism incident�! metab-
olism will continue .
PRINC IPAL METABOLIC SYSTEMS OF MIC ROORGANISMS
There are three principle metabolic systems used by
microorganisms in the me tabolism of pesticides . These are
oxidat' ive systems , reductive systems , and hydro lytic
processes (8, 48).
OXIDATIVE SYSTEMS: Oxidative reactions are the
- mos t numerous . They may cons ist of one or a combination of 9
the following reactions on the pesticide mol � cule : hydrox ylations of aromatic or aliphatic components , N-dealkyl ation , beta -oxidation , decarboxylation, ether cleavage, epoxidation, oxidative coupling, aromat ic ring cleavage , heterocycli c ring cleavage or sulfoxidation (8, 48).
REDUCTIVE SYSTEMS: Reduct ive systems are not widely reported for pest icide met aboli sm. As pesticide degrad�tion studies continue it is certain that more reduc- tive systems ·will be dis covered . The mos t common type of reduct ive system that occurs is dehalog enat ion (48). Other common reductive reactions are trans form at ion of a nitro to an amino group , reduction of sulfoxide to sulfide, and the r�du ction of me t als (8).
HYDROLYTIC SYSTEMS: Hydrolytic react ions are cleavage of the pesticide molecule with · the conco mitant cleavage of wat er. Enzymes are commonly involved, but the reaction can also be readily caused by abiotic factors such as pH, moisture· and heat (8). The c-ause of the reaction is som etimes difficult to determine . To prove this process as a biotic reaction and not a secondary abiotic react ion re quires that the respons ible enzyme be isola ted and , subse quently , in vitro degradation with the pest icide as the substrate, · demons trated (8). 10
CATABOLIC METABOLISM
PESTICIDES SERVING AS A SOURCE OF CARBON AN�
ENERGY: Pentachlorophenol (PCP) is a wide-spectrum herbi- cide and an insecticide commonly used for termite control
( 7 4 ) • It is als o used extens ively as a wood preservat ive in the lumber industry (69). Stanlake and Finn obt ained several bacterial isola tes able to use PCP as a carbon and energy source (69 ). Several of these organisms res embled members of the g �nus Art hrob acter. Growth was sust ained in a chemically def ined med ium �ith pure PCP serving as the only source of carbon and energy . Growth at steady state in a chemostat showed that 97% of the PCP disappeared; Cl was released quantitat ively . A str ain designated as NC had / a growth rate of 0.05 to 0.1 hr-1 , depend ing on the initial concentrat ion of PCP . As PCP was ut ilized , the pH de- creased . When the pH dropped to 6.1 5 PCP became toxic and growth of NC ceased. Increasing the pH to 7.1 resulted in growth and subs equent degradation of_ PCP. Further investi- gat ion revealed that growth of NC was inhibited at concen- trations greater than two �g /ml PCP in its undissociated form, wh ile no inhibit ion occurred with PCP as a sod ium salt at a concentration of 200 pg/ml.
An unidentified bacterium was isolated by Tewfik and Hamd i that decom posed Sevin in the absence of an 11
addit ional carbon and ene rgy source (70) . Growth exper- ime nts in a chemically defined med ium containing 350 ug/ml
Sevin as the only source of carbon and energy. The degra- dation of Sevin was influenc e d by the presence of a nitro- gen sou rce. When this med ium was supplemented with ammonium sulfate, complete degradat ion of Sevin required 21 days . If the nitrogen source was omitted from the medium ,
Sevin was comple tely degraded wit hin five days .
2,4-dichlorophenoxyacetic acid (2,4-D) is a widely use d her bicide that is easily degraded by a wide variety of soil microo�gani sms (27, 36 , 58 ). The �etabolic pathway and �o rresponding enzyme s for 2,4 -D degradat ion have· been studied and known for some time for an Arthrobacter species
,.} (27, 65) . Pemberton et al . and Sha rpee et al . have re- ported several Pseudomonas ba cterial isolates capab le of us ing 2 ,4-D for their sole carbon and ene rgy source (58,
59, 65) .
An herbicide similar to 2,4-D, but cons ider ed much more persis tent is 2,4,5-trichlorophenoxyacetic acid
(2,4,5- T). This herbic ide is slowly incidentally me tab- olized·, and no micro organism prior to 1981 had been described capab le of using it as its sole source of carbon and energy (27, 37). Using a plasmid-assisted molecula r breeding technique develop ed by K� llogg et al� (37),
K ilbane et al. isolated a strain of Pseudomonas cepacia � . 12
that was able to use · 2,4,5-T for its sole carbon and ene rgy source (39). With 2,4,5-T at a concentrations of 2 mg/ ml
P. cepacia rel eased 100% of the chlorine as Cl- from
2,4,5-T, and 97% or more of the 2,4,5-T phys ically disap- pea red . 2,4,5-T became toxic at concentrations greater than 3 mg/ ml (39).
Th e technique used by Kilbane et al . in isola ting this pseudomonad is based on the assum ption that degra dat ive gene s for xenobiotic compounds evolve by recru itment of variou s· gen es from degradative plasmids (37). In order for ·me t abolic pathways to evolve in th is way , the micro organs ims need to be stressed with the organic compound in q G estion (13, 37). Often organic pollutants occur in the environment at a very low concentrat ion and prevents met a bolic pathways from developing (37) , or metabolic enzymes that exist . for these pathways are not induced due to their low concentrat ion (13 ). �o overcome this , Kellogg et al . inocul ated a chemostat with microorganisms obtained fr om various waste-dumping sites , in addition to microorganisms that harbored previously characterize d degradative plasmids
( 3 7 ) • Th e plasmid -containing microorganisms suppli� d requ is ite gen es for the natural development of a degra dative pathway fo r 2,4,5-T , wh ich at this time was not 13
known to be used as a carbon and energy source by any m- icro- organs ims . Initially , the chemostat contained plasmid · sub- strates at a hig her. concent ration than 2,4,5-T . Over time the 2 , 4 , 5 -T con centrat i o.n was increased wh i 1 e dec rea s ing the plasmid substrate con centrat ion. After ten months a str ain of P. cep acia was isolated that was capable of using
2,4,5-T for its sole carbon and ene rgy source (39).
PESTIC IDES AS A NITROGEN OR PHOSPHORUS SOURC E:
Various pesticides have been described as recalc itrant and , as such, are not susceptibl� to microb ial att ack by pure cul tures of �icroorgani sms . Various laboratories have reported that they were not able to isolate cul tures that we re able to degrade the pesticide in question . The data have been confus ing and variab le (18). For instan ce, some inv est igators have described symmetrical triaz ines c �-triaz ines) as recalcitrant ; others suggest that these pesticides · are readily degrad able ; and other researchers abstain from commenting on any s-triaz ine degradation potential (18).
Cook and Hutter emphasized that primary enrichments are a necessary pre requisite for isolation of organi sms . able to med iate either s �triaz ine or other recalcit rant pest icides (17). Where others have failed in isolating microb ial populations able to degrade s-tr iazines, Cook and ·
Hutter have succeeded by using the s-triazines as the sole
43SSOS KOTA Sl SOUTH D 1 4
nitrogen source. Cook and Hutter i�olated various strains of Pseudomonas and Klebsi ella pneumon iae that were able to utilize various s-triazines for a nitrogen source . The growth yields , measured as protein, were similar to yields obtained when ammonium ion served as the nitrogen source.
Cook and Hutter reported that aromat ic ring carbon was released as co 2 , signify ing that the pesticide was degra- datively metabolized . The pesticide was cons idered cata - bolic ally metabolized since growth is not poss ible in the pesticide 's absence under certain conditions (e.g. , when nitrogen is �o t available from any source other than the pest icid e) . An additional carbon source is necessary when · a pest icide provides phosphorus or nitrogen.
Campacci et al . found simila r results usi ng amitrole as the nitrogen source (14)� Us ing enrichment tec hniques, ten isolates we re obtained . Grow th of these isolates in· a medium that contained amitrole as the only source of nitrogen was proportional to the conc entr ation of amitrole present . Degradat ion of amitrole was non existent when the growth medium cont a ined other available forms of nitrogen (ammonium sulfate, peptone, yeast extract). These sources of nitrogen appare ntly are more readily ut ilize d by these isolates than the nitrogen available from amitrole .
To cont inue the synthesis of am itrole degrading enzyme s would be unnecessary and was teful for these microorganisms , 15
therefore enzyme production for amitrole degradation is
"s hut down". Cam pacci et al . reported that amitrole was not capable of simult aneou sly providing the carbon and energy as well as nitrogen to the organisms (14).
Tewfik and Hamdi reported that the degradat ion of
Sevin by an unident ified bacterium was influenced by the presence of a nitrogen source (70). When ammonium s�lfate was present in the growth med ium Sevin degra dat ion was inhibited .
Several bacteria described as belonging to the genus Pseudomonas we re cap able of utilizin g . organophosphates as the sole phosphorus source (60). Using en'richment techniques , the fo llowing organophosphates· we re added to a defined med ium in wh ich t�e organophosphate was the only source of phosphorus : NPD (aspon) , Az odrin
(monocrotop�os), Dasanit (fensul foth ion) , diazinon
(dianon) , mal athion , Orthene (acephate) , parath ion (ethyl parath ion) , Trithion (carbopheno th ion) , dimethoate, Dylox
(trichlor fon), methyl parath ion (parathio n, me thyl) , and
Vapona· (di chl orv os ). The degradative enzym e(s) responsible had a broad specificity �s each isolate was cap able of us ing a vaiiety of the organophosphates for its ph osphorus sou rce. 16
PESTICIDES INCIDENTALLY METABOLIZED
The preceding discuss ion focused on cata bolic me�abolism of pesticides . Us ing selective defined culture media, growth of isolated microbial populations occurred only in the pre s ence of the pesticide; thus the pesticide served as a limit ing nutrient in some aspect , i.e. , carbon , nitrogen or phosphorus source. Wh ile cataboli sm is an
important metabolic activity and important in solv ing toxic waste problems , the ability of the microorganism to trans- form a pesticide wh ile gaining its supp·ly of carbon from anoth er source should not be overlooked and may be more significa�t in nature than catabolic processes . The importance of inc idental met abolism of pest icides will be emphasize d and the important role that it can play will be a component of a subseque nt section �f this thesis .
The following reaction types are commo n example s and do not ·pertain solely to inc idental transform ation reactions ; indeed many of the following reaction exa mples may occur when a pest icide is being used for a carbon and energy source.
A comm on initial step in pesticide oxidation is hydroxylation of an alkyl side chain or aromatic ring (8). Th is increases the polarity of the .pes ticide mole cule and r esults in greater water solubility and , hence, accessi- bility to microb ial att ack ( 40 ). 1 7
Krause et al. studied the transf orm ation of metol achlor by an unidentified a�t inomycete isolated from a soil with a previous his tory of metolachlor use (40). Eight metabolites were ident ified by nuclear magne tic resonance
(NMR) spectroscopy. The metabolites we re produced by the parent compound undergoing benzylic hydroxylations and/or demethylation of the N-alkyl subst ituents. The actin omy cete was not capab le of using met olachlor as a carbon and ene rgy source. Hydroxylation of metolachlor increased the metab olite's polarity as determined by high pressure liquid ch�om atography (HPLC) retention times . Th is result ed in a prbduct with greater water solu b ilit y and availabi li ty to other soil microorganism s . Krause et al . screened various s6il sample s for isolates capable of degrading met olachlor and conclud ed that the ability is no.t widespread among soil organi sms .
Four met abol ites were produced during the inciden dal metabolic transformatio n of alachlor by the fungus
Chaetomiu� glo busum (71). The N-alkyl substituent groups were removed from alac hlor. Chloride ion was released in the transformation of alachlor, but it was not determined if this was a dehalogenation reaction or. if Cl- was released a�iotically after dealkylation of the N-substi- tuent side chain. No hydroxylated met abo l ites were reported. 18
Smith and Phillips reported that alachlor was
incidentally met abolized by Rh izoctonia solani (68).
Alac hlor degradation was proportional .to the concentration
of sucrose present in the grow th medium.
Liu and Bol lag isolated a fungus from soil treated
with the carbamate insect icide, carbaryl, that was capable
of hydroxy lating the parent compound (45). Three metab-
elites identified had been hydroxy lated at the N-methyl
subst ituent group or on the arom a tic ring . Qu a ntities of
the met abol ites produced were inf luenced by the growing
conditions .
Similar met abolites to Liu's (45) wer� found by
Bo llag and Liu studying the degradation of carbaryl by 18
f u n g i ( 9 ) • After five days of incubation an unin- s· o i 1 oculated control had sig nificant levels of !- naph thol, a
chemical hydrolysis product. It was found that the fungi
possessed varying levels of ability in transforming this
- hydrolysi s product, although its met abolites were not
characterized.
Bollag and Liu obtained three fungal isolat es by - ·enrichment in a culture medium cont·a ining Sev in, of wh ich
one was Fusarium solani (1 0 ). It was shown that a chemical
hydro lys is reaction readily occurs with Sevin in a defined
culture medium to form 1-naphthol. Each . of the three iso-
lates were able to accelerate the hydrolysis but it was not 19
determined if this was due to biot ic or abiot ic factor s· .
Fusar ium solani also rapidly transformed !- naphthol when · includ ed in the culture medium. The two remai ning isolates
either accum ulated !-naph thol or on ly slow ly transformed
it . A comb ina tion of the three isolates was able to de-
grade Sevin more rapid ly and extens�vely than either of the
three isolates separately. One of the isolates cou ld toler-
ate 100 ppm of !�naphthol, another could not toler ate 30
ppm or greater, and the third or ganism cou ld on ly tol erate
20 ppm or less.
Bauer et al. isolated a ys eudomon as sp. by enrich-
ment that was capab le of in cidentally me tabolizing 2,4-D
when cul tured on sod ium benzoate (6). Met abo lit e studies . demons trated that the acetate moie ty was cleaved of f and
that the p-ch lorine atom was removed from the arom atic rin g of 2,4-D.
Serdar et al . isolated and identified a strain of
Pseud omonas diminuta that. was ab le to hydrolyze parathion
incidentall y to p-nit rophenol and diethylthio p hosp horic
acid (63). Parathion or it s hydrolysis products were not used as carbon and ene rgy sources . 20
2,4-D PLASKIDS AND PERSISTENCE
It is well known that the degradative pathways for
many hydrocarbons are borne on pla smid gene s . rat her than
chromosomal DNA (8, 37, 56, 58, 63 ). Pemberton et al.
were the first to report the discovery of pestic ide degrad-
ing pla smid s (57, 59). They isolated a Pseudomonas strain
capable of utilizing 2,4-D as the sole carbon and energy
source . Early attempts at isolating bacteria capable of
using 2,4-D as carbon and ene rgy source were described as
unsuccessful due to the instabil ity of the genetic trait .
The strain isolated by Pember ton was stable in using 2,�-0
as its carbon and ene rgy source, unless cul tured in th e
presence of mit omyci n C, a mutagen wh ich is commonly used
� �o r curing bacteria of plasmids . He found that 2,4-D
utilization only occurred in the presence of the plasmid .
Fis her et al. designated a plasmid he is olated from
th is bacterium as pJPl and found that the plasmid facili-
tated the utilization of 2,4-D as a carbon and energy
source (24). The pla smid was determined to be approxi-
mat ely 90 kil obases in length and carr ied genes that
facil itate conjugation . No other traits typically associ-
ated with the plasmid DNA (e.g. , carbohydrate utilization ,
heavy metal resistance, ultravi olet light resistance or
antibio tic resi stance) were borne by pJPl. · On further
taxonomic characterization the genus designation of the 21
bacte rium was changed to Alcaligenes from Pseud omonas.
This iso late was also able to utilize 2-methyl-4-ch loro-
phe noxy acetic acid as its sole carbon and ene rgy source but not the parent com pound phe noxy acetic acid . The plasmid
carries genes for the firs t step in the uti liza tion of
either compound, converting the parent molecule to the
correspond ing phenol . In the absence of the plasmid this did not occur. Conceivably, the plasmid cou ld carry the
genes for fust the firs t step or for the degradat ion of the 24 . entire molecule ( ) The Ps eudomonas sp . isolated by Serdar et al. cap- ab le of hydrolyzin g parathion was shown to harbor a plasmid wh ich facilitated parathion degradation There was a / (63). 100% correlat ion between presen ce of the plasmid and hydro-
lysis of parath ion to p-nitrophenol and die thy l thio-
phosphoric acid .
