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(Agency for Toxic Substances and Disease Registry 1998). The additional effect for the toxicity of phenol may be the formation of phenoxyl radicals (Hanscha et ...
Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Enhancement of phenol degradation by soil bioaugmentation with Pseudomonas sp. JS150 A. Mrozik1, S. Miga2 and Z. Piotrowska-Seget3 1 Department of Biochemistry, University of Silesia, Jagiellon´ska 28, Katowice, Poland 2 Institute of Materials Science, University of Silesia, Bankowa 12, Katowice, Poland 3 Department of Microbiology, University of Silesia, Jagiellon´ska 28, Katowice, Poland

Keywords Bioaugmentation, biodegradation, FAMEs, phenol, Pseudomonas sp. JS150, survival. Correspondence Agnieszka Mrozik, Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellon´ska 28, 40-032 Katowice, Poland. E-mail: [email protected]

2011 ⁄ 1025: received 21 June 2011, revised 12 August 2011 and accepted 17 August 2011 doi:10.1111/j.1365-2672.2011.05140.x

Abstract Aims: To test whether bioaugmentation with genetically modified Pseudomonas sp. JS150 strain could be used to enhance phenol degradation in contaminated soils. Methods and Results: The efficiency of phenol removal, content of humic carbon, survival of inoculant, number of total culturable autochthonous bacteria and changes in fatty acid methyl esters (FAME) profiling obtained directly from soils were examined. Bioaugmentation significantly accelerated phenol biodegradation rate in tested soils. Phenol applied at the highest concentration (5Æ0 mg g)1 soil) was completely degraded in clay soil (FC) within 65 days, whereas in sand soil (FS) within 72 days. In comparison, phenol biodegradation proceeded for 68 and 96 days in nonbioaugmented FC and FS soils, respectively. The content of humic carbon remained at the same level at the beginning and the end of incubation time in all soil treatments. The number of introduced bacteria (2Æ50 · 109 g)1 soil) markedly decreased during the first 4 or 8 days depending on contamination level and type of soil; however, inoculant survived over the experimental period of time. Analysis of FAME patterns indicated that changes in the percentages of cyclopropane fatty acids 17:0 cy and 19:0 cy x10c and branched fatty acids might be useful markers for monitoring the progress of phenol removal from soil. Conclusions: It was confirmed that soil bioaugmentation with Pseudomonas sp. JS150 significantly enhanced soil activity towards phenol degradation. Cyclopropane and branched fatty acids were sensitive probes for degree of phenol utilization. Significance and Impact of the Study: In future, genetically modified Pseudomonas sp. JS150 strain could be of use in the bioaugmentation of phenol-contaminated areas.

Introduction Industrial activities such as oil refineries, gas stations, and production of pesticides, explosives, paints, textiles, wood preservatives and agrochemicals release phenol and its derivatives into the environment. These compounds are also the products of auto exhaust, and therefore, areas of high traffic likely contain increased level of phenol (Budavari 1996). Although there is no consistent evidence that phenol causes cancer in humans, it is stated that long-

term or repeated exposure may cause harmful effects on the central nervous system, heart, liver, kidney and skin (Agency for Toxic Substances and Disease Registry 1998). The additional effect for the toxicity of phenol may be the formation of phenoxyl radicals (Hanscha et al. 2000). This is a reason why cleaning up of phenol-contaminated sites is of a great ecological concern. There are many methods for the detoxification of phenol from contaminated soils. As an alternative to physico-chemical treatments, the use of micro-organism

