ISSN 0003-6838, Applied Biochemistry and Microbiology, 2009, Vol. 45, No. 4, pp. 384–388. © Pleiades Publishing, Inc., 2009.Original Russian Text © O.N. Voinova, G.S. Kalacheva, I.D. Grodnitskaya, T.G. Volova, 2009, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 4,pp. 427–431.

384

INTRODUCTION

A rapid development of chemistry and the transitionof agriculture to intensive technologies led to theappearance and application of a vast variety of chemi-cals for fighting against pests, weeds, and pathogenscausing diseases of cultivated plant species. Pesticides,which are commonly used in the form of powder, sus-pensions, and emulsions often do not ensure targeteddelivery of agents, which leads to the dissipation of thelatter and their subsequent accumulation in the bio-sphere. Unfortunately, the large-scale use of pesticidesdid not completely ensure the protection of agriculturalcrops. Numerous insects and weeds remain uncon-trolled and continue to inflict serious damage to agri-culture. A strong disadvantage of modern agents is theiraccumulation in the biosphere and the acquisition ofresistance to them by adverse organisms. Pesticidesexhibiting mutagenic and carcinogenic effects areingested by humans with food and pose a threat forhuman health [1–3]. Thus, the conventional use of pes-ticides is inconsistent with solving the global problemof environmental protection. This determines thenecessity of searching for more efficient means andmethods for plant protection having no detrimentaleffect on humans and the environment in general.

A new direction aimed at reducing the risk of theuncontrolled distribution of xenobiotics in the environ-ment is the development of environmentally friendlyformulations of a new generation with a targeted andcontrolled release of the active component owing to theuse of specific coats and/or carriers made of biodegrad-able materials. In recent years, papers reporting the

obtaining of pesticides embedded in polymeric materi-als have been published. Several examples of usingpolymeric carriers, such as ethyl cellulose [4, 5], poly-urethane [6], sodium alginate [7], shape-memory poly-mers [8], for delivery of a number of weed and pest-killer chemicals (norfurol, ethyl benzoate, and simazin)were described. The possibility of the incorporation ofherbicide alachlor into ethyl cellulose microparticles[9] and avermectin and validomycin into porous quartznanoparticles [10, 11] was shown. Other authors dem-onstrated the ability of controlled delivery systemsbased on sodium alginate and lignin to reduce the wash-out of isoproturon, imidacloprid, and cyromazine fromsoil [12]. The study with the use of polymers (meth-acrylate and hydroxymethyl methacrylate) as carriersfor the delivery of herbicide simazin showed the possi-bility to regulate the herbicide release rate by changingthe polymer composition [8].

The key moment in designing formulations of thistype is choosing the material for the carrier, whichshould meet specific properties, the principal of whichare environmental friendliness and compatibility withthe global biospheric cycles, i.e., biodegradability,safety for living and nonliving nature, long-term (forweeks and months) retention in nature, controlleddestruction with the formation of nontoxic products,and the possibility of transformation by available meth-ods compatible with the technologies used for the pro-duction of these formulations.

The group of materials that may be suitable for thesepurposes includes the degradable polyesters of micro-bial origin (polyhydroxyalkanoates). These polymers

Microbial Polymers as a Degradable Carrier for Pesticide Delivery

O. N. Voinova

a

, G. S. Kalacheva

a

, I. D. Grodnitskaya

c

, and T. G. Volova

b

a

Institute of Biophysics, Siberian Branch Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036 Russia

b

Siberian Federal University, Krasnoyarsk, 660041 Russia e-mail: volova45@mail.ru

c

Institute of Forestry, Siberian Branch Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036 Russia

Received February 27, 2008

Abstract

—The possibility of use of polyhydroxyalkanoates (PHAs), biodegradable microbial polyesters, as acarrier for pesticides (

α

-hexachlorcyclohexane and lindane) for targeted and controlled delivery of these com-pounds to soil was investigated. The kinetics of polymer degradation and the dynamics of pesticide release fromthe extended-release formulations was studied. It is shown that pesticides embedded in a degradable polymer(PHA) carrier are released gradually and slowly, without surges, as the polymer is degraded by the soil micro-flora. The microbial soil component actively responded to the addition of the polymer as an additional nutrientsubstrate: the latter was degraded and then utilized. The rate of the pesticide release to the soil can be regulatedby varying the polymer–pesticide ratio.