A Pseudomonas cepacia strain isolated was capabl e of using 2,4,5-T for the sole carbon and ene rgy source
• This trait was presumed to be plasmid mediated due (3 9 )
to it s inst abi lity. (39). Plasmids could represent a means for transfer of pestic ide degradation abi lit y among so il microorganisms .
Pem berton et al. reported how mic roor ganisms might evolve new degradative pathways for xenob iotic compounds. He is o- . la ted an Alcaligenes eutrophus strain that was capable of 22
utilizing 2,4 -D, but not phenoxyacetic acid (PAA) for its
sole carbon and energy source (58). Mutants of this organ-
ism were isolated tha t were capab le of growth on PAA at a
mutation freque ncy of 10-6, whi le retaining the 2 ,4-D
degrada tion phenotype, indicating spontaneous mutat ion of a
sing le gene . Pemberton et al. concluded that the 2, 4-D
gen e wh ich is carried on a plasmid is first duplicated and
then a mutat ion occurs in on e of the genes enabling it to become PAA+ . The duplication of on e gene, then mutation of
one of the duplicated genes generates mut ants with dual sub-
strate speci� icit y rather than sing le substrate spe cificity
wh ich wou ld be expected if there wa� on ly one gene present
( 5 8 ) .
De gradat ive plasmids appear to evolve by recruit-
me nt of variou s genes from other plasmids (37). Plasmids
also may represent a means for novel degradativ e pathways
to develop for xenobiotic compounds. For instance 2 ,4,5-
trich lorophenoxya cetic acid (2,4,5-T ) is a recalcit rant
molecule and is on ly slow ly transformed by co- oxid ative
mechanisms . No known organisms are capab le of using
_ 2, 4, 5 -T for the sole carbon and ene rgy source (39).
Ke llogg et al. deve loped �� tec hnique -called plasmid -
ass isted mo l ecular breeding to enhance biodegradation of
toxic compounds (37). This techni�ue was based on the
ass umption that microbia l systems need to be stressed in 23
order to develop enzyme sys tems capab le of degrading
com p lex compnunds (13, 37). Microorganisms not capable of
using 2,4,5-T for carbon and ene rgy were grown in the
presence of 2,4,5-T . These organisms did have the degra-
dative pathways to utilize a number of hydrocarbons in
wh ich the pathway was carried on plasmid genes. A chemo-
stat was ino culated with microorgani sms ob tained from var-
ious toxic waste dum ps . The chemos tat was sta rted with an
initial concentration of 50 pg/ml 2,4,5-T and�p lasmid sub-
strates in order for grow th of the organisms to occur .
Over a per iqd of 8-10 mon ths the 2, 4,5-T conc entrat ion was
increased wh ile the plasmid substrates were decreased. At
the end of 10 mon th� 2,4,5-T was the sole carbon and ene rgy ( . source susta- ining the growth of the microbial community in
the chemos tat (37). Th is culture cons isted of 3-4 dif-
ferent colony types . Eventua lly a pure culture of
Pseudomonas cepacia was isolated that was capab le of utilizing 2,4,5-T as the sole carbon and ene rgy source
(39).
SIGNIFIC ANCE OF MIC ROBIAL COMMUNITIES
Typically microb iolog is ts initiate degradation studies intend ing to isolate pure culture s of micro- organisms capab le of us ing a pesticide as the sole carb on and ene rgy source (67)� Researchers common ly init iate such 24
experiments by enriching the samp le with the pesticide �o
increase the numbers of organisms ab le to cata bolically metabolize it. This is then follo wed by attempt ing to
isolate pure culture s of the organism by phys ically sepa-
rating them . This is commonly done by plat ing the samp le on an agar cul ture med ium containing the pesticide as the
sole source of carbon and energy .
The soil environment is com posed of many diffe rent types of microorganisms . It is safe to assume that in the many different possible types of microenvironments present, different re� ation ships would develop and exist between different microorganisms (67) . Microorganisms that ut ilize pest icides for carbori and energy as pure cultur�s in the ( laboratory may not respond the same way in soil. As Slater and Lovatt have stated " ... the time . has now arrived when biochem ists ought to give serious thought to fundaree ntal microbiological principles wh ich could have important reper - cuss ions for our unders tanding of biodegra dation princ�ples" (67).
As was just stated, most degra dation studies are initiated ignoring the fact that the soil supports the growth of many different ·.microorganism s wh ich have many different metabolic capabilities . It is possible to iso- late stable associat ions of microotganisms if the proper isolation techniques are used (67 ) . 25
The following seven different microbial communities have been described and are discussed in dept h by Slater and Lovatt (67 ).
1. Interactions based on the provision of
specific nutrients.
2. Interact ions based on the removal of
growth-inhib itors .
3. Inter act ions base d on the modification �f
ind ividual organism basic growth param eters .
4. Interactions based on combine d (concerted)
metabolic attack.
5. Interact ions based on come tabolism.
6. Interact ions based on hydrogen (or electron) transfer.
7. Interactions based on the pre sence of more
than one pr imary subst rate utilizer.
Microbial commun it ies based on interactioris 4) and
5), above , could have the most significant impact for micro biolo gists working on pest icide degradation . · While the remaining communities are importa nt , the y revolv e around the concept of a member providing another member that pos sesses the met abolic cap�bility but is unable to grow, with a selecti ve · advantage for growth .
The following example of ati interaction based on the provision of spe�i fic nutrients wi ll illustrate these 26
pr incip les . A microbia l community capable of degrading
trichloroacetic acid (TCA) consisted of an unidentified
bacterium and a Streptomyc es sp. (67). Th e bacterium was
the primary user of TCA, using it for a carbon and ene rgy
source, but was not capab le of growing in the absence of
the Streptomyc es species . It was found that the strepto-
myc ete provid ed vitamin B 12 for the bacterium. If vitamin
B 12. was _provid ed for the bacterium from an exogenou s source , the community dis solved . This community doe s not
depend on increasing the metabolic versatilit y of the mi c ro-
organisms invo l ved , while commun ities based on in teractions
4) and 5), above do.
The on ly requirement for a community based on in ter-
action 4) �s that the ind ivid ual members of the communit y
are not capable of totall y mine ralizing the pesticid e ,
whe reas the entire microbia l community working
synergis tically, is . As early as 1968 it was shown that
two or more bac terial is olates may be needed to degrade a
pest icide mo lecule. Gunner and Zuckerman described the
effects of concerted microb ial degradat ion of diaz inon ( 3 1 ,
"6 7). They describ ed an association between a �treptomyc es
sp. and an Ar throbacter sp. that was ab le to mineraliz e dia-
zinon to co 2 . Either of the isolates by itself was not 14 ab le �o incorporate or convert ring labeled c- diazinon to 2 7
co 2 , together the diazinon was converted to co 2 and two
unidentified met abolites .
MIC ROBIAL COMMUNITIES BASED ON CO-METABOLISM
Usually biodegradat ion is cons idered from the stand-
point of the pesticide mol ecule serving as the carbon and
energy source for an organism. The pesticide may be miner-
alized or a por t ion of the pesticide mo l ecule may serve as
the carbon and energy source, (e.g. , an alkyl side chain on
aromat ic ring) (48). Assuming this , many experiments are
not designed to elucidate the numerou s potential trans for-
mat ion s and potential mineralization s of these chemic�ls.
Frequentl y , microorgani sms are capab le of transforming pes r ticides but not capab le of grow th us ing the pest icide as a
source of carbon and energy (8, 48). Therefore , many mi cro-
organisms are not isolated and characterized that are capa-
ble of transforming pesticides . Unfor tun ately , there is
not an esta blished protoco l for isolat ing and char acter-
izing mic roorganisms that are capable of· trans forming pesti-
cides withou t using them for a carbon and ene rgy source
( 6 7 ) • i t is evident that incidental met abol ism �s a major .
decompos it ion process fo� pest icides in soi l (8, 48, 6 7) . . Un fortun ately , its significance has been underes t imated due
to the difficulty involved with the isolat ion and character-
izat ion of indigenou s microo rganisms . For examp le, there 28
are difficult ies in devising suitable enrichment procedure s
for the isolation of responsible microorganisms that trans
form but do not use the compound of interest for a carbon
and energy source (8, 67). Enrichments cannot be ach ieved
on the basis of growth . If the compound does not act as a
nutrient , no selecti ve adva ntage is gained by the
microorganism (8, 67).
The preced ing inform ation underlines the basic
principles for a community based on com etabolism. This
commun ity depends on a microQrganism that has the capa
bi�it y of altering a substrate, even though the organism
gains nothing beneficial. The new compound formed is hi�hly likely to be used as a subs trate for growth by other microorganisms (8, 67).
Parathion degradation is an examp le of an insec
ticid.e that has been shown to be biotically degraded by a microb ial commun ity that is based on a cometabolic step.
Initially th is microbial commun ity was is olated in a chemo
stat from samples of sewage used as inoculum (54, 67 ).
Growth was sustained initially by adding 0.1% glucose to
the broth wh ich contained parathion at 10 pg/ml . Parath ion . was in cre ased to 3 mg/ml over a 30 day period wh ile glucose was decreased to zero . The init ial community contained nine microorganisms (one Gram' s posit ive Brevi bacterium and
eigh t Grams 's negative pseudomonads). This was considered 29
an unsimp lified commun ity because the community was subse- quently characterized as having three distinct memb e rs :
Pseudomonas aeruginosa, !· stutze ri, and an unidentified coryneform bacterium (21, 67 ). P. stutze ri cons titutively produced a hydrolase enzyme that degraded parath ion pro- ducing p-nitrophenol and die thyl thiophosph oric acid.
Neither of these two products could support the growth of
P. stutze ri. P. aeruginosa was capab le of utilizing p-nit ropheno l for carbon and ene rgy , but not parathion or diethyl thiophosp horic acid . A carbon source must have been available for- P. stu tzeri to use which may have been made available through cell lysis or from exogenously-produced metabolites of P. aeruginosa (67). No function was ( described for the coryneform bacterium which was elimina ted in subsequent studie s (67).
The practical implica tion of the preceding examples is that many pesticides that are considered recalcitrant may be perceived so on ly because the experime ntal design was confined to pure cultures of organi sms . It is often demons trated that a compound is degraded more slo wly in the laborator y than in the fie ld where concerted mic robial attack occurs (1, 8, 67 ). Da lapon is such an examp le (67).
degraded Da lapon 20% f4ster than the A _ mic robia l communit y tot al combined rates of the in dividual popula�ions makin g u p the mi c robia l communit y. 30
It may be possible to fabricate microbial commu
nities to actively degrade recalcit rant pest icides . In
effect, the gene products from many different microor
ganisms are pooled together and a me tabolic pathway is
cons tructed that is capab le of mineralizing the pesticide.
Slater and Lovatt constructed a two-member ed microbial com mun ity in the laboratory wh ich was capable of using 1-mono
chloroacetic acid (MCA) that was dependent on a cometabolic
step (67). Pseudomonas putida PP3 was isolated from a mic.robial commun ity growing on the herbicide Dalapon that
produced a broad substrate specificity dehalogenase enzyme
Th is pseudomonad was capable of using 2-monochloro-
propionic acid (2MCPA) for its carbon and energy source.
The dehalogena se enzyme that it produ�ed for 2MCPA utili zation also dehalogenated monochloroacetic acid (MC A) , prod ucing glycolate. When P. pu tida was grown in a mixture of 2MCPA and MCA , both substrates we re dehalogenated, but glycolate accum ulated . A stab le microbial community was con str ucted using a Fla vobacterium sp. that used the gly
. colate produced, but not 2MCPA or MCA for its carbon and ene rgy source. Whether t h is associat ion exists in the soil o r c an operate in the soil , if introduced , is not known . 3 1
SOIL AND APPLIED DEGRADATION STU DIES
The studies described thus far have been conducted with pure cultures in vitro . If an organism is ca� ab le of rapidly degrading a pest icide in vitro , the quest ion arises : Can the pure culture degradat ion process occur in a soil milieu where competit ion among nat ive populat ions occurs in the presence of alternate carbon sources? The soil system is a mu ch more complicate d sys tem than what occurs in broth studies with pure culture ·techniqu es .
Addition ally, degradation of pest icides by cell-free extracts coul� play important roles in detoxification of pesticides industrially (3). A thorough review on enzy- mo logy of pesticides is presented by Munnecke et al . (50). ( Kilbane et al . isolated a bacterium identified as
Pseudomonas �a cia that was capable of using up to 3 mg/ml
2,4 ,5-T in broth mediu m as sole carbon and ene rgy source
(39). At concentrations greater than 3 mg/ml no growth was observed . At the end of the 6th day of incubat ion, there was greater than 97% loss of 2,4,5-T and essentially 100% release of Cl
Cb atterjee et al. showed that this �· cepacia is capable of degrading 95% �f 2,4,5�T at a concentration as hig h a s 1 mg/ml in the soil with in one week (16). This bac terium was able to grow and mu ltiply in so·il with
2 ,4,5-T serving as the sole carbon and ene rgy source . The 32
P. cepacia titer increased as 2,4,5-T was utilized and decreased after 2,4,5-T was used up. Degradation was
carried out in the presence of indig enous microorganisms at 8 a concentration of 2.0 x 10 cells/s of soil. Add itionally
the 2,4,5-T seTved as a carbon and energy supply in the presence of various other carbon sources that would normally be present in soil.
Further studies by Kilbane et al . showed that the bacterium was able to remove 90% of 2,4,5-T in conta minated soil that contained 20,000. ppm of pest icide (38). The long term survival of th is pseudomonad was also inv est igated .
Soil lacking 2,4,5-T that had been inoculated with 1.0 x
107 cells of P. cep acia showed no detectab le titers after
12 weeks of incubation. 2,4,5-T was then added at a concen- trat ion of two mg/gram of soil. Afte.r a two week lag peri- od , P. cepacia increased its titer until no more substrate existed .
Barles et al . tes ted the ab� l ity of an acclimated culture that was known to degrade para t h ion in situ (4).
The acclimated culture was composed of Pseudomonas stutzeri and P. aerugin osa. P. stutzeri was responsible for hydro- lyzing parath ion to p-nittophenol and ionic diet hylthio
,phosphoric icid. The p-nitrophenol was then used as a carbon and energy source by � aeruginosa. Th is culture was capable of com pletely degradin g parath ion in 35 days at 33
a concentration of 1250 kg/ha (557 pg/g ram) . At hig her �on-
centrations , the degradation was much slower and inhibited, probab ly due to a lowering of pH by p-nitrophenol wh ich is known to be toxic to many microorganisms . A slo wer rate was observed when the emul sif iable con cent rate was used
ins tead of technical grade parathion .
The use of cell-free enz yme extracts capable of detoxif ying pesticides has cons iderable potential for· waste waters containing pesticides or for soil conta minated b y chemical spills · (3). Bacterial enzymes could be useful for de�oxify ing �as te waters by pass ing the contaminated water through a column containing an immobilized enzyme (52) , or the enz yme could be added to the water-pesticide mixture r maintained in bulk holding vessel (51, 53). Munnecke has shown that an enzyme extract is able · to degrade emulsi - fiable parathion (51, 53). Enzymat ic degradat ion was determined to be faster than chemical hydrolysis when the form ulat ion was dilute d to 1%, and a carbonate buffer used to control the pH . 34
CHAPTER 2
SCREENING FIELD ISOLATES FOR INCIDENTAL
AND CAT ABOLIC PEST ICIDE METABOLISM
MATERIALS AND METHODS
Tech nical grade 2-chloro-2 ',6'- diethyl-N-
(meth oxy-meth yl) acetanilide (alachlor) , 2-methoxy-3 ,6-di-
chlorobenzoic acid (dicamba ), and 2,3-dihydro- 2 ,2-di methyl-
7-� enzo-furan? l methylcarbamate (carbofu ran) were used in the following degradation studies . Alachlor is a pre eme r-
gence herbicide with a mo lecular weight of 269.77 (74). ( Dicamba is a herbicide with a mo lecular weight of 221 .04,
and carbofuran is an insecticide with. a molecular weight of
221 � 26 (74). These pest icides are hydrophobic and there-
fore are limited in their water solubility . Their water solu bilities are 140, 700 , and 75 �g/ml respective ly for alachlor, carbofuran , and dicamba (74).