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processes has become the most promising approach in remediation technology. Bioremediation is really suited to vast and moderately contaminated soils and decreases usually high energy demand and consumption of chemical reagents (Khan et al. 2004). In recent years, there has been an increasing interest in developing new techniques for bioremediation of soils contaminated with toxic organic pollutants. One of the ways to enhance the efficacy of contaminant removal is bioaugmentation. This strategy is based on the inoculation of given soils with micro-organisms either being pure or mixed cultures characterized with desired catalytic capabilities (Heinaru et al. 2005; Silva et al. 2009; Guimara˜es et al. 2010). Moreover, genetically modified micro-organisms exhibiting enhanced degradative potential are considered to be attractive for soil bioaugmentation. It is thought that bioaugmentation should be applied when the biostimulation and bioattenuation did not bring expected results (Vogel 1996). Soils that need to be clean may be inoculated with both wild indigenous or allochthonous strains or laboratory-constructed strains carrying necessary degradative pathways. Several bacterial strains have been reported to posses the metabolic pathways for the degradation of phenol. The most effective bacteria studied are represented by strains from genera Burkholderia (Schro¨der et al. 1997), Pseudomonas (Kargi and Serkan 2004; Yang and Lee 2007; Mrozik et al. 2010), Acinetobacter (Paller et al. 1995; Mazzoli et al. 2007), Serratia (Pradham and Ingle 2007), Rhodococcus (Goswami et al. 2005; Nagamani and Lowry 2009) and Ralstonia (Chen et al. 2004). Effective hydrocarbon-degrading strains are often used as commercial inocula to enhance the bioremediation of hydrocarbon-contaminated sites. For example, significant increase in aromatic compounds biodegradation rate was achieved by using commercial products such as Sybron 1000, Biozyn 301 and DBC-plus (Dott et al. 1989; Sobiecka et al. 2009). Several studies have successfully applied this strategy for cleaning up polluted soils; however, results of other experiment indicated its major limitations (Simon et al. 2004). A success of bioaugmentation depends on both biotic and abiotic factors. The most important is a strain selection (Thompson et al. 2005). Bacteria for bioaugmentation should survive and multiply in soil as well as to compete with autochthonous micro-organisms for nutrients and oxygen. Moreover, after soil inoculation, they should not lose their degradative capacity. Mineralization rate of organic contaminants is also strongly influenced by many physico-chemical environmental parameters. They include chemical structure, bioavailability and concentration of pollutants accompanied with soil type, pH, temperature, salinity, water and oxygen content (Leahy 1358

and Colwell 1990; Davis and Madsen 1996; Stalwood et al. 2005). The presence of phenols shows harmful effect on the biological properties of bacterial cell membrane. Particles of phenolic substrates partition into phospholipid bilayers resulting in the changes in cytoplasmic membrane fluidity, stability and permeability (Weber and de Bont 1996). As a response to phenols, many bacteria can adapt to unfavourable conditions by the modification of fatty acid composition. The adaptive mechanisms include de novo synthesis of fatty acids, cis to trans isomerization, the increase in branched and cyclopropane fatty acid content and alteration in lipid-to-protein ratio (Diefenbach et al. 1992; Heipieper and de Bont 1994; Kaur et al. 2005; Fischer et al. 2010). Based on these considerations, changes in bacterial fatty acid composition may be used as a marker for monitoring the process of bioremediation. Materials and methods Bacterial strain and culture conditions Bacterial strain Pseudomonas sp. JS150 was kindly provided by Dr J. Spain from Air Force Civil and Engineering Support Agency, Tyndall Air Force Base, Florida, USA. Pseudomonas sp. JS150 is a nonencapsulated mutant of strain JS1 obtained after ethyl methanesulfonate mutagenesis. It is known as an efficient degrader of phenol and other aromatic compounds such as toluene, benzene, benzoate, salicylate and naphthalene (Haigler et al. 1992). This strain was routinely grown at 30C on nutrient agar medium and in Kojima mineral liquid medium (Kojima et al. 1961) supplemented with phenol at the concentration of 752 mg l)1. Soils Soil samples were collected from the top layer of 5–20 cm at two distinct sites localized close to Sosnowiec (Upper Silesia, Poland). Soils came from mixed and pine forests and were signed as FC and FS, respectively. No phenol contamination was determined in these soils. Prior to experiment, the air-dried soil samples at room temperature were sieved (2 mm) and transferred to plastic pots (150 g). Physical and chemical properties of each soil are presented in Table 1. For experiment purpose, triplicate portions of FC and FS soils were amended with phenol at three concentrations: 1Æ7, 3Æ3 and 5Æ0 mg g)1 and pre-incubated for 1 day. Such phenol concentrations were significantly (1000 times) higher than in similar biodegradation studies. Part of soil samples was additionally inoculated with