DOI:

10.1134/S0003683809040061

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2009

MICROBIAL POLYMERS AS A DEGRADABLE CARRIER 385

are not prone to rapid chemical hydrolysis and aredegraded as a result of true biological degradation. Forthis reason, their complete degradation in biological(including natural) media takes months, which is veryimportant for designing prolonged-action formulations[13–16].

The goal of this work was to investigate the possibil-ity of using biodegradable polyhydroxyalkanoates(PHAs) for constructing environmentally friendly pes-ticides.

MATERIALS AND METHODS

The copolymer of hydroxybutyrate and hydroxyval-erate (PHB–PHV), which, as was shown earlier, isdegraded in soil more readily than the homopolymer(PHB) [7], was used as a carrier for pesticide delivery.Polymer samples were synthesized at the Institute ofBiophysics, Siberian Branch, Russian Academy of Sci-ences [14, 15] (hydroxyvalerate incorporation intocopolymer, 11 mol %; crystallinity degree, 58%;molecular weight, 168 kDa).

The study was performed with

α

-hexachlorcyclo-hexane (HCCH) and lindane. The former is a complex-action insecticide used for fighting against variousharmful insects in general and as an insecticidal addi-tive to seed protectants in order to protect seedlingsfrom damage by soil-inhabiting pests; the latter (

γ

-iso-mer of HCCH) is a highly effective insecticide with abroad spectrum of action used for many agriculturalcrops.

The polymer was synthesized by the

Wautersiaeutropha

strain B5786 under optimized culturing con-ditions. The polymer was isolated from the bacterialbiomass in the form of flakes. Insecticides were embed-ded in the polymeric matrix according to the followingscheme. The polymer was ground in a laboratory mill.The granulometric composition of obtained particleswas as follows: 0.50 mm, 70.6%; 0.32 mm, 23.4%. Theground polymer (fraction 0.14–0.20 mm) was thenmixed with the pesticides. The thus obtained homoge-nous powders were cold-compacted under a pressure of120 kgs/cm

2

with an AutoPellet 3887 laboratory press(Carver, United States) to form bulky pellets 3 mm indiameter weighing

21.0

±

1.3

mg. As a result, weobtained experimental forms of pesticides (HCCH andlindane)—polymeric matrices with different proportionof pesticides. The polymer–pesticide ratio in the pel-leted form was 60 : 40 for HCCH and 40 : 60 for lin-dane (percent of total mass).

The microbiocenosis was studied in samples of gar-den soil (100 g) placed in plastic containers (125 cm

3

).The samples were weighed on analytical balances(accuracy class IV, Metler, United States) and incu-bated in a constant-temperature cabinet at

25°ë

at con-stant moisture (60%). The microbial soil componentwas characterized using the conventional soil-biologymethods [18]. The abundance of microorganisms was

analyzed in soil samples at the end of the experiment.Pesticide-free soil exposed under the same conditionswas used as a control. The main groups of microorgan-isms (ecological–trophic, systematic, and physiologi-cal) were determined by the dish method in a series ofdiagnostic media: beef-extract agar (BEA) for bacteria,wort agar (WA) for micromycetes, starch–ammoniaagar (SAA) for prototrophs utilizing mineral nitrogenforms, and soil agar (SA) for oligotrophs. Media wereinoculated with a soil suspension (1 :

10

3

) in triplicate.Petri dishes were incubated at

25°

C for 3 and 5 days inthe case of bacteria and micromycetes, respectively.