The obj�ctive of this study was to determine the ab ility of bacterial isolates to catabolically or inciden- tally metabolize - pesticid�s . Isolates evaluated were p rev iou sly obtained from cultivated field pesticide biodeg- radation studies. Catabolic metabolism was determined by i n o c ulating each of the microorganisms into a medium 35
contain ing the pesticide as the sole source of carbon and ene rgy . If growth occurred , this would indicate that the
· pesticide was � erving as a carbon and energy source for the microorganisms . If no growth occurred, but the pest icide was degraded in the presence of an alternate carbon and ene rgy source, such as nutrient broth , this would be an indication of incidental metabolism.
CULTURE MEDIA: Culture media used for growth of actinomycete or bacterial isolates typically made use of tryptone-yeast extr act (TYE ) broth (tr ypt one , 5 g; yeast
·e x t .r a c t , 3 g ; . g 1 u c o s e , 3 g ; p e r 1 i t e r o f d i s t i 1 1 e d w a t e r
(pH=7 .2)) or nutrient broth (nutrient broth (Difco) , 8.0 g; glu 9ose, 3.0 g; per liter of distil led water (pH=7 .2)).
Glucose was included as a carbon and ene rgy source to en sure that rapid growth was achieved .
STORAGE CONDIT I ONS USED: Is�lates were sto red at
-70° C to mini mize genetic mutations of the microorganisms .
In addition , frozen suspensions of the isolates provided an imm ediate and re liable source of inoculum .
Bacterial isolates were sto red at -70° C in freezing medium (62). . Actinomycete isolates were sto red by susp e nd- ing their mycelial spores in freezing medium and then frozen as above . Malt extract-yeas t extract (ME-YE) agar
ion med ium for the w a s use d as sporu. lation induct actinomycetes (66). 36
Phosphate buffer (2.77 g, Na2 HP04 ; 2.77 g, KH2 P04 ; per liter, pH 6.8) cont�in ing 20 ml Hutner's vitamin -free . mine ral base and 1.0 gram NH4 C l/liter was used for sus pend ing the actinomycete spores, providing a properly buf fered isotonic diluent (26, 30 ). Th is was referred to as Hutner's basal (HB) medium . Actinomycete spores have an outer sheath that is highly hydrophobic and res ists being sus pended in aqueous solutions (23). HB contain ing 0.05%
Tween 80 was used as a surfactant to facilitate uniform sus pension of the spores (28).
After sporulat ion had taken place (3-7 days ), 5 ml of HB supplemented with Tween 80 was use d as a suspension med ium for the spor es . This spore suspension was trans- ferred to sterile, capped culture tubes. . One volume of freezing medium was added after wh ich the spores were sto red frozen (-70° C) . Additional suspens ions of spores were made by taking thre e drop s of the initial spore sus pension and inoculating the surface of YE-ME agar slants .
These we re allo wed to sporulate and then treated as just describ ed. This allowed a reason able supply of spores to be used as required for inoculating various culture media�
DETERMINING PESTIC IDE CATABOLIC AC TIVITY:
Pesticide media were prepared for studies designed to assess the ability of bacterial i�olates to utilize carbo furan as the sole source of carbon and energy . Agar and 37
broth media were supplemented with carbofuran that provided
the sole source of carbon and ene rgy . Growth of microor-
ganisms occurring on · these media would indicate that they
were capable of metab oliz ing carbof uran.
Commercial agar contains contamin ants that are not
removed during it 1s process ing and can interfere with
growth studies (12, 42). Therefore , growth studies we re
performed comparing media that contained unw ashed, washed, and no agar . A stock solution of technical gr ade carbo-
furan was made by dissolving carbofuran in 95% ethanol (2-5 mls' ). Th is was filter sterilized (Ge lman Acrodisc, 0.2
�m) , and stored at S ° C. Th is stock was used when adding
ther carbofuran to broth or liquified agar.
PREPARATION OF SO LID MEDIA: HB broth conta ining
1.5 to 2.0 % (W/V) unwashed Bacto agar (Difco) was aut o claved and allowed to cool to 55° C after wh ich filter ste rilized carbofuran dissolved in 95% ethanol (2-5 mls) was added to ach ieve a fina l concentrat ion of 300 pg/ml.
The agar pesticide medium was thoroughly stir red with a mag net ic stirrer to achieve uniform distribut ion of the · p est icide throughou t the medium before pouring .into sterile pet ri dishes . Agar plates · containing 2% unwashed agar but not con taining carbofuran or any other addit ional carbon a nd ene rgy source were prepared as n�tritional controls . 38
Pestici de-containing washed agar, and washed agar
containing no additional source of carbon and energy , wer e
prepared in the same manner as for unw ashed agar. Filter
sterilized carbofuran dissolved in 95% ethanol was added to
a final concentration of 300 pg/ml after cooling the HB
agar solution. Washed agar medium containing glucose was
prepared to ascertain the growth obtainable from these
concentrations of organic carbon compounds us ing washed
agar. Memb rane filter (Ge lman 0.2 pm) sterilized glucose
was added to a final concentration of 300 pg/ml to auto
cla � ed and cooled (55° C) HB containing washed agar. Bacto
agar (Difco) was washed using a standard procedure (42) to
( remove the contaminants (12, 42). Pesticide-containing
agar us ing the washed agar were prepared in the same manne r
as described above .
PRE PARATION OF BROTH MEDIA: Broth medium con-
taining carbofuran as the only added exogenousl y availab le
source of carbon · was prepared by adding membrane filter
(Ge lman 0.2 pm) sterilized carbofuran· to autoclaved HB
broth to achieve a concentrat ion of 150 and 300 pg/ml .
Th is was t hen dispensed into sterile, capped 16 x 150 mm
cul ture tubes.
Growth experiments using glucose as the sole source
of carbon and ene rgy at similar concentr atio ns to carbo-
furan were performed to determine the yield of growth 39
obtainable from this limited supply of carbon in broth
mediu m. Memb rane filter (Gelman 0.2 pm) sterilized glu�ose
was added to achie ve a final con centration of 20 pg/ml as
the sole source of carbon and energy in HB broth .
PREPARATION OF INOC ULUM: Extrane ous carbon
sources originating from the freezing mediu m that would
interfere in determining carbon and energy requirements
were removed from the inoculum by centrifuging 1.0 t6 1.5
mls of 1, Streptomyces pilosus , Isolates 5, 9, 16, 17, 22, and 26 at 12,000 x g for 10 minu t es . The resulting pellet
was washed one or two time s with sterile HB medium , and
resuspended in sterile HB med ium . One drop of this
sus�ens ion from a sterile Pasteur pipette was use d to
inoculate culture med ia. These were incubated at 26° C and
then scored for growth or absence of · grow th.
SCREENING ISOLATES FOR INC IDENTAL PESTICIDE
ACTIVITY: · Based on the results of the preceding exper-
iment , a study was cond ucted to determine the incidental
metabolic activity of the same bacterial isolates with
alac hlor , carbofuran and dicamba . Th is was ach i eved by
. preparing· cul ture medium contain ing sources of carbon and . ene rgy used for growth by· the microorganisms supplemen ted
· with the pesticide. After inoculat ion and incubation at
26° C on a culture tube roller, pesticide res idue levels 4 0
were determined either by high perform ance liqui d chrom� ·
atography (HPLC) or gas chrom at�graphy (GC) .
PESTICIDE-CONTAINI NG BROTH MEDIUM:· TYE broth
suppleme�ted wit h 0.3% (w/v) glucose and supplemented with
pest icide was used as a culture medium in determining inci-
dental metabolic activity of the isolates . Stock solutions
of alachlor and dicamba were prepared as described above
for carbofur an. Pesticide was added to sterile broth
medium to a final conc� ntration of approxim ately 100 �g/ml .
Five ml of pesticide broth was then aseptically trans ferred
to � terile 16 x 150 mm culture tubes , capped and stored at
5 ° C.
After thawing the frozen spore and bacterial sus
pens ions , one drop from each suspens ion was added to TYE
broth containing either dicamba , alachlor, or carbofuran to
determine each organism 's ability to incidentally metab-
olize the pesticide. Four uninoculated tubes wer e used as
negative controls . One ml aliquots we re removed from each
tube of broth after inocul�t ion to determine the initial
pesticide concentration . Tubes were incubated at 27° C on a
tube roller (New Brunswick Scientific) for a period of 30
days . At the end of the .incubation period, 1.0 ml was
. withdrawn for post-incubation res idue analysis . Act ino-
my cete isolat es that had a well developed myceliu m and
could not be samp led by pipetting we re subjected t o 41
sonication (Br anson Ins truments Sonifier; Model SllO) a� a
power setting of four to five for 15-30 seconds to break up
the mycelium . These sam ples were sto red frozen at -20° C
and res idue analyses performed at a later time .
PESTICIDE RESIDUE ANALYSES: Alach lor was
extracted from broth us ing ethyl acetate (reagent gra de)
when sam ples were quantified us ing a flame ionizat ion
detector (FI D) . Hexane (reagent grade) was used as the
solvent for extraction of alachlor when detection was
performed using either a Hall or nitrogen-phosphorus (N-P )
detector. One to five ml of solvent was used for the
extraction . Carbofuran was solvent-extr acted from the
bro� h with ethyl acetate in a similar manner. Samp les conta ining dicamba were acidified to a pH of 2.0 by adding
1 N HC l (four drops ) followed by sol vent-extract ion with
t-b utyl alcohol (reagent grad e ).
Carbofuran and dicamb a were quantified by HPLC and alachlor con centr ations by GC . The HPLC was fitted with a
Waters Associates M-6000 pump , Waters Associates U6K injec
tor, and a Beckman 153 UV detector set at 280 nm . An
All tech 018 column (25 em x 4.6 mm id) run at ambient tem perature was used to separate the pesticides using a mob ile phase of 65 :35 methanol:water at a flow rate of 1.5 ml/min.
Sensitivity was set at 0.01 absor bance units full scale.
When quantifying dicam ba, the mobile phase was acidified 42
with 1% glacial acetic acid. A Varian 3700 gas chrom ato -
graph was configured any one of several ways under dif-
fering conditions to measure alachlor levels . When using a
FID the ins t rument was fitted with a 6' x 4 mm id column
and packed with 3% OV- 101 ads orbent on Chromosorb W-HP sup-
port medium . The oven temperature was maintained between
200-225° C, the injector temperature held at 230° C, and the
detector temperature operated at 350° C.
When a Hall detector was used it was operated in
its halogen (reductive) mode with a reactor temperature of
850° C us ing �ydrogen gas . Identical column and temperature
parameters were emp loyed as with the FID , except the �etec r tor tempe rature was 350 ° C. When a nitrogen-phosphorous
(N-P ) detector was used , either 3% OV- 17 adsor bant on
Chrom osorb W-HP support in a 6'x 2 mm id column or 3% OV-25
on Chr omosorb W-HP in 6'x 4 mm id column , was use d . Oper -
ating temperatures were 200-225° C, 350° C, and 230 ° C for the
oven, detector and injector , respective ly.
CHARAC TERIZATION OF AC TINOMYCETE ISOLATES
CE LL WALL ANALY SIS: Based on the proceed ing
study , the cell walls of r·solates 5, 16, and 26 were
- characterized qualitatively for .diaminopimelic acid (DAP )
content by a modified vers ion of Be�k er, et al. thin layer
chrom atography procedure (7, 25). Actinom ycete cells were 43
cultivated in 250 ml of TYE broth supplemented with 0.3%
glucose (7). Cells were incubated on rotary shaker at 26° C
unt il ad�quate mycel ium was present , (four to six days) and
then harv ested by filtering through Wh atman· #4 filter
paper. The cells were washed three times wit h distilled
water, twice with 95% ethanol and allowed to air dry . Ten
mg of dried cells were placed in two-ml glass ampules , to wh ich 1 ml of 6 N HC l was added, at which time the a�pule was sealed with a propane torch . Following this ampules were combus ted for 24 hrs at 100 ° C in a sand bath. The
resulting lysate was filtered through a 0.45 �m (Ge lman
Acrodisc) memb rane filter into the original ampule. A 1 ml ( rinse with distilled water was passed through the filter to
ens ure com plete rec over y of lysa te. Samp le and rinse
filtrates were comb ined and lyophili�ed with subsequent
rehydration in 0.3 ml of distilled wa ter. Th irty-five pl of the rehy�rated sam ple was spotted on Whatman #1 chroma-
tography paper and descend ing ly developed for eight hrs �n a mobile phase of .methanol:water: lO N HCl:pyrid ine
(80: 17. 5:2. 5:10). The paper was removed from the chamber, allowed to dry for 15-30 minutes, and developed in the same direction for another eight hrs (61) . . Developing the chrom- atog ram twice after intermittent drying improves the sepa-
ratio n and resolution of the different DAP isomers (61).
Following separation , the paper was dried , and sprayed with 44
acetonic ninh ydrin (0.1%, w/v) , followed by heating for - 2
minutes at 100 ° C. Streptomyces cacao i ATCC #3082 and
Nocardia autotrophica ATCC #19727 were included as refer-
ences to differentiate bet ween the meso- and 11-isomers of
DAP .
CARBOHYDRATE UTIL IZATION: Taxonomic data wer e
collected by determining wh ich of the fol lowing compounds
could be uti lized as sole carbon and ene rgy sources : ·
2-aminoethanol, 1-arab inose, betaine , cellobiose, citric
acid , fructose, glucose, lactose� malon ate, mannitol,
oxalate, 1-proline , 1-tyr osine, sarcosine , succinate,
D-sor bitol, sucrose, and xy lose. The carbon and ene rgy ( source was added to a basal salts (HB) mediu m at a concen-
tration of 0.3% and filter steril ized . Spore suspens ions
of Isolates 5, 16, and 26 we re harv ested by cent rifugation
and washed twice with HB . The fina l spore pellet was resus-
pended in one ml HB . One drop of spore suspension of each
isolate was aseptic ally added to each of the carbohydrate
utilization media and allo wed to incubate at room temper-
ature for a period of two weeks . After this incubation
period ea�h of the cultures were assessed for growth using
HB containing no carbon and ene rgy as a negative control.
· Weak pos itives we re incubated an additional two weeks and
their growth reva luated . 45
DEGRADATION OF ALACHOR IN COMPLEX, SEMI-DEF INED, AND
DEFINED MED IA
Based on the previous experiment, a study was under-
taken to determine the extent of alac hlor degradation by
Isolates 5 and 26 in complex (tr yptone-yeast extract), semi- defined (Hutner's basa l-yeast extract), and defined
(Hutner's basal) cul ture media. This study provided an
underst and ing of nutritional requirements of the bacteria
to degrade alachlor .
CULTURE MEDIA: A field isola te, identified as
St�ep t omyces _pilosus , and wh ich had previously demonstrated
an ability to grow on carbamates and organophosphates·, was ( inc luded for com parative pur poses in the following studies
( 2 5) . To as s e s s the rate and extent of the d i .s appearance
of alachlor in complex (TYE), semidefined (HB + 0.05% yeast
ext ract) , and def ined (HB) med ia, alach lor dissolved in two
mls 95% ethanol was filter sterilized and added to 50 ml of
each broth media to ach ieve a fin al concentrat ion of about
125 pg/ml . Also each med ium was supplemented with 0.3%
(w/v) glucose. One hundred pl of Isolates 5, 26 and S.
pilosus were added to each medium to achieve a concen- 5 tration of approxim ately j x 10 spores/ml . These were . incubated at 26° C wit h shaking on a rotary shaker (130
rpm). One ml sam ples we re withdrawri at 24 hr intervals for
gas chrom ato graphic measurement of pesticide res idues . 46
BIOMASS DETERMINATION : On completion of the
degradatio n study in the three types of culture media, . the
biomass of each isolate was determined in order to compare
the rates of degradation with the biomass produced by each
x isolate. The broth media were centrifuged at 6,000 g for These we re washed with dis- 20 minutes to harv est cells .
tilled water, transferred to weigh ing tins and dried for 24 hours (70 ° C) and weigh ed.