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Table 1 Characteristics of soils

Soil property Sand (%) Silt (%) Clay (%) Density (g cm)3) pH (H2O) Organic matter (% d.w) Total organic carbon (% d.w) C hum (% d.w)* CEC (cmol + kg)1) P2O5 (mg 100 g)1) K2O (mg 100 g)1) Conductivity (lS cm)1)

Clay (FC)

Sand (FS)

Method ⁄ source

59 31 10 0Æ57 6Æ02 29Æ5

96 4 0 1Æ17 6Æ89 1Æ9

PN-R-04032:1998 PN-R-04032:1998 PN-R-04032:1998 PN-88 ⁄ B-04481 PN-ISO 10390:1997 Combustion

7Æ9

0Æ61

1Æ38 13Æ7 0Æ05 41Æ5 99Æ3

0Æ24 1Æ9 0Æ06 5Æ0 30Æ3

PN-Z-15011-3 Lityn´ski et al. (1972) ISO 23470:2007 PN-R-04023:1996 PN-R-04022:1996 PN-ISO 11265 + AC1:1997

*Total humic and fulvic acids.

phenol-degrading Pseudomonas sp. JS150. For soil bioaugmentation, bacteria were cultured in 250 ml of nutrient broth medium (Becton Dickinson, Franklin Lakes, NJ, USA) at 28C on rotary shaker at 125 rev min)1 to reach the mid-logarithmic growth phase. Then, cultures were centrifuged (8000 g), and pellets were washed twice (0Æ85% NaCl). After this, the pellets were resuspended in sterile NaCl. Next 15 ml of this suspension was poured into pot resulting in 2Æ50 · 109 bacteria per gram of soil. The final water content of the soils was adjusted to about 50% of the maximum water-holding capacity. All pots were kept in a chamber cabinet at room temperature. Biodegradation and survival experiments To estimate phenol removal in bioaugmented and nonbioaugmented FC and FS soils, samples were taken on 1, 4 and 8 days and next at 8-day intervals. Phenol was extracted from soil with methanol, and its concentration was determined by colorimetric method with diazoate p-nitroaniline at the wavelength 550 nm (Lurie and Rybnikova 1968). For monitoring the survival of inoculant, the spontaneous rifampicin-resistant mutant of Pseudomonas sp. JS150 was used. On the sampling days, the numbers of inoculant and total heterotrophic bacteria were calculated in bioaugmented soils, whereas in nonbioaugmented, only total heterotrophic bacteria were determined. For this purpose, 5 g of soil was placed into Erlenmeyer flasks containing 45 ml of 0Æ85% NaCl for shaking (30 min, 125 rev min)1) and preparing serial 10-fold dilutions for plate counts. Nutrient agar supplemented with rifampicin at the concentration of 100 lg ml)1 and nutrient agar