The experiment continued for 84 days. The experi-mental forms of pesticides embedded in the polymericmatrix were periodically taken from the containers.After purification from soil and weight stabilization,the appearance of pellets was analyzed under a micro-scope (Meiji Techno Co. Ltd., Japan) equipped with adigital camera (Infiniti I Luminera Corp., Canada),after which the weight of the pellets was determined.Then, after preliminary methanolysis of samples [15],the residual content of polymers in pellets was deter-mined by the standard procedure. To study the dynam-ics of pesticide release from the polymeric matrix,HCCH and lindane were extracted from soil with aseries of solvents. Then, after a series of standard pro-cedures (concentration, washing, pH stabilization, anddehydration) [19], the content of these pesticides wasdetermined in a gas chromatograph equipped with aGCD plus mass-spectrometric detector (Hewlett-Pack-ard, United States). The chromatogram and mass spec-trum of lindane extracted from a soil sample are shownin Fig. 1.

RESULTS AND DISCUSSION

The polymer containing the pesticides was activelydegraded in the course of the experiment (Fig. 2). Themain mass of the polymer in both variants wasdegraded 40–50 days after the beginning of the experi-ment. At the end of observation, the residual content ofthe polymer in compacted pellets was approximately5–10% of the initial content (Fig. 3a).

It is known than many soil microorganisms candegrade PHAs and utilize them as a growth substrate[20]. The microbiological analysis showed that soilsamples used in the study are characteristic of culti-vated rich chernozem soils. The main ecological–trophic microbial groups, as well as certain functionaltaxonomic groups of microorganisms, were highlyabundant and diverse. A comparative analysis of thecontrol soil sample and soil containing pesticide formsshowed that the total titer of aerobic microflora in thecontrol was lower than in the soil containing the poly-meric carriers with HCCH and lindane (

4.9

×

10

6

, 5.9

×

10

6

,and

7.2

×

10

6

CFU/g soil, respectively).

It should be noted that the presence of the PHB–PHV copolymer as an additional substrate in the soil

386

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VOINOVA et al.

stimulated the activity of microorganisms. The miner-alization of organic matter was more active in theexperimental soil samples, as judged by the abundanceof microorganisms capable of active primary destruc-tion of organic matter. The presence of the polymer inthe soil stimulated the activity of this group of microor-ganisms, whose abundance in the soil samples withHCCH and lindane (

10.0

×

10

6

CFU/g soil) was higherthan in the control (

8.7

×

10

6

CFU/g soil. This group ofmicroorganisms included bacteria (spore and nonsporeforms), micromycetes, and actinomycetes.

The abundance of oligotrophs—microorganismsthat can utilize various low-molecular-weight productsof polymer hydrolysis and live in the presence of lowconcentrations of mixtures of various compoundsformed as a result of the degradation of barely availablecompounds—was greater in the presence of the poly-mer in the soil [21]. Oligotrophs are the ultimate utiliz-

ers of organic matter getting into the environment. Theabundance of oligotrophs is indicative of a higher activityof these groups of microorganisms in soil samples con-taining the polymers with HCCH and lindane (

9.5

×

10

6

and

8.4

×

10

6

CFU/g soil, respectively) compared to thecontrol (

7.0

×

10

6

CFU/g soil).

Thus, the decomposers actively responded to theaddition of the polymer as an additional nutrient sub-strate.

The pesticide was slowly released to the soil fromcompacted polymer pellets as the polymer underwentdegradation. This was especially pronounced in exper-iments with HCCH, the content of which in the initialsamples was 40 wt % (Fig. 3b, panel I). In the first 30–40 days, the HCCH release to the environment was lowand accounted for only 2–3% of its content in the pellet(minimum release rate, 0.2

µ

g/day per gram soil). Thecontent of this pesticide in the soil increased only at fur-

Intensity

140000

120000

100000

80000

60000

40000

20000

040 60 80 100 120 140 160 180 200 220 240 260 280 300

(‡)

m

/

z

51

61

75

85

109

113145

133158

181

187

219

216

233

254290292

(b)

Intensity

2000000

1500000

1200000

800000

400000

6.70

350 450 550 650 750 min

Fig. 1.