METABO LITE CHARACTERIZATION
The � egradat ion of alachlor by the various isolates
was determined by measuring the disappearance of parent com ( pound by us ing chrom �tograph ic me thods . A study was under-
taken to determine if the ring aromat icity was maint ained
after degradation by the isolates . Thin layer chrom atog-
raphy was performed on a solvent-extracted broth medium con-
taining me tabolites pro duced by growth of Isolate 5 in
glucose supplemented (0.3�, w/v) tryptone-yeast extract One liter of (TYE) broth containing 150 pg alach l or/ml . this A broth mediu m was inoculated with Isolate 5. sepa- . rate, uninoculated flask of TYE + 0.3 % glucose containing
alachlor at the same con c entration served as an uninocu-
late d control, and a�other flask of the TYE medium without
alachlor served as a control to monitor for cell products
or medium constit uents that would partition into the 47
8 solvent . Inoculated flasks received 1 . x 10 spores of
Isolate 5. Leve ls of al achlor were monitored dai l y. When
the alachlor concentration dropped to (10 pg/ml, the broth
was solvent-ext racted by the addition of 300 ml reagent
grade methy lene chloride and stirred for 4 hrs . The
methylene chloride was separated from the aqueous phase
using a separatory funnel, and then reduced in volume to
approximately 5 ml by rotary evapor�t ion . Th is volume was
quantitat ive ly trans ferred to a test tube and further
re�uced in volume to 1-2 mls by evaporat ion over nitrogen
gas . Twenty-.five pl aliquots we re spotted and developed on
alupinum oxide thin-layer chromatog raphy plates (Ba ker -Flex
IB-F , 30 min) with a mobile phase of ethyl acetate and
me thanol (25:0.5, v/v) . The plates we re dried and then
viewed us ing an ultraviolet lig ht.
SC REENING ISOLATES FOR PLASMID DNA
The genes for the degradat ion of chlorinated com
pounds and xenobiotics have been shown to be borne on
plasmid DNA , and more recently some pesticides have been
shown to be degraded by a plasmid mediated pathway (24, 57,
59, 63). Isolates that we re capab le of alachlor degra-
·dat ion were screened for the presence of plasmid DNA. Alka- · l i ne and neutra l lysis procedure we re carried out and
compared with one another. 48
ALKALINE LYSIS: Approxim ately 1 x 107 spores �f
Streptomyces pilosus , Isolates 5, 16, and 26 were inocu- late d into 50 ml YE-ME bro�h (yeast extract, 4.0 g; malt extract, 10.0 g; glucose, 4.0 g; distilled H 2 o, 1.0 1; pH=7 .3) . This was incubated with shaking (rotary ) for 24
The mycelium was centrifuged for 10 min utes at 10,000 x g (So rva11 SS-34 rotor) in 50 ml sterile Oak
Ridge centrifuge tubes and washed once with 10.3% ' sucrose solution in TE buffer (50 mM TRIS , 50 mM Na2 EDTA, pH=8.5). Each of these isolates were screened for the presence of plasmid DNA using an alkal ine lys is procedure (15). The
DNA was concentrated and residual SDS removed by repeated ( ethanol precipitation (46). Electrophoresis of the DNA was performed in 0.7 % agarose (SeaKem HGT) at 70 volts constant volta ge in a horizontal electrophore�is bed (Model 850 elec- trophoresis apparatus, Aquebogue Mach ine Shop , Aquebogue ,
NY) for 14 hrs in TRIS-phosphate buffer (22). Streptomyce s pilosus is known to harbor a 9.7 Kb plasmid (25) and was included along as a positi ve control. The gel was stained with ethid ium bromide for 15 minutes followed by 30 minutes of destaining in TRIS -phosphate buffer (62).
NEUTRAL LYSIS: P·lasmid DNA was isolated us ing another procedure by inoculating aproxim atel y 5 x 106 spores of �· pilosus , Isolates 5, 16, and 26. into YE-ME containing 34% sucrose and 0.005 M MgCl (15). This was 49
incu bated with shaking for 24 hrs at 30° C. The mycelium was centrifuged for 10 minutes at 10,000 x g (Sorvall SS-34
rotor) in 50 ml sterile Oak Ridge centrifuge tubes and
suspended in 4 ml 0.05 M TRIS HC l containing 15% sucrose
(pH=8.0). For each of the result ing plasmid preparations electrophoresis was carried out as described above .
DEGRADATION OF ALACHLOR IN SOIL
A study was undertaken to determine the degradat ion . of alachlor at a concentrat ion of abou t 550 and 3700 pg/g
1n soil slurr ies by Isolate 5.
STERILE AND NON-STERILE SOIL: The soil used in th is stu dy was obtained from the Plant Science Department at South Dakota State Univers ity and was cla ssified as a loacr . The soil was obtained from fields used in res earch at South Dakota State Univers ity. Soil was sterilized by autoclaving for 30 minutes at 121° C and 15 psi. After this initj al sterilization treatme nt , the soil was incubated at amb ient room temperature for 48 hrs and resterilized a second time to ensur e asepsis . A samp le of the presumab ly
· s terile soil was serially diluted in HB diluent and plated o n nutrient agar plates to determine if autoclaving was effe ctive . In a similar manne r, viable plate counts were made in the uns terilized soil to determine initial concen t r at i ons of ind igenous microbial populat ions . so
SOIL SLURRIES: The degradation studies were
performed in a soil slurry prepared by adding 100 ml of HB
containing 0.05% yeast extr act to �hree 250 ml Erlenmeyer
flasks containing 100 gra ms sterile soil and to two flasks
containing 100 grams of soil that was not sterilized.
Membrane filter (Ge lman 0.2 pm) sterilized alachlor was
added to each of the flasks to ach ieve a final concen-
tration of approxim atel y 550 pg/ml . To one flask con-
taining sterile soil and one flask containing non-sterile
soil, filter sterilized gluco�e was added to a final concen- ·
tration of 0.3% (w/v) . After addition of the alachlor ,
flasks were shaken for 12 hrs before inoculating with 8 spore s of Isolate 5. Approximately 1.5 x 10 spores were
inoculated into the two flasks containing sterile soil, the
remaining one was not inoculated and served as an uninoc-
ulated control. Imm ediately follo wing inoculation , approx-
imately four grams of soil were samp led fro m each flask.
Additional samp les were collected weekly and placed in
screw- c apped tubes . At day 21, the soil slurries that were 8 initially inoculated were reinoculated with 1.5 x 10
_ spores of Isolate 5. At day 56, ten mls of Isolate 5 grown
i n TYE + 0.3 % glucose was added to each of the flasks that
� ad been inoculated with spores . After each sam pling,
t h ree rnl of HB was added to replenish liquid lost via
s a mp ling. 51
An anal ogous stu dy was devised in a similar fashion as that just described except that the alachlor concentra tion was elevated to abou t 3700 pg/g soil .
. Degra dation studies by Isolate 5 in a s�il slurry contain ing alachlor originating from a chemical spill was also perform ed. One hundred mls HB containing 0.05% yeast extract was added to 100 grams spill soil in each of four flasks . Filter sterilized glucose was added to two flasks to a final concent ration of 0.3% (w/v) , and to one of these flasks 1.5 x 108 spores of Iso late 5 was added . The two remaining flasks received no spores of Isolate 5. These two flasks were used in com paring alachlor degradation by an ind igenous population with the degra dation of alachlor by Isolate 5 in nonsterile soil. Of these two remaining f 1 asks , one was supp 1 e me nte d with g 1 u·c o s e to a fin a 1 con c en tration of 0.3% (w/v) ; the remaining one did not receive an add itiona l carbon and ene rgy source .
RESIDUE ANALYSIS: Residual alachlor levels 1n the samples we re either measured gas chrom atograph ically the same day that they were sam pled , or stored frozen for a nal ysis at a subsequent time . All samples were solve nt e x t ract ed by vigorously shaking with 10 ml of ethyl acetate
· ( reagent grade). GC analysis was performed using either . the Hall or NP detector as stated above . After GC ana l ysis , the soil - slu rry sam ple was placed in drying tins , 52
° and dried for 24 hrs at 100 C. Alachlor concentrations were adjusted for soil moisture and expressed on a dry weight basis (pg alachlor/g dry soil). 53
RESULTS AND DISCUSSION
HB is a basal salts medium and does not contain
organic compounds that could serve as a carbon and ene rgy
source for microorganisms . A source of carbon and ene rgy
mus t be present if microorganisms are to grow . If gro wth
of microorganisms occurred in HB medium at the expense of
carbofuran or other organic compounds , this would signify
that the micro�rganisms were ass imilating carbon and obtain-
ing energy from the substra te. Consequently no growth of
microorganisms should occur when inoculated into HB basal
broth medium not containing a carbon and ene rgy source.
GROWTH IN UNWASHED AGAR : Growth occurred with
Streptomyces pilosus , Isolates 1, 5, 9, 16, 17, 22, and 26
in unwashed agar containing carbofuran and unwashed agar
not containing any addit ional source of carbon and energ y
(Table 1). In other wor ds, growth of the isolates on the
pestic ide-containing medium was not necessarily due to the
pesticide, as unwashed agar by itself contains s �me com
pounds that allo w growth of these isolates . Growth of
. the se isola tes did not occur on wa shed ag ar, whether it had b e e n supplemented with either pesticide or glucose (Table
1) . The carbon and energy source that was present
( glu cose) was not capable of being ut ilized . The washin g p roc e ss removed background grow�h wh ich was present in the Tab la 1. Growth of Isolates on unwashed and washed agar 21 In HB broth supp l emented with var ious carbon end anergy sources as shown .
Unwashed Weshad Washed . Iso l ate with with with Coda Unwashed Ca rbofuran* Weshad Carbofuran* Glucose**
*** 1 + + 5 + + 9 + + 16 + + 17 + + 22 + + 26 + + St reptomyces p IIosus + +
SUpp le.. nt ed with 388 carbofuran/• 1 • . • PI ** SUpp le.. nt ed with 388 1'11• 1 aluco•e . for ..tl on of vl•lb le co lon ie• after four week• Incubat ion. Vl *** + +:"- - no vl•lb le arowth after four week• Incubation.
' 55
unwashed agar, apparently removing a nutrient that is
required for grow th . These results indicate the confus ing
and misleading interpretat ions that can occur when using
unwashed or washed agar when assessing substrates to be
used as carbon and energy sources .
Marshall et al. obtained similar results using agar
as a solidifying agent when enumerating microbial popula
tions from soil capab le of using herbicides for carbon and
energy (47). -They observed that the number of bacteri a iso
lated on an agar med ium supp lemented with a carbon and
ene rgy source was only rarely more than two fold that of
bacteria isolated on agar wi th an inorganic basal medium
base, and at other times the number of colonies were less.
Marsha ll et al., commonly found that plat ing out serial
10-fold dilutions of field soil onto an inorgan ic salts
medium containing Bacto agar supported many bacteria.
Consequently, it was found that agar is not the inert
gelling agent that it is commonly thought to be (47)
Marshall et al. , suggested that erroneous results would
result in determining carbon and utilization needs of
- b a c teria.
Agar is com posed of 70-85% agarose and is generally
c·ons idered inert to the action of microorganisms (12, 47).
Agaropectin com prises the remaining port ion a�d does not
c o n tribute to the ge lling action of agarose. It is plau- 56
sible that microorganism s may affect agaropecti n more
read ily than agarose. Agaropectin may also serve as a con
taminating source of carbon compounds providing D- and
L-galactose, D-glucuronic ac� d , and 3,6- anhydro-L-galactose by hydrolys is of itself or agarose (12, 48).
Based on the preceeding results it was decided to determine catabolic metabolic activity of the isolates in broth medium to eliminate the erroneous res ults that may occur with solid medium. Glucose was added to HB broth as
the sole source of carbon and ene rgy at a concentrat ion of
20 pg/ml . The growth obtained from th is concentrat ion would be indicat ive of the growth expected from a similar concentration of carbofuran. No growth occurred when carbof uran was the sole source of carbon and ene rgy in HB broth at a concentrat ion of 150 pg/mi (Tab le 2) . Good growth was obtained with glucose as the sole source of carbon and energy and sli ght growth was obtained even in the ab sence of a carbon and energy source (Table 2). Acti- nomycete spores contain an intracellular reserve of trehalose wh ich may be used for ene rgy to initiate germ ination under the correct cond itions ( 23). Th is may offer· an explanation as to why t here was slight or questionable
·growth in HB brot h not containing a source of carbon and ene rgy and in th e HB broth supplemented with carbofuran
(Ta ble 2). T�b le 2. Growth of Isola tes In broth med ia conta lnlng · ve r lous carbon and- energy sources .
Nutr ient Broth HB* HB HB Iso late with Glucose HB Cerbofuran Ce rbofu ren Coda Carbofur an** Broth** Broth Broth** Broth***
NAI NA s 1 + 511 5 NA + + - I NA g + + - 16 NA + + - · S s s 17 + + 22 s NA + + 26 + + s St reptomyces p IIosus s + + NA
lt.ttner Beee I Hed lu• • '• 388 1'1 · ** SUpp leMnted with cerbofuren o·r alucoee/•1. SUpp le.. nt ed _with t58 pa cerbofuren/•1. *** Ul I NDt epp llceb le beceuee teet not perfor�d. ""-J II Sl ltht or queet loneb le arowth. 58
SCREENING FOR INCIDENTALLY METABOLIZED PESTICIDES
One objective of these experiments was to screen
isolates for their potenti al use for environmental pesticide waste disposal. The refore the concentrations of pest icide used in these screening studies are considerab ly higher than what occurs for norma l field applicat ion rates .
Twenty-three filamentous and nonfilam entous bac teria were scr eened for their ability to degrade dicamb a, carbofuran and alachlor incidentall y . As a group , �lachlor was the mos t susceptible toward degradat ion and dicamb a was the most recalcitrant (Figure la, lb, lc) . Six of the iso- lates reduced the alachlor concentrat ion to less than one pg/ml , and 13 of the organisms decreased the concent rat ion to 36 pg/ml or less after one month of incubat ion
There was no appreciable amount of dicamba degra dat ion by any of the isolates (Figure la, lb, lc). Dicamba contains two ch lor ine atoms on its ring , wh ich offers an exp lanation for it's recalcitrant behavior . Arylhalides are commonly considered to be chemically ine rt to nucleo phi llic diplacement react ions (20). Because of the hig h activation energy �equired for displacem ent of the halogen it is unlikely that bacteria have evolved adequate pathways for direct remo val of the halogen atom. More likely what happens is that bacteria use other enzymes to break open 1 40 - -- LEGEND
1 20 000 " Alaohlor Q � c 888...... !18 1 00 :.- c Dlcamba ...0 R &E 80o ·� Furadan
• - -· :J 60 :2 ! 40 � 20
0 I "\ffY 'flY "\ffY- Control 1 2 4 5 7 9 Isolate Code
Figure 1a. Screening for pesticide de�radation by bacteria after four weeks incubation . 100% = 104 pg/ml alachlor, 134
pg/ml dicamba , and 75 pg/ml carbofuran. U1 \0 · ------.;,;,..__;______140 , --- LEGEND
1 20 0 " 000 " � Alaohlor Ot Ill �... �.. c • • 8... II! 1 00 c i mmml Dloamba ... �� 80 � Furadan
• :I '"0 60 . 1 Q: 40 � 20
0 I n 16 17 19 Isolate Code
Figur� lb. Screening for pesticide degradation by bacteria after four weeks i�cubation . 100% = 104 pg/ml alachlor , 134 ()\ pg/ml dicamba , and 75 pg/ml carbo furan. 0 140 LEGEND
...... 1 20 aao � Alachlor at ...... , ...... a1 c 888 0 ... · i c 1 00 a ... i milDlcamba u ·�
E• 80 i Hi Furadan � � • :J , 60 i IX 40 � 20
1 ! 0 • Control20 22 23 2 4 26 28 31 pll. S. Isolate Code
Figure 1c. Screening for pesticide degradation by bacteria
after four weeks incubation . 100% = 104 pg/ml alachlor , 134 0'1 pg/ml dicamba , and 75 pg/ml carbofuran. � 6 2
the aromat ic ring producing nonaromat ic metabolites with a
less stable carbon-halogen bond (20).
Isolate 24 was the only isolate capab le of degra
ding carbof uran to 48 pg/ml or less, as shown in Figure lc.
