Enhanced of Phenol Degradation by Soil Bioaugmentacion With Pseudomonas SP JS150

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, Jagiellonska 28, Katowice, Poland 2 Institute of Materials Science, University of Silesia, Bankowa 12, Katowice, Poland 3 Department of Microbiology, University of Silesia, Jagiellonska 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, Jagiellonska 28, 40-032 Katowice, Poland. E-mail: agnieszka.mrozik@us.edu.pl
Abstract Aims: To test whether bioaugmentation with genetically modied Pseudomonas sp. JS150 strain could be used to enhance phenol degradation in contaminated soils. Methods and Results: The efciency of phenol removal, content of humic carbon, survival of inoculant, number of total culturable autochthonous bacteria and changes in fatty acid methyl esters (FAME) proling obtained directly from soils were examined. Bioaugmentation signicantly accelerated phenol biodegradation rate in tested soils. Phenol applied at the highest concentration (50 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 (250 109 g)1 soil) markedly decreased during the rst 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 conrmed that soil bioaugmentation with Pseudomonas sp. JS150 signicantly enhanced soil activity towards phenol degradation. Cyclopropane and branched fatty acids were sensitive probes for degree of phenol utilization. Signicance and Impact of the Study: In future, genetically modied Pseudomonas sp. JS150 strain could be of use in the bioaugmentation of phenol-contaminated areas.
2011 1025: received 21 June 2011, revised 12 August 2011 and accepted 17 August 2011
doi:10.1111/j.1365-2672.2011.05140.x
Introduction Industrial activities such as oil reneries, 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 trafc 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 detoxication of phenol from contaminated soils. As an alternative to physico-chemical treatments, the use of micro-organism
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2011 The Authors Journal of Applied Microbiology 111, 13571370 2011 The Society for Applied Microbiology
Soil bioaugmentation
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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 efcacy 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; Guimaraes et al. 2010). Moreover, genetically modied 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 (Schroder 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, signicant 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 inuenced 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
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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 uidity, stability and permeability (Weber and de Bont 1996). As a response to phenols, many bacteria can adapt to unfavourable conditions by the modication 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 efcient 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 520 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: 17, 33 and 50 mg g)1 and pre-incubated for 1 day. Such phenol concentrations were signicantly (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

Clay (FC) 59 31 10 057 602 295 79 138 137 005 415 993 Sand (FS) 96 4 0 117 689 19 061 024 19 006 50 303
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)
Method source PN-R-04032:1998 PN-R-04032:1998 PN-R-04032:1998 PN-88 B-04481 PN-ISO 10390:1997 Combustion PN-Z-15011-3 Litynski et al. (1972) ISO 23470:2007 PN-R-04023:1996 PN-R-04022:1996 PN-ISO 11265 + AC1:1997
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 Kozdroj (2000) and identied using the Microbial Identication 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, 022 mm ID, lm thickness 033 mm) and ame ionisation detector. Peaks from chromatograms were identied 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 632%. The exponential function describes very well all our temporary data (uncertainty of estimated parameters is relatively low, and R2 coefcient 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 tting the exponential function to an experimental data. This way values of g0 and s and their uncertainty were estimated.
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*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 (085% NaCl). After this, the pellets were resuspended in sterile NaCl. Next 15 ml of this suspension was poured into pot resulting in 250 109 bacteria per gram of soil. The nal 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 asks containing 45 ml of 085% 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
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5 4 3 2
(a1)
FC17 FC17+B
= 108 18 days = 36 11 days
(a2)
FC33 FC33+B
= 12 1 days = 73 08 days
(a3)
FC50 FC50+B
= 334 32 days = 253 30 days
Phenol (mg g1)
1 0 5 4 3 2 1 0 0 20 40 60 80 0 20 40 60 80 Time (Days) 0 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 17 mg g)1 (a1, b1), 33 mg g)1 (a2, b2) and 50 mg g)1 (a3, b3).