(a) Mass spectrum and (b) ion chromatogram of lindane assayed after extraction from soil. The

m/z

ratio is the ratio of themolecular weight of the fragment to its charge.

(a) (b) (c) (d)

Fig. 2.

The appearance of compacted forms of the PHB–PHV copolymer and lindane undergoing degradation of the polymericmatrix in the soil: (a) the initial form and partially forms on day (b) 28, (c) 42, and (d) 70 of exposure in the soil. Scale, 1 mm.

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MICROBIAL POLYMERS AS A DEGRADABLE CARRIER 387

ther stages and reached 30

µ

g/g soil on day 70–80 ofthe experiment (i.e., the release of HCCH accounted for10–12% of its initial content in the pellet). The rate ofHCCH release to the environment was maximum onday 40–55 and reached 0.73

µ

g/day per gram soil. Theprofile of the lindane release to the soil, whose contentin the sample was greater than the content of the poly-mer (60% of the total pellet weight), was more pro-nounced (Fig. 3b, panel II). The maximum rate of thelindane release recorded during the entire observationperiod was 1.43

µ

g/day per gram soil. At the end of theexperiment, soil samples contained 50

µ

g of lindaneper gram soil, which corresponded to 30% release ofthe initial lindane content in pellet, at a mean releaserate of 0.77

µ

g/day per gram soil.

The release of the active ingredient from the poly-meric matrix largely depends on the polymer type andthe initial agent–polymer ratio [5]. Our studies showedthat, the lower the content of pesticides in the PHB–PHV pelleted form, the lower their release rate. Thisfinding is consistent with the data obtained by theauthors of [4], who studied the use of ethyl cellulose-based microspheres as a polymeric carrier for norflua-zon delivery.

Similar slow rates of pesticide release were recordedfor herbicide simazin embedded in methacrylate andhydroxymethyl methacrylate. On day 25 of the experi-

ment, the release of simazin from methacrylate andhydroxymethyl methacrylate accounted for 2 andapproximately 27%, respectively, of the initial contentof this agent in the polymeric matrix [8].

Thus, using HCCH and lindane as examples, weshowed that pesticides embedded in a degradable PHA-based polymeric carrier are slowly and gradually (with-out surges) released to the environment as the polymeris degraded by the soil microflora (the mean rate of pes-ticide release was 0.49 and 0.77

µ

g/day per gram soilfor HCCH and lindane, respectively). The analysis ofthe abundance of ecological–trophic groups of micro-organisms showed that the content of microorganismsin the experimental soil samples was higher than in thecontrol. The rate of pesticide release to the soil can beregulated by varying the polymer–pesticide ratio.

Thus, these experiments were the first to demon-strate the possibility of using degradable polyhydroxy-alkanoates as a matrix for embedding pesticides toensure their targeted and controlled release to the envi-ronment.

ACKNOWLEDGMENTS

This study was supported by the Ministry of Educa-tion and Science of the Russian Federation and theU.S. Civilian Research and Development Foundation

%100

75

50

25

0 20 40 60 80day

(‡)

(b)

I II

0

%100

75

50

25

0 20 40 60 80day

0

0 20 40 60 80day

µ

g/g

30

20

10

00 20 40 60 80

day

µ

g/g

20

0

60

40

Fig. 3.

(a) The dynamics of degradation of the PHV–PHB copolymer (%) and (b) the release profile (

µ

g/g soil) of (I)

α

-hexachlo-rcyclohexane and (II) lindane during exposure of pellets in the soil.

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(grant no. P1Mé002), joint program of the RussianFoundation for Basic Research and the KrasnoyarskKrai Science Foundation (project no. 07-08-96800-r_yenisei_a), the Krasnoyarsk Krai Science Foundation(project no. 18G142), and the Russian Science SupportFoundation.

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