Two isolates that degraded alachlor to less than 1 �g/ml
were chosen for further study .
DEGRADATION OF ALAC HLOR IN COMPLEX , SEMI-DEFINED , AND
DEFINE D MEDIA
The degradation of alachlor by Isolates 5 and 26 in
complex (TY g) , semi-defined (HB + 0.05% yeast extract), and
defined (HB) was determined . Th is stu dy gave an indicat ion
of the nutritional requirements of these actinom ycetes for
degrading alachlor . A field isolate, identified as
Streptomyc es pi losus was included in the study for com
parat ive purposes .
The three isolates demo nstrated success ive ly less
growth in the semidefined and defined culture media than
An uninoculate d control, t�e com plex med ium (Tab le 3). depicted in Figure 2, shows that the leve l of alachlor
remains constant, except for variations due to experimental
error . The se differences we re determined not to be stat is-
- tically significant . Streptomyces pilosus did not reduce
the alachlor concentrat ion in any of the culture media
The differences noted were due to experimental (Figure 3). Tab la 3. Mi llig rams of myc al l�l . blo�ess harvested from each cu l ture fla sk fo llo wing degradat ion of ele ch lor In three types of cu lture med ia.
Is.o eta I Coda Comp lex Sam ldaf lnad Def ined ' i
5 119 79 39
26 89 . 59 69 St reptomyces p IIosus 89 89 49
(7\ l.tJ 1 20 r-�------�------UEDIUU
1 00 A + . a Complex E . ::s • g <> .A------· ------1 ...E ------80 --- __. __.----- __. 0)( :2 ------:- I D Deflneci+YE 't o 60 8 -- � 8
•
• 40 <> :I -1- Defined , ... 0:I 20 .
0 �--�----�----�--�----� 0 3 6 9 12 15 Time Days In
Figure 2. Degradation of alachlor in · complex (tr yptone yeast extract) , defined+YE (Hutner 's basal + 0.05% yeast extract) , and defined (Hutner 's basal) media . Uninoculated (J'\ .p. control. 100%= 106 pg/ml alachlor. 120 �EDIU�
• /::a Complex 1 9 () ¢ E::J o 90 - ¢ - -- - ... 0 a - EX 0 --� � rz���x-�-o����----- 0 Deflned+YE /::a .... 0 0 60 � .. CD ::J <> Defined -o -- (1) 30 CD 0::
0 ... + -+ ... 0 3 6 9 12 1 5
Time In . Days Figure 3. Degradation of alachlor in· complex (tryptone yeast extra ct) , defined+YE (Hutner 's basal + 0.05% yeast
extract), and defined (Hutner 's basal) media by Streptomyces (J'\ pilosus. 100%= 111 pg/ml alachlor. V1 66
error and were determined not to be sta tisticall y signif
icant . Apparent ly, the S. p ilosus isolate does not have
the me tabolic diversity needed for the deg�adation of ala
chlor. A i achlor degradat ion by Isolate 5 was determined to
be aignificant at the p= 0.002 and p� 0.0 01 levels in the
complex and semidef ined med ia, respectively (Figure 4).
Degradation of alach lor by Isolate 5 in the complex medium
had decreased the concent ration to less than one �g/ml by
the sixth day, and to two pg/ml by the tenth day in the
semidefined medium . Correspond ingly , Isolate 26 was not
ab le to decrease the herbicide leve l to below ten �g/ml for
the duration of the trial in complex medium (Figure 5).
Th is degradation wa s cons idered significant (Figure 5).
Alac hlor degradation by Isolate 26 w as significant in the
chemically defined cul ture medium (p=0.00 9), but not signif-
icant in the semidef ined culture med ium (Figure 5). Th is was considered unusua l since it was expected that the addi
tion of yeast extr act would provide needed vitamins and cofactors wh ich would enhance the growth , and thus alachlor degrad ati�n. Under the condit ions imposed by this stu dy
raded alach�or more qui.ckly an d extens iv Isolate 5 deg ely than did Isolate 26 in complex and semidefined media.
Apparently, if the media l·acked required growth factors alachlor degradation did not occur for Isolates 5 and 26. It i� plaus ib le that this occurred for either of 120 A �EA DIU� r, l Complex 100 [] E [] .<> <> :J � <> <> ...E ,,._ <> X 80 0 0'<>' <> \ " � " Deflned+YE \ [] Y=-1 0.285X + 1 05.0SO [] 't- I \ �D p<0.001 0 60 t \ " r=-0.891 ·-- � \ A " .. CD \ " 40 " <> Defined -o:::J =-18.108X+111 488 I· ... " � \ p=0.002 (I) " CD \ r=a0.965 [] " � 20 \ " \ " " 1::. ' " I • c O l 0 3 6 9 12 1 5
Time In Days Figure 4. Degradation of alachlor in complex (tryptone yeast extract), defined+YE (Hutner's basal + 0.05% yeast
extract) , and defined (Hutner 's basal) media by a-. Streptomycete Isolate 5. 100%= 136 �g/ml alachlor. "'"-J 120 r-----�------�EDIU�
100 ·� A Complex t A I E c ::J c
-- EX 0 80 ' � ' Deflned+YE ' -- [] 't- ' � -- c.. _. T ' 0 60 . a-8.9-41X +85.1 · Ia [] ft!:Strl IHI... ----- ·-· - pa0.002 ' a � ' .. r=0.822 CD ' A ' 40 <> Defined :::J ' I "tJ-- ' A ' CDen � 0:: ' ·20 . A ' ' A A ' A ' ' 0 0 3 6 9 12· 15
Time In Days Figure 5. Degradation of alachlor in complex (tryptone yeast extract), defined+YE (Hutner 's basal + 0.05% yeast extract), and defined (H utner 's basal) media by Steptomycete ()'\ Isolate CX> 26. 100%= 126 pg/ml alachlor. 69
the follo wing reasons :
1. A min imum concentrat ion of enzyme needs to be
produced. As overall growth (biomps s) of the iso-
lates decreased, so does the enzyme concentration
unt il eventually alachlor degradation did not take
place.
2. The medium becomes limiting and nonessential enzyme production sto ps and/or can not continue .
Th is · type of degradat ion is typical of incidental
degradation (48). Inciden� al degradat ion is
dependent upon the activity of microorganism� , and
the activity of microorganisms is generally en
hanced by the addition of glucose or in nutritious
media.
PLASM ID ISO LATIO N
No plasmid DNA was detected in Isolates 5, 16, or
26. A plasmid has been reported in Streptomyces £ilosus
(25) and this organism was included as a posit ive control
for the plasmid scr eening protocol. Plasmid DNA was detected much more rea4ily with the polyethylene glycol
(PEG) procedure than the alkal ine procedure as depicted in
The PEG procedure utili�es a high osmot ic broth Figure 6. med ium (34% sucrose) in wh ich stable protoplasts are
form ed , and then lysed by the addition of sod ium dodecyl 70
Figure 6.- Rapid plasmid DNA screening of Isolates using either an alkaline or polyethylene glycol (PEG) procedure . From left to right: HIND · til STD , � · pi losus alkal ine , �· pilosus PEG , Isolate 5 PEG , Isolate 5 alkaline, Is6late 26 alkaline , Isolate 26 PEG , � · pilosus plasmid isolated · by cs-C l-et hidium bromide centrifugation procedure. 71
sul.fate (SDS) detergent . The alkaline lysis procedure
rel ied more on the ability of lysozyme to gene rate protop lasts , wh ich are then disrupted by SDS . If proto- plast form ation is impeded in any way , lys is would be
inadequ ate. This would reduce the amount of DNA available
for detection during electrophoresi s . Use of a high os- mot ic broth medium seemed to be more cons istent in detect ing the plasmid. present in S. pilosus .
METABOL ITE ISOLATION
The fate of the alachlor molecule after degradat ion
.by these isolates is not known . A stu dy was undertaken to determine if the arom aticity of the compound was maintained or lost.
There were many · quenched spots on the thin layer chrom atog raphy (TLC) plate not present in the control, indi cating the spots had orig inated fro m the alachlor metabo- lites . This quenching indicates the arom atic nature of the ring was retained for sever al of the metabolites.
These findings are similar to Krause et al. , and Tiedje and
Hagedorn who independentl y observed . that metabolites they identified in studies on alachlor and me tolac hlor, respec tive ly, retained the arom atic ring (40 , 71). Krause et al. identitied me tab olites produced from metoalachlor by ben zylic hydroxy lations of the ara lkyl side chains and/or 72
demethylations at the N-alkyl subst ituents (40) . Tiedje
and Hagedorn reported four identifiable metabolites pro
duced from alac hlor ; three of them produced by N-dealky l
ation reactions occurring on the parent molecule molecule
( 7 1 ) •
The se metabolites had smaller R f values than the parent mo lecule, indicating tha� the biotransform ation
produced a more polar compound . The sym bol R f stands for - "ratio to front" and is expr essed as a deci m al. Mathemat -
ically it is expr essed as R f = distance traveled by sub stance divided by the distance traveled �y the solvent
. front (55). The me tabolites identified by Krause et al.
and Tiedje and Hagedorn were also more polar as determined
by GC retention times (40, 71).
ALAC HLOR DEGR ADAT ION IN SOIL
That degradation occurs in broth us ing pure culture
techni ques does not mean degradat ion occurs in soil in a
similar manner. It is pla usible that _ in a soil slurry
growth could be limited and thus alachlor degradation would be affected . This may b� due to limited availability of oxygen in a soil slurry , or the soil not providing ade quate nutrients for growth .· The preceeding alachlor degradation studies were performed in broth mediu m with the her bicide
concent rat ion ver y near its solub ility level of 140 �g/ml 73
( 74 ) · . The availability of alachlor in soil degradation
studies is determined by the sorption coefficients between
soil water and soil organic matter (29, 64). This stu dy was conducted with the following objectives using Isolate 5
as the test organism.
1. To determine if alachlor degra dation could
indeed occur in a soil slurry mixture by a
microorganism that degraded alachlor in a
brot h medium.
2. To generally �etermine at what alachlor con
centration lev els any observed degra dation
occurred .
STERILE SOIL: Th is �tudy involved comparing alachlor degradat ion of Isolate 5 in sterile and nons terile -2 soil. No colonies devel oped on a 10 dilut ion from autoclaved soil indicat ing that aut oclaving reduced the number of organisms / gram of soil to less than 100 . A
x 6 microb ial count of 5.9 10 bacteria/gram of soil was enume rated in the nons terile soil. It was . ass umed , the re-
fore , that autocla ving the soil was effective .
ALAC HLOR DEGRADATIO N IN SOIL : Soil was spiked to achieve about 550 pg/g and about 3joo pg/g alachlor to determine what level of the herbicide could be degraded , if at all; by Isolate 5. Degradation was also monitored in a soil slurry where the soil was contaminated with approx- 74
· imatel y 4000 �g/g alach lor from an alachlor chemical s pill.
The slurry containing the higher concentratio� of spiked alachlor and the ch�mical spill slurry did not show any significant degradation (Figures 7a, 7b, 8a, and 8b) .
It is plausible that the pest icide was toxic at these con centrations or not enough time was allowed for degradation to occur . The spiked soil contain ing the low concentration oE alachlor did show a los s of parent compound in some of the flasks as depicted in Figures 9a and 9b. Signif icant degra dation was observed i� the sterile slurry supplemented . with glucose inoculated with Isolate 5 (p= 0.004) and the sterile slurry not supplemented with glucose but inoculated with Isolate 5 (p= 0.046· ). Signific ant degradation occur- red in the nonsterile slurry supplemented with glucose (p=
0.00 9). Th is slurry was not inoculated with Isolate 5. In other words , alach lor degradation was mediated by Isolate
5. Degradation also occurred due to the indigenous popula- tion when supplemented with glucose� Apparently the soil harbors bacteria capab le of degrading the alac hlor , but requires some enrichment for this to occur. Only when supplemented with an addit ional carbon and ene rgy source were they capabale of degrading the herbicide. These microorganisms apparantly naturally produce enz y mes that are capable of degrading alachlor , but need to have an exog enously supplied source of carbon and energy to stimulate 120 . TREATMENT
A IIN +GW 100 a <> ll. . E:J 0 liN ...EX 0 80 TO � 0 - 't <> IUNIN __ --- �------...... _ o 60 - ... - . u -0ll. A .,U_ - --a-- ($" - - - - - c <> o � • o a a • ' A CD.. il. A :1 40 <> , ... CD., 0:: 20
0 . . 0 30 60 90 120 Time In Days
Figure 7a . Alachlor degradation by Isolate 5 in a soil slurry supplemented with alachlor. 100%= 3732 pg/g ...... , alachlor. S-sterile ; IN-ino culated ; UNIN-uninoculated ; V1 Glu-glucose. 120 TREATWENT
+ 1 NSUNII 100 * E :J + + I X -- I NSUNIN+GI.U EX 0 s :;i o + r, · + 't- _,... - ...... - --- 0 60 t\ - + � \ * + CD.. + 40 + + :J + �, -- tx + "'0 I ·· CD., 0:: 20 +
. 0 + . 0 30 60 90 120
Time In . Days
Figure 7b. Alachlor degradation by Isolate 5 in a soil slurry supplemented with alachlor. 100%= 3732 p g/g -....J alachlor. NS-not sterile; UNIN-uninoculated ; Glu-glucose. "' 120 TREATMENT
A II
T b. c 100 I! c E::J A. A 0 II+GW ...E - X 80 ----- 0 c ---- t --- - c A ------� _ _ _ - ---�.. - � • 0 't c A 0 o 60 t . b. c A 0 � .. A Q) 40 A ::J -o... en Q) 0::: 20.
- 0 . -- '. - 0 30 60 90 1 20
Time . In ·oays
Figure Sa . Alachlor degradation by Isolate 5 in a soil g/g ...... slurry from an agriculture chemical spill. 100%= 3979 p ...... alachlor . IN-inoculated ; GLU-glucose .
'\. 120 TREAntENT
<> UNII CTRL I + E <> ::J + <> . I + .... + + UNII + GW E -- X --t + 0 80 ±------<> � <> + <> .... 0 60 f • + <> � <> 41' CD I · 40 ::J "0... (I) G) 0::: 20
_I. . 0 __._ --.- 0 30 60 90 120 Time tn Days
Figure 8b . Alachlor degradation by Isolate in a soil slurry 5 from an agriculture chemical spill. 3979 p g/g alachlor. 100%= ""-.J UNIN-unin6culated ; CTRL-control ; GLU-glucose. (X) 120 - TREATYENT
. /::a liN + GW 100. [] l::a ·E ::J <> ... <> [] SIN EX 0 80 � <> � A [] <> <> SUNIN .... 0 0 60 <> - <> <> SIN <> � � ' , ?ri�:--�0--� , Ya-0.283X+82.87-4 .. ' A Iii . . ' ' 0 - p=0.048 Q) .40 D , - 0 ::J . - - ' , -Q_ --- . -- i - , SIN +GLU , - �::a .... -f=I0;512._- (It l::a .... D Q) . Y•-0.523X+8.1.518 /::a ' 0:: 20 p-0.004 /::a .... [] ' t:...... r-0.738 ...... /::a /::a �----�------� 0 -- � � 0 6 120 30 0 90 Time tn Days
Figure 9a . Alachlor degradation by Isolate 5 in a soil slurry supplemented with alachlor. 100%= 570 pg/g alachlor. " S-sterile; IN-inoculated ; UNIN-uninoculated ; GLU-glucose. \0 120 TREANENT
+ NSUNIN I 100 X + E:l + I X ...E NSUNIN+GW X 80 + 0 + -- ---*- � T ...... + 0 + ...... X .. + ...... 60 ...... + + ...... � x ...... + � x X ...... CD "')( ...... _ . X ...... :l 40 X ...... X ...... •· -o...... CDen ·x NSUNIN+CLU ...... � ...... X � 20 ...... Ya-0. 433X+87 .228 p=-0.009 r=0.893 0 0 30 60 90 120 Time In Days
Figure 9b . Alachlor degradation by Isolate 5 in a soil slurry supplem ented with alachlor . 100%= 570 pg/g alachlor. 00 NS-not sterile; UNIN- uninoculated ; GLU-glucose. 0 81
nonspecific and general metabolic activity of soil micro-
organisms . This is similar to the model of inciden�al
metabolism propose d by Matsumura (48). Matsumura states
that a characteristic of incidental metabolsim is that it
1s increased by the add ition of glucose to the system.