(b1)
FS17 = 262 27 days FS17+B = 61 07 days
(b2)
FS33 FS33+B
= 244 17 days = 166 16 days
(b3)
FS50 = 334 32 days FS50+B = 253 30 days
Table 2 Selected FC soil parameters at the beginning and the end of experiment
FC soil FCP17 Soil parameter Organic matter (% d.w.) Organic carbon (% d.w.) C hum (% d.w.) pH Day 1 2950 790 139 582 Day 24 2878 766 137 612 FCP17+B Day 1 2984 799 138 591 Day 8 2923 780 139 655 FCP33 Day 1 2961 804 140 554 Day 36 2871 744 142 633 FCP33+B Day 1 2991 814 141 566 Day 24 2876 768 143 618 FCP50 Day 1 3021 832 142 542 Day 68 2812 720 145 627 FCP50+B Day 1 3031 837 141 551 Day 56 2791 751 145 623
Values are the means of three replicates (standard errors <5%).
Table 3 Selected FS soil parameters at the beginning and the end of experiment
FS soil FSP17 Soil parameter Organic matter (% d.w.) Organic carbon (% d.w.) C hum (% d.w.) pH Day 1 199 066 024 659 Day 56 189 062 023 672 FSP17+B Day 1 206 068 022 651 Day 32 197 062 021 682 FSP33 Day 1 216 068 022 644 Day 64 207 059 022 664 FSP33+B Day 1 222 071 022 640 Day 48 200 067 023 669 FSP50 Day 1 221 079 022 619 Day 96 197 069 025 661 FSP50+B Day 1 228 070 023 621 Day 72 184 062 025 671
Values are the means of three replicates (standard errors <5%).
Results Biodegradation studies Phenol degradation experiments were carried out in clay (FC) and sand (FS) soils varied in their physico-chemical parameters (Table 1). Such soils were chosen to compare
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the degradation rate in soils distinctly differed in organic matter and carbon content. Owing to phenolic compounds participate in forming of humic and fulvic acid structure, additionally the content of total humic carbon was determined at the beginning and the end of the experiments (Tables 2 and 3). To assess the impact of phenol-degrading bacteria on phenol biodegradation rate,
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a part of soil samples were inoculated with Pseudomonas sp. JS150. This allowed us to compare the rate of phenol removal by indigenous micro-organisms exhibiting tested soils and enriched with inoculated strain with high catabolic potential. The results clearly showed that bioaugmentation significantly accelerated phenol degradation in tested soils and was correlated with the type of soil. In FC soil contaminated with the concentrations of 17, 33 and 50 mg g)1 soil (FC17, FC33 and FC50) and inoculated with Pseudomonas sp. JS150 (FC17+B, FC33+B and FC50+B), phenol was completely degraded within 8, 24 and 56 days, respectively (Fig. 1a1,a2,a3). The same doses of phenol in contaminated and inoculated FS soils (FS17+B, FS33+B and FS50+B) were degraded slower, within 32, 48 and 72 days, respectively (Fig. 1b1,b2,b3). In contrast, in both nonbioaugmented FC and FS soils, phenol removal lasted 23 weeks longer (Fig. 1). Moreover, in soils inoculated with Pseudomonas sp. JS150, the removal of 50% of phenol (DT50 = )sln 05) proceeded signicantly faster as compared to nonbioaugmented soils. For example, DT50 for FC17+B and FC50+B were 25 and 71 days, whereas in nonbioaugmented FC soils, FC17 and FC50 were 75 and 14 days, respectively. In comparison, in FS17+B and FS50+B treated with the same phenol concentrations, DT50 reached the value of 42 and 175 days for bioaugmented soils and 182 and 232 days for FS17 and FS50, respectively. Figure 2 shows the estimated values of the relaxation time (s) for biodegradation processes. Bacterial inoculation of soil with phenol at the concentration of 17 mg g)1 soil increased 3- and 4-fold the rate of phenol removal for FC17+B and FS17+B, respectively. In turn, in bioaugmented FC and FS soils with higher phenol dosages, the biodegradation rate was lower; however, it was