were used for counting the number of inoculant and total number of bacteria, respectively. Inoculated plates were incubated at 28C for 48 h. Data are representative of three individual experiments. At the beginning and the end of experiments, organic matter, organic carbon and humic carbon contents were determined in all soil treatment. The procedure of humic substance extraction from soil was described in detail in previous article (Mrozik et al. 2008). midi-FAME analysis Fatty acid analyses were performed on the same days when phenol concentration and survival of inoculants were determined. Duplicate samples of 5 g of each soil were extracted according the procedure by Kozdro´j (2000) and identified using the Microbial Identification System (Microbial ID Inc., Newark, Delaware, USA) standard protocol (Sasser 1990). The procedure of fatty acid extraction and methylation was carried out as described previously (Mrozik et al. 2010). Fatty acids were analysed by gas chromatograph (Hewlett-Packard 6890, Santa Clara, CA, USA) equipped with capillary column Ultra 2-HP (5% phenylmethyl silicone; 25 m, 0Æ22 mm ID, film thickness 0Æ33 mm) and flame ionisation detector. Peaks from chromatograms were identified using midi software (Sherlock aerobe method and TSBA library ver. 5.0). Data analysis Decay process can be described by several types of functions, e.g. exponential, bi-exponential, stretched exponential, inverse logarithmic and power law (Dec et al. 2007). The simplest function is the exponential one. This function g(t) = g0e)t ⁄ s (where g0 and g(t) are the initial and after time t concentrations of phenol, respectively, and s is relaxation time of described process) depends on two parameters g0 and s only. It is important for the analysis of experimental data containing a few points only (see Fig. 1a1). Relaxation time has very clear interpretation – during this time, phenol concentration decreases by about 63Æ2%. The exponential function describes very well all our temporary data (uncertainty of estimated parameters is relatively low, and R2 coefficient is close to the unity). Additionally, similar function was successfully used for the analysis of degradation rate constant, rate of disappearance and disappearance time for phenol in different soils inoculated with Pseudomonas sp. CF600 (Mrozik et al. 2010). Therefore, for quantitative analysis of phenol degradation process, the exponential function has been chosen. The least square method was used for fitting the exponential function to an experimental data. This way values of g0 and s and their uncertainty were estimated.

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5

(a1)

4

τ FC1·7 τ FC1·7+B

3

A. Mrozik et al.

(a2)

(a3)

τ FC3·3 τ FC3·3+B

= 10·8 ± 1·8 days = 3·6 ± 1·1 days

τ FC5·0 τ FC5·0+B

= 12 ± 1 days = 7·3 ± 0·8 days

= 33·4 ± 3·2 days = 25·3 ± 3·0 days

Phenol (mg g–1)

2 1 0 5

(b1)

4

(b2)

τ FS1·7 = 26·2 ± 2·7 days τ FS1·7+B = 6·1 ± 0·7 days

3

(b3)

τ FS3·3 τ FS3·3+B

τ FS5·0 = 33·4 ± 3·2 days τ FS5·0+B = 25·3 ± 3·0 days

= 24·4 ± 1·7 days = 16·6 ± 1·6 days

2 1 0

0

20

40

60

80

0

20

0

40 60 80 Time (Days)

20

40

60

80

Figure 1 Dynamics of phenol degradation in bioaugmented (j) and nonbioaugmented (h) FC and FS soils contaminated with phenol at the concentrations of 1Æ7 mg g)1 (a1, b1), 3Æ3 mg g)1 (a2, b2) and 5Æ0 mg g)1 (a3, b3).

Table 2 Selected FC soil parameters at the beginning and the end of experiment FC soil FCP1Æ7+B

FCP1Æ7

FCP3Æ3

FCP3Æ3+B

FCP5Æ0

FCP5Æ0+B

Soil parameter

Day 1

Day 24

Day 1

Day 8

Day 1

Day 36

Day 1

Day 24

Day 1

Day 68

Day 1

Day 56

Organic matter (% d.w.) Organic carbon (% d.w.) C hum (% d.w.) pH

29Æ50 7Æ90 1Æ39 5Æ82

28Æ78 7Æ66 1Æ37 6Æ12

29Æ84 7Æ99 1Æ38 5Æ91

29Æ23 7Æ80 1Æ39 6Æ55

29Æ61 8Æ04 1Æ40 5Æ54

28Æ71 7Æ44 1Æ42 6Æ33

29Æ91 8Æ14 1Æ41 5Æ66

28Æ76 7Æ68 1Æ43 6Æ18

30Æ21 8Æ32 1Æ42 5Æ42

28Æ12 7Æ20 1Æ45 6Æ27

30Æ31 8Æ37 1Æ41 5Æ51

27Æ91 7Æ51 1Æ45 6Æ23

Values are the means of three replicates (standard errors