That no degradation occurred with alachlor at a concen
tration of 3000 pg/g or _ greater are similar to those re
ported by Junk et al. (34). Junk et al. reported that no
degra dation -occurred in soil containing 4000 pg/g alachlor wh ile alachlor was degraded to less than 10% when the soil
contained 200 pg/g alachlor (34). Junk reported that the
extent of alachlor degradation was enh anced by addition of
0.01% pept one . These findings are also similar to those of
Matsumura and others who report that pesticide res idues may
persist for longer than norma l field application degrada
tion rates when high concentrat ions are encountered (3,
48) .
TENTATIVE .TAXONO MIC CHARAC TERI ZAT ION OF ALACHLOR
DEGRADING AC TINO MYC ETES
CEL L WA LL ANALYSIS : The order Actinomyc etales - conta in� bacteria that form branching filaments, and in some families these deve lop into a myc elium (11). An aid to the cla ssif ication of these organisms is chemical anal- ysis of their cell wal ls . Cell walls of all families 1n 82
the Act i nomy cetales order fall into one of four categories
(Types I, II, III or IV) dependin g on the presence or ab-
sence of diaminopimelic acid (DAP ) isomers (7, 11). Type I
cell wall contains LL-DAP and 1s characteristic of gene ra
in the famil y Streptomy cetaceae, wh ile cell wall types II ,
III , and IV contain mes o-DAP and is characteristic of
gene ra in the families Micromonosporaceae, Act ino
my cetaceae, and Nocard iaceae. Strept o myces species form
well developed branch ing and aerial mycelia (7 , 11). This
aerial myc � lium typifies the str uctura l features of the
organism durin g sporu lat ion and is an important criterion
in the identification of Streptomyces spp . Nocardia spp.
occasion ally pro duce aerial spores, wh ich then makes di·f fer
entiat ion between the two difficult.
DAP IS OME RS IDENTIFIE D: LL-DAP migrated the
furthest on the chromatogram , wh ile DL(meso)-DAP lagged behind LL-DAP by approxim ately 3.5 em for a TLC run of 16 hours . Steptomyces cacaoi and Nocardia autotrophica, wh ich we re used as sta ndards , had dist inctly different DAP iso- me rs . Isolates 5, 16, and 26 were shown to contain LL-DAP wh ich bel ongs to cell w� ll Type I, and theref ore these organisms were members of the gen us Stre ptomyces .
MELANIN PIGMENT: These three isolates (Isolates
5, 16, and 26) produced a dark brown me lanin pig ment wh ich is also characteristic of some Streptomy ces spp . Isolate 83
5 and 16 produced aerial mycelium characteristically wh ite
in appearance, while Isolate 26 produced a gray aerial mycelium .
CARBOHYDRATE UTILIZATION: Isolate 5 was able to / use a wider variety of carbon and energy sources than either Isolates 26 or Streptomyces pilosus (Tab le 4).
Tentative placement of these isolates into a species - cla ssification was not possible with the data collected.
The carbohydrate utilization study was sufficient to conclude that Isolate 5 and 26 are not the same micro organisms . 84
Tab la 4. Summa ry of Texonam lc Cher ectar lstlc s far Alech l or Degred lng Bectar le.
/
ISOLATE ISOLATE 5 26
Hutner's Bese l (nag contro l) A I ech I o ·r 2-Am lnaethena l + Arab inose + +
Bate lne + Ca l I ob I ose +
CItr Ic Ac Id +
Fructose +
Glucose + +
Lactose + Me l onete
Mann itol + Oxe lete
L-Pro I I ne + +
L-Tyros lne +
Sa rcos ine +
Succ inate +
D-Sa rb ltol +
Sucrose + +
Xy lose + . 85
CHAP TER 3
PESTICIDE DEGRADATION BY BACTERIA ISOLATED FROM AN
AGRICULTURE CHEMICAL SPIL L /
MATERIAL S AND METHODS
The objective of this study was to determine the
ability of bacterial isolates obt ained from an agricultural
chemical spill to catabolieally or incidentally metabolize
alach lor a � d/or trifluralin . Catabolic metabolism was de-
termined by inoculating each of the microorganisms into a med ium containing the pesticide as the sole source of
carbon and energy . If growth occurred , this would indicate
that the pest icide was serving as a carbon and ene rgy
source for the mic�oorganism. If no growth occurred , but
the pest icide was degraded in the pre sence of an alt ern ate
carbon and energy source such as glucose, th is would be an
indication of incidental metabolism.
Tech nical grade alachlor (Lasso) and trifluralin
(Treflan) were used in _ the following degradation studie� .
Alachlor and trifluralin are her bicides with molecular weights of 269 .77 and 335. 29 re spectively (74). These pesticides are hydrophobic and therefore limited in their 86
water solubility . Their solu bilities are _140 and 24 g/ml p in distil led H 2 o, respectively (74).
DETERMINING CATABOLIC METABOLISM AC TIVITY :
Pesticide media were prepared using an inorganic basal salts medium that contained the pest icide as the sole source of carbon and energy . Growth . of the isolates in this medium would indiciate catab6lic metabolism. The basal- salts medium contained (per liter) 2.77 g, Na2 HP04 ;
2.77 g, KH2 ro4 ; 1.0 g, NH4 Cl; 20 ml Hutner' s vitamin-free mineral base; pH=6 .8 (30). _ Th is was referred to as Hut- ne r' s basal (HB ) medium . Fifty mls of HB broth was dispensed into 250 Erlenmyer flasks and sterilized by auto claving for 20 mins . at 121° C and al-lowed to cool . . Sterile pesticide was then added as the sole source of carbon and ene rgy . Stock solutions of pest icides were made by dis- solving tech nical grade alachlor and trifluralin 1n 95% ethanol. Th is was filter sterilized (Ge lman , 0.2 and p m) kept re frigerated unt il used . Alachlor and trifluralin were added as the sole sourece of carbon and energy to HB media to acheive a final concentration of approxim atedly
150 and 25 pg/ml , respectively. Isolates HA- 001 and
H 2 0-002, obtained from a previous study in this laboratory, we re inoculated into each HB medium containing alachlor or trifluralin to determine their catabolic activity. HB medium containing neither of the pesticides was inoculated 8 7 .
and served as a negat ive control. The aff�ct of degra-
dation, if any , by a mixture was assessed by inoculat ing
these two isolates as a mixture into each of the pesticide
containing mediu m. In additi on , the mixture of HA-001 and
H 2 o-oo2 were inoculated into HB broth containing no carbon and energy to serve as a negative control. Each of the
media were inoculated with 100 pl of a washed cell suspen-
sion of the isolates . The washed cell suspension was pre-
�n pared by growing the cells over night (18 -24 hrs . ?
nutrient broth ( Difco) . One. ml of each isolate was centri-
x fuged for 10 min (10,0 00 g ) in sterile 1.5 ml polyproply- ene microcentrifuge tubes . The bacterial pel let was
resuspended in HB broth and washed once more by centri
fugation and resuspended in 1.0 mls of HB brot h . The media
containing either Isolates HA-001 or H 2 0-002 were inoc ulated with 100 �1 of each isola te, wh ile the media contain
ing the mixture of isolates were inoculated with 50 pl of
each isolate to obt ain the same approx i m ate beginning titer
of cells as the former media. After inoculat ing these ° media we re incubated at 26 C on a gyratory shaker ( New
Brunswick Scient ific Company) at 130 rpm . DETERM INATI ON OF GROWTH ·: If the pesticide was catabolically metab olized , an incre ase 1n bio mass would
An increase in viable cell counts of each Erlen- meyer flask was considered an indication that g£owth 88
occurred . The initial bacterial titer of �ach flask was determine d by withdrawing one ml from each of the flasks after inoculating with the washed cell suspension . Each of the sam ples were diluted serially by ten-fold dilutions and
0. 1 ml was plated onto duplicate nutrient agar plates
(Difco) . These plates we re incu bate d at 26° C for 48 hrs and enumerated. Subsequent samp les we re taken at specific intervals to determine the viable cell count of the flasks .
PESTIC IDE RESIDUE ANALYSIS: Pesticide res idue concentrat ions were quanti fied by gas chromatography (GC) .
A decrease in pesticide res idue levels occurring with a concomitant increase in bacterial titers would be an indi cation of catabolic metabolism of alach lor by the isolates .
To determine the initial residue concentration , one ml samp. les were withdrawn from each of the flasks containing the pesticide at the time of inoculat ion. These samp les typically were frozen at -20G C and analyzed by GC when time permitted . S�bsequ ent samples we re taken at specific time intervals and quantified by GC . If alachlor was being cata bolically metabolized a decrease in the alachlor concentra tion would be observed . . An indication of incidental metabolic activity was determined by the addition of an altern ate carbon and ene rgy sour�e to each of the media con- taining alachlor . Membrane filter (Gelman 0.2 pm) steril- ized glucose was added to the med ia to achieve a fin al 89
concentration of 0.1 %. After addition of the glucose, the
flasks were incubated an additional seven days , at wh ich
time 1.0 ml was removed and the alachlor concentrat ion was
determine d by GC .
Alachlor was removed from the broth samp le by
solvent extr action using two - five ml ethyl acetate
(reagent grade), depending on conditions of the chromate-
graph . This was vigorously shaken to ensure that all
alachlor was extrgcted into the solvent . This was quanti
fied using a Varian 3700 gas chrom atograph fitted with a
fla me ionization detector (FI D). The GC was configured with a 6' x 4 mm internal diam eter (id) colu mn and packed with 3% OV-101 adsorbent on Chromosorb W -HP support medium .
The oven tem perature was maintained between 200-225° C, the
injector temperature held at 230° C, and the detector temper ature operated at 350° C.
Trifluralin res idue leve ls were not determined .
The trifluralin had gone out of solution and had conglom erated on the bottom of the Erlenmeyer flask , giving a nonhom ogeneous solution.
Based on the results of these preceding studies , it was deemed necesary to determine the incidental metabolic ability of these isolates and others obtained from the same ch�mical spill. Th is was carried out by deter mining if decreases in alachlor concentrations occurred during growth 90
of the isolates· on an alternate carbon and ene rgy source other than alachlor .
SC REENI NG ISOLATES FOR ALACHLOR DEGRADATION BY
INC IDENTAL METABOLISM: Based on the results of the / preced ing study , a stu dy was conducted to deter�ine the incidental metabol ism of alachlor by these isolates . This was achieved by preparing pesticide media that con�ained a growth substrate (gl ucose, 0.3%) other than the pesticide for a carbon and energy sou rce. After incubation of the media with the isolates , pest icide res idue levels were determined by gas chrom atog raphy as just described. De creases in alachlor res idue levels would be an ind ication of incidental metabolism.
PES TIC IDE-CONTAI NING BROTH MEDIA :
Tryptone-yeas t extract broth suppleme nted with 0.3% (w/v) glucose a.nd with 100 pg/ml alachlor was used as the culture med ium for determining incidental met abolism of the pesticide by ihe bacterial isolate� . A stock solut ion of alachlor was prepared as described above and aseptically added to autocla ved and cooled TYE broth media to a fina l concentrat ion of appro� i m ately 100 pg/ml . Glucose was filter sterilized and added to the medium at a final concentration of 0.3% (w/ v). Glucose was . added to ens ure rapid growth of the isolates . Five mls of th is pest icide 91
broth media were aseptically transfe rred t·o sterile 16 x
0 1 50 mm tubes , · capped and stored at 5 C .
The isolates from the chemical spill were main- tained on TYE broth that was not supplemented with pesti- / cide. Isolates H 2 0-001, H 2 0-002, HA-001, HA-002 ,
HA-INOC-001 , HA-INOC-002, H 2 0-INOC-001 , and H 2 0-INOC-002 (obtained from a previous study) were inoculated into the pesticide containing broth media with one drop from a pas- teur pip ette from 24 hr TYE broth cultures of · each of the isolates obtained. A one ml samp le was withdrawn from each of the freshly inoculated tubes to be used in determining the initial concentrat ion of alach lor by GC . These inoc- ulated tubes were then incubated for four weeks on a tube
0 roller at 26 C. After incubation one ml was withdrawn for use in determining pos t-incubat ion alachlor concentrations .
The alachlor concentrations were determined by GC as des- cribed above . Decreases in pos t-incubation alachlor resi- due levels from the initial alachlor leve ls would be an indication of incidental me tabolism.
PLASMID DNA ISOLATION
All of the isolates were scr eened for the presence of plasmid DNA using various scr eening procedu res (5, 32,
35 , 49 ), and total DNA was isolated using an ethid ium dye-isopycnic cesium chl oride gradient procedure (43). 92
STATISTICAL ANALYSIS
Statistical analyses (regression and T-test) were performed on an IBM-PC fitted with and 8087 Numeric Data I Processor using the Stat is tics Program , SYST AT (73). 93.
RESULTS AND DISC USSION
Significant growth occurred fo� Isolate HA-001
(p= 0.03), and the mixture of Isolates HA-001 and H 0-002 2 /
(p= 0.02) in the medium containing alachlor as the sole source of carbon and ene rgy (Figure 10). No signifi cant growth occurred with Isolate H 2 0-002 in this medium (Figure 10) . · It seems reasonab le to assume that the increase in titer of the mixture is due to Isolate HA-001. No signif- icant growth occurred with � either of these two isolates separately or as a mixture �n the basal medium not contain ing a carbon source or the basal medium containing triflur alin (Figures 11 and 12). These two media showed a trend for the titers of Isolate HA-001 and the mixture of Iso lates HA-001 and H 2 0-002 to sli ghtly increase with time , wh ile the titer of H 2 0-002 remained relatively constant . It seems like ly that the incre ase in titer of the mixture may be due only to the increase of Isolate HA-001. Th is trend was not stat ist ically significant .
Alach lor levels significant ly decreased �n concentrat ion in the media that were inoculated with Iso- lates HA-001 (p� 0.00 1), H 2 0-002 (p= 0.02 5), and the mixture (p=0 .009) of these two isolates (Figure 13). �------���----� 9 ISOLATE ID
A . HA-001 <> I
8 I �
L " , <>A - & ...• , -- .,. Y•0.07.tX+I• .,. A .t53 c H20-002 t- , I p•0.021 .,. .,. 0 , r-0.0.180 , Y•O.OI5X+I.317 .... " I- 7 t , - <> , - p-0.033 , , 0) 6 r=aO.U7 : .3 <> MIXTURE �� - --- - I � - c - ..._, ______------. ------6 D t c c
5 �----�----�----�------�----� 0 5 10 15 20 25 Time In Days
Figure 10. Growth of two isolates obtained from Brookings chemical spill in basal medium with alac hlor as sole source \0 of carbon and energy . +"
' �------� 9 ISOLATE ID
A HA--OOf
8 A
.... • <> ...... <> D H20-002 .... A 0 � 7 A
0) <> <> MIXTURE ..9 c - �------o------C I 6 n
5 �----�----�----�------�----� 0 5 1 0 15 ·20 25 Time In ·Days
Figure _ 11. Growth of two isolates obtained from Brookings chemical spill in basal medium with trifluralin as sole 1.0 source of carbon and energy. Ln
' 9 ISOLATE. ID
A HA-001
A s '- I <> .! • H20-002 . J= c 0 ...... ' 7 , , , m <> .3 c <> MIXTURE
. 6 ------� ------A�-- --- 0
[]
5 �----�----�----���--�----� 0 5 10 15 20 25 Time In Days
Figure 12. Growth of two isolates obtained from Broo kings \0 chemical spill in basal medium not containing an add itional 0\ source of carbon and energy . 120 ISOLATE ID MIXTURE Y•-2.3UX + 1 1 0.-453 p•O.OOI A 0 H20-002 HA-001 1.00 Adg oo I ICI A , ' r-o.n2 · Y•-0.181 X+I2.255 0) � ' A . t: -- 00 p•0.025 ... --. -- 0 c � """'- -- ...... r-0.81&<> ... ' 0 0 80 0 ' -- 0 -- ' ' -- 0 ------. -- c H20-002 ECD ' ------I Q! ' ' ' 60 ' ·-- CD ' --- ::J HA-001 ' "0 Y•-2.110X+101.71t -- • ' ., ' p 0 �------+------�------�------� 0 7 14 ' 21 28 Time fn Days Figure Alachlor degradation by two isolates obtained 13. . from Brookings chemical s pill. The basal medium contained alachlor as sole source of carbon and energy . Glucose added to fina l concentration of on day pg/ml \0 0.3% 21 . 100%= 156 -....! alachlor. " 98 Glucose was added on day 21 and then incubation carried out an additional 7 days , at wh ich time a sample was taken for GC analysis . Much of the decrease in a1achlor occurred during this final 7 days of inc� bation . It is likely that / the significance of the decre ase 1n alachlor concentration is due to the decrease in alachlor concentration wh ich occurred after the addition of an alternate carbon and ener- gy source wh ich the organisms were able to use for growth . Had the glucose not been added a significant decre ase in - ala chlor might not have occurred. Matsumura states that the incidental metabolic activity of microorganisms 1n- creases with the addition of a readily availab le carbon and energy source (48). These results seem to be in agreement with Matsumura. Each isolate fro m the chemical spill degraded alachlor inciden tally, although to varying extents (Tab le . 5). In t-test anal ysi s , the post-incubation alachlor con- centratioris as a who le had signifi�antly decreased from the pre-incubation concentrat ions (p� 0. 001 ). Five of the iso- lates degraded alachlor to less than 40% of the beg inning concentrat ion (Tab le 5). PLASMID DNA ISO LATIO N: CsCl cen trifugat ion gradi- ents yielded only one band of DNA wh ich in�icated only chro- mosomil DNA was pres ent . This correlated with the results of the scr eening procedur es wh ich indicated that plasmid Tab le 5. Alech lor degradat ion af�er four weeks by eight becter le Isola ted from en agr icultura l chem ical· sp i ll. Iso l ate· Pe rcent Alech lor Code Res idue Remain ing * HA-BB1 22 .7 HA-BB2 35 .2 H2B-BB1 34 .1 H29-992 89 .4 HA-INOC-BB1 37 •5 HA-I NOC-992 87 .1 H29-INOC-991 38 .7 H29-I NOC-BB2 61 .4 1881 •que Ia 93 • pal•'· \0 \0 " 100 DNA was not present . Apparently , any degra dation of ala- chlor is not dependant or mediated by plasmid DNA . It was deemed necess ary to isolate total DNA since it has been shown that certain Pseudomonas pla smids are not always detectab le us ing rapid screening methods due to their instability (72). 101 CHAPTER 4 ENRIC HME NT FOR MIC ROORGANISMS UTILIZING PESTICIDE S FOR CARBON , NITROGE N, OR PHOSP HORUS SOURC ES. MATERIAL S AND METHODS If microorganisms are to evolve metabolic pathways for pest icides or recalcitrant compounds , they need to be stressed by high concentrat ions of that compound (54). Soils where pesticide spills have occurred would serve as stressed environm ents for microorganisms . It seems plau sible that these soils would have a higher probabiiity of harboring bacteria that have developed pathways to dissim ulate pest icides . Th is was the rat ionale for e �riching for these microorgani sms from pes ticide contamina ted soils . The enrichment med ium incorporated the pesticide serving as a carbon and energy , nitrogen, or phospho�us sou rce. Cam pacci and others have isolated bacteria by enrichme nt techniques capab le of ut ilizing pesticides as the sole source of nitrogen (14,. 17� 18) . Other laborator ies have isolated bacteria capab l� of ut ilizing pesticides as the sole source of phosphorus or carbon and ene rgy using similar techniques (19, 60) . The enrichment medium for phosph orus or nitrogen uti lizers would need to have an 102 additional organic compound added to serve as the carbon and ene rgy source, while the pest icide would serve only as a nitrogen or phosphorus source (17, 19) . These media would favor growth of microorganisms that had developed dissimulatory pathways for the p esticide. Other micro organisms present from the sample would eventually be eliminated due to their inability to utilize the pesticide as a nut rient . Eventually pesticide degrading bacteria would become the dominant population growing in the broth mediu m. ENRICHMENT MEDIA: An inorganic mine ral base was used to provide the ne cess ary mineral requirements wh ile being free of nitrogen, phosphorus , and organic comounds . A vitamin free min eral base referred to as modified But · ne r 's basal (MHB) medium was used . Th is consisted of (per liter) ; Na2 HP04 , 2.77 g; KH2 P04 , 2.77 g; modified Hutner' s vitamin free mineral base, 20 ml ; NH4 Cl, 1.0 g; pH=6 .8. Hutner's vitamin free mine ral base (30) was mod ified to be free of any nitrogen, phosphorus , or organic compounds . It contained per liter: MgS04 .7H 2 o, 1 4.45 g; Cac1 2 , 2.5 g; Feso4 .7H 2 0, 0.35 g; ZnS04 .7H 2 o, 0.5 5 g; MnS04 .H2 0, 0.077 g; Cuso4 .5H 2 0, 0.0196 g; Na2 B 4 o 7 .10H2 0, 0.0089 g; NaMo04 .2H 2 o, 0.0014 g; CoC 1 2 .6H 2 o, 0.010 g. The vitamin free mine ra l base formed a precipitate upon com bining the cons tituents · to distilled H 2 o. To dissolve th is , 6 N HC l was added 103 unt il the precipitate went into solution (10 -15 mls). The volume was brought up to 1.0 1 with distilled H 2 o and was sterilized by autoclaving (15 min, 121° C) . To enrich for microorganisms capab le of using the / pest icide as a carbon and ene rgy source, MHB was used as a basal med ium . Th is med ium did not cont ain a source of carbon and energy . Five ml of this med ium was aseptically transfered to sterile 16 x 150 mm glass culture tubes that had been washed thoro ugh ly and rinse d to remove res idual organic or inorganic comp�unds (18) . . This basal medium was used in enriching for microorganisms utilizing pest icides as a carbon and energy source. The nitrogen source (NH 4 Cl) was omitted from MHB medium when the pesticide served as the nitrogen source . Thus the only availab le nitrogen source availab le would be the nitrogen contained within the pesticide 's structure . Alternate sources of carbon and ene rgy were provided ; conse quently , any growth observed occurred because nitrogen came from the pest icide . Glucose, sod ium succin ate, and glycerol were filter sterilized by membrane filtration (0.2 urn Gelman Acrodisc) and added to the sterile base to achieve a final concent ratio n of 0.0 6% (w/v) . Th is medium was then aseptically added to sterile cul ture tubes that had been baked for 24 hrs . at 230° C to decom pose and volatilize any contaminating nitrogen salts present (2). 104 The phoshpate buffer was omitted from MHB and replaced with 50 mM tris C tris[ hydroxymethyl]aminome thane) buffer when enriching for microorga nism s capab le of using the pesticide as a phosphorus source. This enrichment base was prepared by subst ituting tris buffer for phosphate buf fer in the inorganic basal medium described above and adjust ing the pH to 7.2. After sterilization, sterile carbon and ene rgy sources were added as descr ibed above to a final concentrat ion of 0.06% (w/v) . This enrichment base was then aseptically adde� to clean �teri le cul ture tubes that we re �ree of conta min ating phophates . These tubes were cleaned by steeping the glassware in hot water for two �n hours , followed by a 24 hour immers ion 15% nitric aci d ( 6 0 ) • The nitric acid was removed by thorough ly rinsing in deionized and distilled water. Pest icides were added to the enrichme nt bases to achieve a final concentration of 0.1 % pesticide (w/v) . Tab le 6 describes the various enrichments (nitrogen, phos phorus , or carbon and energy ) performed for each of the pest icides . The enrichment bases that contained a source -3 of nitrogen (NH4 Cl) or phosphorus (P0 4 ) were supplemented with a tech nical grade pesticide to serve as the source of carbon and ene rgy . Tab le 6. En r i chments perfq rmed with ve r lnu� pest icides added es �lt her c�r�on end energy , nitro gen , . or phosphorus · '· f sou.rces . Ca rbon · end Pest icide Nitr ogen Pho sphorus Energy Alech lor * + + Atrez lne * + + EPTC * + + r f I u r e ·n * T I I I + + Pe.reth on ** : I + + Ch lorpyr l fos ** + + ' Fonofos ** + + * Herb ic ide In•ect l�lde •• ...... 0 \Jl 106 These we re alachlor (Lasso) , atrazine (Aatrex) , carbofuran (Furadan) , chlorpyrifos (Dursban) , EPTC (Eptam) , fonofos (Dyfonate) , parath ion (Ethyl para t h ion) , and triflurali n (Tre flan) . These pest icides were dissolved in 95% ethanol / added to sterile basal salts media to (10% w/ v) and 50 ul achieve a final concentrat ion of 0.1 %. The enrichm ent bases that cont ained sources of carbon and ene rgy (gl ucose, glycerol, sod ium succinate) but absent in nitrogen or phosphorus we re supplemented with pest icides to serve as th�t source . Alachlor , atrazine , E PTC , and trifluralin contain nitrogen atoms with in their str uctural makeup . These chemicals were added to enrich ment bases absent in NH4 Cl to achieve a 0.1 % (w/v) concen trat ion; thus if growth was observed nitrogen would have been derived from the pest icide . Parath ion, chl orpyfif os , and fonofos were added to enrichment bases that did not con tain a phosphorus source since these pest icides contain phosphorus atoms within their molecular str uctur es . SO ILS USED AS INOCULUM: Soils wer e obtained from South Dakota State University's Pesticide Laboratory . These soils contained varying levels of comme rcial formu- lations of pesticides resulting from 1n situ spill. It was plaus ib le that a population of m.icroorgani sms had been enriched for in these soils due to the high concentrat ion of pesticide availab le. Pesticiie spill soils we re 107 obt ained for alachlor , atrazine , EPTC , parath ion, and trifluralin. Two non-spill soi ls were used as sourc�s of inoculum . These two soils, obtained from the South Dakota State University Plant Science Departm ent , wer e classif ied as loa ms and had previous his tories of fonofos and chlor- pyrifos use. On these soils, the insecticide had failed to control corn roo tworm (O.D. Walgenbach , SDSU Plant Science Dept .; personal communication) . These soils were thought to contain _ a population of microorganisms capable of rap idly degrading fonofos and chlorpyr±fos . One-half to one gram of soil was used as inoculum for each of the following enrichments . Soils obt ained from the alac hlor , atrazine , EPTC , trifluralin , and parath ion spills were inoculated into each enrichment medium contain ing alachlor, atrazine , EPTC , trifluralin , and parathion, as the sole source of carbon and ene rgy , respectively. These same soils were then inoculated into each enrichm ent medium contai�ing alachlor , atrazine , EPTC , and trifluralin as the nitrogen source , respective ly . Soil obtained from the parath ion spill was used as inocu lum for enrich i ng for microorgani sms using para t hion as either a carbon and ener- gy source or as a phosphorus source . Soil from fon ofos and chorp yrifos failure fields we re inoculated into enrichm ent media _ containing fonofos and ch lorpyrifos as the sole source of carbon and energy respective ly . These two soils 108 were also inoculated into enrichment media containing fono fos and chl orpyrifos as the sole source of phosphorus . These enrichment media were then incubated at amb i ent tem perature (22 -23° C) on a tube roller (40 rpm) for an incubation period of three w�ek� . Two drops of each enrich ment broth was transferred to a secondary enrichment cul ture tube containing the same components and allowed to incubate for two weeks under the same conditions . After incubation they were subcul tured again by transferring one drop to a new enrichment c_ulture tube . After another two-week incu bation period an additional subculture was made by transferring one loop of inoculum to fresh enrich- ment culture tubes . Bacteria we re streaked for isolation when growth was apparent by visual turbidity 1n the culture tubes . 109 RES ULT S AND DISCUSSION Grow th was evident in final enrichm ent media that contained the pest icide as sole source of carbon and ene r gy . Modified Butner's basal ( MH B ) medium not supp lemented with a carbon and ene rgy source served as a negative con- trol for growth . Grow th did not occur in these negative controls when subcultured from enrichment broths that exh ib- ited growth . This was expected . . Growth occurred in MHE supplem ented with 95% eth�nol sugges·ting that ethanol may have serve� as a carbon and energy source for these micro- organisms . Typicall y, these organisms were Gram' s negat ive rods . It 1s possible that these microorganisms could be members of rh e genus Pseudo monas , although no taxonomic anal ysis was performed . Bergey 's · Manua l of Syste mat ic Bacteriology list several pseud omonads capab le of growth with ethanol provided as the only source of carbon and ene r- gy (41). No growth was evident 1n enrichment media contain ing the pesticide as a sole source of nitrogen or phos- phorus . Two explanat ions for this observation are possible: 1. Appare ntly these soils did not harbor micro or�anisms capab le of using the pesticides as nitrogen or phosphoru s sources . This 1s inconsistent with results of 110 investigato rs who have isolated microorganisms utilizing pest icides for either nitrogen or phosphorus sources with relative ease (14, 17, 18, 19). 2. The inocu lum contained nitrogen or phosphorus / that allo wed growth of most soil microorgani sms present , thereby overgrowing and eliminating the bacteria capable of ut ilizing the pesticides for a nitrogen or phosphorus source. In effect, the nitrogen and phosphorus enrichments were unsucc�ssful. 111 CONC LUSIONS Alachlor was found to be more easily degraded than e� ther carbofuran or dicamb a with isolates obt ained from / cultivated fields . These organisms degraded alachlor by incidental metabolism and as such were not capab le of us ing alachlor as a source of carbon and ene rgy (48). Two of these· isolates used in subsequent studies were capab le of degrading a ·lachlor in in vitro broth and in in vitro soil slurries at concentr ation� less than 550 ppm. Higher ala- chlor concent rat ions of alach lor in the soil slurry were not degraded. Whether th is was due to alachlor being toxic or its degradation not being detected is not known . These findings are similar to those reported by Matsumura and others (34, 48). Indigenous soil microbial populations were deter mined to be capable of degrading alachlor when their meta bolic activity was increased by the addit� on of glucose. Isolates obtained from an agricu ltural chemical spill were also capable of incidentally degrading alachlor; but were not capab le of using alachlor as a sole source of carbon and ene rgy . It seems likely that these organi sms were selected on the basis of surviving ext reme conditions imp�se·d by the chemical spill, rather than on the basis of being enriched for alachlor degradation capability. These 112 same microorganisms were not able to use alachlor as a carbon and ene rgy source and therfore were probably not selectively enriched for on this basis . Furthermo�e , alachlor degrad ation by these mic roorganisms see ms to be / chromosoma lly mediated since plasmid DNA was not detected in any of these isolates . FUTURE WORK: The fate of alachlor needs to be studied futher by characterizing metab olite(s) result ing from its de�radation. The goal of such research would be - to enhance degradation of environm ental pollutants so as to eli minate their presence in the environm ent . Comp lete min- eralization of the target comp ound would be desirab le since me tabolites migh t also pose enviro nmental concerns . Resea rch by many investigators has shown that com- plete mineralization of some enviro nmental pollutants oc curs by microbial commun ities . One mem ber of the community incidentally metabolizes a compound wh ich then is subse quent ly used as a source of carbon and ene, rgy for other microorganisms . Many researchers initiate degradt ion stud- ies using pure culture techniques . Since soil contains a mu ltitude of organisms� it seems likely that many more microbial community interactions remain to be discover ed. The possibility of minera lizing alachlor by the action o f two or more microorgani sms would be a worthwh ile endeavor and should be inv estigated. Th is aspect has been ignored 113 in the past but may play an important role in the degra dation and mineralizat ion of many other enviro nmental pollutants as well. 114 LITERATURE CITED 1. Alexander, M. 1977 . Soil Microbiology , 2nd ed. John Wiley and Sons , New York . 2. Bailar, J.C. Jr. , T. Moeller, J. Kleinberg, C.O. Guss, M.E. Castellion, and C. Metz . 1978 . Chemistry. Aca- demic Press, New York . 3. Barik , S., and D.M. Munne cke . 1982 . Enzym atic hydrol ysis of concentrated diaz inon in soil. Bull. Environ. Contam . Toxicol. 29 : 235-239. 4. Barles , R.W. , C.G. Daughton, and D.P.H. Hsieh. 1979 . A�ce lerated parath ion degradation in soil inoculated with acclimated bacteria under field conditions . Arch . Env i r on· . C ont am . T ox i c o 1 . 8 : 6 4 7 - 6 6 0 . 5 . Barn es , W.M. · 1977. Flasmid detection and sizing in s i n g 1 e. c o 1 ony lysates . Science 195: 393-394 . 6. Bau er, S.R. , E.M. Wood , and R.W. Traxler. 1979. Co oxidation of 2,4 -dichlor9phenoxyacetate by Pseudomonas sp. Int. Biodeterior . Bull. 15: 53-56. 7. Becker, B. , M.P. Lechev alier, R.E. Gordon, and H.A. Lecheva lier. 1964 . Rapid differentiation between Nocardia and Streptomyces by �aper chrom atography of whole-cell hydrolysat es . App l. Microbial. 12: 421-4 23 . 8. Bollag, J.-M. 1982. Microbial me tabolism of pesti cides , p. 125-168 . In J.P. Rosazza (ed.), Microbial trans formations of bioactive compounds , V. 11. CRC Pre ss, Boca Raton . 9. Bollag, J.-M. , S.-Y. Liu. 1972. Hyd� oxylations of carbaryl by soil fungi. Nature 236 :177-178. 10. Bollag, J.-M. , and S.-H. Liu . 1971. Degradation of sevin by soil mic roorganisms . Soil Biol. Biochem. 3:337-3 45 . 11. Buchanan, R.E., and N.E . Gibbons (ed. ). 1974 . Bergey 's man ual of determinat ive bacteriology . '8th ed. The Williams and Wilkins Co. � Baltimore . 115 12. Bridso n , E.Y. , and A. Brecker. 1970. Design and formu lation of microbial culture med ia, p. 229-295 . In J.R. Norris, and Ribbons (ed.), Met hods in Microbiology:3A. Academic Pre ss, New York . 13. Bumpus, J.A. , M. Tien, D. Wright, and S.T. Aust. 1985 . Oxidation of persistent envi ronmental pollutants by a white rot fungus . Science �28 : 1434-1436. / 14. Campacci, E.F. , P.B. New , and Y.T. Tehan . 1977. Isola tion of ami trole-degrading bacteria. Nature 266:1 6 4-165. 15. Chater, K.F. , D.A. Hopwood , T. Kieser, and C.J� Thompson. 1982. Gene cloning in Streptomy ces, p. 69-95 . In P.R. Hofschneider, and W. Goebel (ed. ), Gene cloning· in organisms other than E. coli. Springer �Verlag , New Y ork . 1 6 . C h a t t e .r j e e , D . K . , J . J . K i 1 b a n e , a n d A . M . C h a k r a b a r t y • 1982. Biodegradation of 2,4 ,5-trichlorophenoyacetic acid in soil by a pure culture of Pseudomonas cep acia. App l. Environ. Microbi ol. 4 4:514- 516. 17. Cook , A.M. , R. Hutter. 1981 . Degradation of s-tria zines : A critical view of biodegradation , p. 237-249 . In T. Leising er, R. Hutter, A.M. Cook , and J. Nuesch "'(;d.), Microbial degradation o·f xenc;>b iotics and recalcitrant compounds . Academi c Press, London. 18. Cook , A.M. , and R. Hut ter. 1981 . s-Triazines as nitrogen sources for bacteria. J. Agric . Food Chem. 29:11 35-1143 . 19. Cook , A.M�, C.G. Daug hton , and . M. Alexander. 1978 . Phosphonate utilization by bacteria. ' J. Bacterial . 133:85-90. 20. Dagley , S. i 981 . New perspectives in aromat ic catabo lism, p. 181-188. In T. Leisinger, R. Hutter, A.M. Cook , and J. Nuesch (ed.), Micro bial degradation of xenobiotics and recalcitrant compounds. Academic Press. , London·. 21 . Daug hton, C.G. , D.P.H. Hsieh. 1977 . Parathion utili za tion by bacterial symbionts � n a chem6stat . Appl. Environ. Microbiol. � :175-184 . 116 22. Davis, R.W. , D. Botstein, and J.R. Roth . 1980. Advanced bacterial genetics . Cold Spiing Harbor , New York . 23 . Ensign , J.C. 1978. Form ation, propert ies , and germina- tion of actinomycete spores , p. 185-219. In M.P. Starr -- (ed. ), Ann . Rev. Microbiol: 32. Ann . Rev. , Palo Alto. / 24 . Fish er, P.R. , J. Appleton, and J.M. Pemberton . 1978 . Isolation and characterization of the pesticide degrading plasmid pJPl from Alcaligenes paradoxus . J. Bacteriol. 135: 798-804 . 25. Gauger, W.K. , J.M. MacDonald, N.R. Adrian , D.P. Matthees , and D.D. Wa lgenbach . 1986 . Charact�rization o£ a streptomycete growing on organophosphate and car bam ate insecticides . Arch . Environ . Contam . Toxicol. 15: 137-141 . - 2 6 . G e r h a r d t , P . 1 9 8 1 . D i 1 u e n t s and b i oma s s me a s u reme n t , p. 504.-507 . In P. Gerhardt , R.G.E. · Murray , R.N. -- Costilow , E.W . Nester, W.A. Wood , N.R. Krieg, and G.B. Phillips (ed.). Manual of me thods for general bacteriology . Am . . Soc. Microbiol. Washington , D.C. 27. Ghosal, D. , I.-S . You , O.K. Chatterjee, and A.M. Chakrabarty . 1985 . Microbial degradation of halogen ated compounds . Science 228 : 135-142. 28. Goodh ue , C.T. 1982 . The me thodology of microbial trans form ation of organic compounds , p. 9-44 . In J.P. Rosazza (ed. ), Microbial trans form ations o�bioactive c omp o· u n d s , V . 1 . C R C P r e s s , B o c a R a t o n . 29. Goring, C.A.I ·., and D.A. Laskowski. 1982 . The effects of pest icides on nitrogen trans f ormat�ons in soils , p. 689-720 . In F.J. Stevens on (ed. ), Nitro gen 1n a g r i c u 1 t u r � s o i 1 s . �� ad i s on , W i s cons in . 30. Guirard , B.M. , and E.S. Snell. 1981 . Bio chemical fac tors in growth , p. 79-111. In P. Gerhardt, R.G.E. Murray , ·R .N. Costilow, E.W.Nester, W.A. Wood , N.R. Krieg, and G.B. Phillips (ed.)� Manual of me thods for general bacteriology . Am . Soc. Microbial. Washin gton , D.C. 31. Gunner, H.B. , and B.M. Zuckerman . 1968. Degradat ion · of 'diaz inon' by synergistic mi crobial action. Nature 217:1 1 83-1184 . 117 32. Hansen, J.B. , and R.H. Olsen. 1978 . Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMGS . J. Bacterial. 135: 227-2 38. 33 . Hargrove , R.S�, and M.G. Merkle. 1971. The loss of alachlor from soil. Weed Sti . 19:652-654. / 34 . Junk , G.A. , J.J. Richard , and P.A. Dahm. 1984. Degra dation of pestic ides in controlled water-soil systems , p. 37-67 . In R.F. Krueger, and J.N. Seiber (ed. ), Treatment and disposal of pest icide wastes . Am . Chern. Soc. , Washington, D.C. 35. Kado , C.I. , and S.-T. Liu . 1981 . Rapid procedure for detection and isolation of large and small plasmids . J. Bacterial. 145 : 1365-1373 . 6 . K e a r n e y , K M 3 P . C . , and D . D . au f man . 1. 9 7 2 . i c r o b i a 1 d e g rad ation of some chl orinated pesticides , p. 166-189 . !...!!, Proceedings of a Conference, Degradation of Syn thetic Organic Molecules in the Biosphere . Natl. Acad . Sci. , Washington, D.C. 37. Ke llogg, S.T. , D.K. Chatterjee, A.M. Chakrabarty . 1981 . Pla smid-assisted molecu lar breeding: new technique for enhanced biodeg�adation of pers istent toxic chemicals . Science 214:1133-1135 . 38 . Kilbane, J.J. , D.K. Chatterjee , and A.M. Chakrabarty . 1983 . Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by a Pseudo monas cepacia. Appl. Environ. Microbial. 45 : 1697-1700. 39 . Kilbane , J.J. , D.K. Chatterjee , J.S. K arns , S.T. Kel logg, and A.M. Chakrabarty. 1982 . Biodegradat ion of 2,4,5-trichlorophenoxyacetic acid by a pure cul ture of Pseudo monas cep acia. App l. Environ. Microbial. 44 :72-78. 40 . Krause, A. , W.G. Hancock , R.D. Minard , A.J. Frey� r, R.C . . Honeycutt, H.M. LeBaron, D.L. Paulson , S.Y. Liu, and J.M. Bol lag . 1985 . Microbial transf orm ation of _ the herbicide me tolachlor by a soil act· inomycete. J. Agric. Food Chem. �: 584-589 . · 118 41. Krieg, N.R. , and J.G. Holt (ed). 1984. Bergey 's man ual of systemat ic bacteriology , V. 1 . Wil liams and Wilkins , Balti more . 42 . Krieg , N.R. , and P. Gerhardt. 1981 . Solid cul ture , p. 143-150. In P. Gerhardt , R.G.E. Murray , R.N. Costilow, E.W. Nester, W.A. Wood , N.R. Krieg, and G.B. Philli p s / (ed. ), Manual of methods · for general bacteriology . Am . Soc. Microbiol. , Washin gton , D.C. 43. Lew is, B.A. 1981 . R plasmids in clinical isolates of Serratia marcescins . M.S. thesis . Univ . Wyoming , Laramie. 44 . Lichtens tein , E.P. 1971 . Environmental factors affect ing fate of pesticides , p. 190-205 . In Proceedi ngs of a Conference, Degradation of synthetic organic mole cules 1.n the biosphere . Natl. Acad . Sci. , Washi ngton , D.C. 45 . Liu, S.-Y. , and J.-M .. Bollag . 1971. Metabolism of car bary l by a soil fungus . J. Agr ic. Food Che rn . 19: 487-4 90. 46. Maniatis , T. E.F. Fritsch , and J. Sambrook . 1982. Mo lecular cloning a laboratory manual, p. 461-463 . Cold Spring Harbor Laboratory . 4 7. Marsha 11, K. C. , J. S. Wh iteside , and M. Alexander. 1960. Problems in the use of agar for the enumeration of soil microorganisms . Soil Sci. Soc. Proc . 24:61 -62. 48 . Matsum ura , F. 1982. Degradation of pestic ides in the enviro nment by microorgan isms and sunlig ht, p. 61-87 . In F. Mats·umura, and C.R. Krish. na Murt.i, (ed.), Bio deg r ad a t i on o f p e s t i c i d e s . P l e n u m P r e s s , N e w Yo r k . 49 . Meye rs , J.A. , D. Sanchez , L.P. Elw ell, and S. Falkow. 1976. Simp le agarose gel electrophoretic method for the ident ification and characterization of plasmid deoxyribonucl eic acid. J. Bacteri ol. 127: 1529-1537. 50. Munnecke , D.M. , L.M. Johnson , H.W. Talbot , and S. Barik . 1982 . Microbial me tabolism and enzymology of selected pest icid es, p. 1-32. � A.M. Chakrabarty (ed . ), Biodegrad ation and detoxification of environ mental pol lutants. CRC Press, Boca Raton . 119 51. Munnecke , D.M. 1980. Enzym atic detoxification of waste organophosphate pest icid es . J. Agr ic. Food Chern. 28: 105-111. 52. Munnecke, D.M. 1979 . Hydrolysis of organophosphate insecticides by an immob iliz ed-enz yme syste m. Biotech . Bioeng. 21: 2247-2 261. / 53 . Munnecke, D.M. 1976. Enzym at ic hydrolysis of organo phosph ate insecticides , a possible pesticide disposal method . App l. Environ. Microbial. 32: 7-13 . 54. Munnec ke, D.M. , and D.P.H. Hsieh. 1974. Microbial decontamination of parathion and p-nitrophenol · in aqueous media. Appl. Microbial. 28: 212- 217. 55 . Pavia, D.L. , G.M. Lampman, and G.S. Kriz , Jr. 1982. Introduction to organic laboratOFY techniques , 2nd ed . p. 571-584 . CBS College Publishing , Philadelphia. 56. Pemberton , J .M. , and R.H. Don . 1981 . Bacterial plas mids of agricultural and enviro nmental importan ce. Agric . Enviro n� 6:23-32. 57 . Pemberton , J.M. 1979 . Pesticide degrading pla smids : A biologica l answer to environmental pollution by pheno xy herbicides. Amb ia 8:202-205 . 58. Pem berton , J.M. , B. Corney , and R.H. Don . 1979 . Evo lution and spread of pesticide degrading ability among soil microorgan isms , p. 287-299. In K.N. Timm is and A. ---; P u h 1 e· r ( e d . ) , P 1 a s m i d s o f me d i c a 1 , n v i r o n me n t a 1 and comme rcial importance. Elsevier/North-Holland Biomedical Pre ss. 59 . Pemberton , J.M. , and P.R. Fish er. 1917 . 2,4-D plas mids and persistence. Nature 268 :.732-733. 60. Rosenberg, A. , and M. Alexander. 1979. Microbial clea vage of various organophosphorus insecticides . App 1. Environ. Microbial� 37: 886-891. 61 . Schaal, K.P. 1985. Identification of clinically significant actinomycetes an4 re lated bacteria us ing chemical techniques, p. 359-381 . In M. Goodfel low, and D.E. Minnikin (ed.), Chemical me thods in bacterial systemat ics . Academic Pre ss, Orlando . 120 62. Schl eif, R.F. , and P.C. Wens ink . 1981 � Practical methods in molecular biology . Springer-Verlag , New York . 63. Serdar, C.M. , D.T. Gibso n, D.M. Munnecke , and J.H. Lancaster. 1982. Pla smid involvement in parath ion hydrol ysis by Pseudo monas diminuta. Appl. Environ. Microbial . 44 : 246-249 . / 64 . Sethunathan, N. , T.K. Adhya, and K. Raghu . 1982 . Microbial degradation of pesticides in tropical soils, p. 91-115. In F. Matsumu ra, and C.R. Kri shna Murti (ed. ), Bio degradation of pest icides . Plenum Press, New York . 65 . Sharpee, K.W. , J.M. Duxbury , and M. Alexander. 1973. 2,4 -Dichlorophenoxyacetate me tab olism by Arthrobacter sp. : accum ulation of a chlorobutenolide . App l. Microbial� 26 :445-4 47_ 66. Shirlirig , E.B. , and D. Gottlieb . 1966. Methods for characterizat ion of Streptomyces species . Intl ·. J. Syst. Bact . 16:313-340 . 67 . Slater, J.H. , and D. Lov att . 1982 . Biodegradation and the sign ificance of microbial communities , p. 439-485 . In D. Gibso n (ed. ), Biochemistr y of microbial degra �t ion . Marcel Dekker, New York . 68 . Smith , A.E. , and D.V. Phillips . 1975 . Degradation of alachlor by Rh izoctonia solani . Agron . J. 67: 347-350 . 69 . Stanla ke , G.J. , and R.K. Finn . 1982 . Isolation and characterizat ion of a pentachl orophenol-degrading bacterium . . Appl. Environ . Microb ial. 44 :1421-1427. 70. Tewfik , M.s· ., and Y.A. Ham di. 1970. Decomposition o f sevin by a soil bacteriu m. Acta Microbial . Polan. Ser:B 2: 133-135. 71 . Tiedje, J.M. , and M.L. Hagedorn . 1975. Degradat ion of a a c h o r by a s o i 1 . fun g u s , C h a e. t om i u m o b o s u m . . 1 1 g 1 J Agr ic. Food Chern . 23 : 77-80 . 72 . Wh e atcroft , R. , and P.A. Williams . 1981 . Rapid meth ods for the study of both stable and uns tab le plasmids in _ Pseu domonas . J . Gen. Microbial . 124:433-437 . 121 73 . Wilkin son, L. 1986 . Systat : The syste m for stat is tics . Systat, Inc. , . Evans t�n, IL . 74. Windholz , M. (ed. ). 1983 . The Merck index, lOth . ed . Merck and Co ., Rahway , NJ . 75. Zimda hl , R.L. , and S.K. Clark . 1982. Degradation of three acetanilide herbicides in soil. Weed Sci. / 30: 545-548 .