still remarkably higher as compared to nonbioaugmented FC and FS soils. In both soils with low phenol concentration, biodegradation rate was almost constant, but above 33 mg of phenol g)1 soil, its degradation rate was lower. In bioaugmented FC and FS soils, time for phenol removal was almost linear function of initial phenol concentration. Microbial numbers During biodegradation studies, the survival of Pseudomonas sp. JS150 and total heterotrophic bacteria was determined in both phenol-contaminated FC and FS soils. The number of total culturable bacteria was also counted in phenol-polluted and nonbioaugmented soils. Obtained data indicated that Pseudomonas sp. JS150 introduced to FC and FS soils survived during experimental period; however, cell number decreased over time. The observed decrease strongly depended on soil type and the level of phenol contamination. In FC17+B soil, the number of inoculant decreased from 25 109 g)1 soil on day 0 to 32 107 g)1 on day 8, when phenol was completely degraded (Fig. 3a1). In comparison, in FS17+B soil on day 8, the number of Pseudomonas sp. JS150 cells reached the value of 59 106 g)1 and on day 32 after phenol degradation, it reached 50 105 g)1 soil (Fig. 3b1). In turn, in FC50+B and FS50+B soils, number of inoculant on day 56 declined to 64 103 and 53 102 g)1, respectively, and nally on day 72, it was equal to 25 102 g)1 in FS50+B soil (Fig. 3a3,b3). Similarly as for Pseudomonas sp. JS150, the number of total autochthonous bacteria decreased in both contaminated and bioaugmented FC and FS soils. The more phenol pollution, the stronger decline in bacterial counts was observed. In FC17+B soil, the number of heterotrophic bacteria was reduced from initial 25 108 to 33 106 g)1 on day 24, whereas in the same soil exposed to the highest phenol concentration, it was reduced to 15 104 g)1 on day 56. However, in FS17+B bacteria, number decreased from 80 105 to 10 104 CFU g)1 soil on day 32 and in FS50+B to 10 102 on day 72 (Fig. 3). Data analysis of bacterial numbers in nonbioaugmented and contaminated soils showed that phenol applied at increasing concentrations in different degree decreased the number of autochthonous bacteria. The highest decline in bacterial counts was observed during the rst 4 days of the experiment in both FC50 and FS50 soils. From that sampling time till the end of phenol degradation, numbers of bacteria maintained at the similar level (Fig. 3a3,b3). In turn, the smallest decrease in bacterial number was determined in soils polluted with the lowest phenol concentration.
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30
(days)
20
10
3 4 Phenol (mg g1)
Figure 2 Phenol concentration dependences of relaxation time. ( FCP; ( ) FCP+B; ( ) FSP and ( ) FSP+B.
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109 107 105 103
FC soil
(a1)
FS soil
(b1)
CFU g1 soil
109 107 105 103 109 107 105 103 0
(a2)
(b2)
(a3)
(b3)
10 20 30 40 50 60
0 10 20 30 40 50 60 70 80 90 Time (days)
Figure 3 The number of introduced and total bacteria in bioaugmented and nonbioaugmented FC and FS soils contaminated with phenol at the concentration 17 mg g)1 (a1, b1), 33 mg g-1 (a2, b2) and 50 mg g)1 (a3, b3). (); control soil; ( ) with phenol; ) with phenol and bacteria (total) and ( ( ) with phenol and bacteria (inoculant).
Fatty acids analysis In the study, the impact of phenol contamination and bioaugmentation with Pseudomonas sp. JS150 on soil fatty acid proles was analysed. To make the comparison of fatty acid methyl esters (FAME) proles, all extracted fatty acids were grouped into ve major classes: straightchain, branched, hydroxylated, cyclopropane and unsaturated fatty acids. Both phenol and bioaugmentation inuenced the soil FAME proles that changed over the experimental period. The most visible changes under phe-
nol exposure between augmented and nonbioaugmented soils involved the abundance of branched and cyclopropane fatty acids. For example, at the beginning of the experiment in FC17+B soil, the percentage of branched fatty acids in FAME proles composed 2247%, while in nonbioaugmented, it was signicantly lower and constituted 348% of total fatty acids only (Table 4). In comparison, in control soil (nonbioaugmented and nonpolluted), their content reached the value of 283%. Over phenol degradation, the content of branched fatty acids increased in FC17+B to 2853% on day 8 and to
Table 4 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FC soil during phenol degradation at the concentration 17 mg g)1
Total fatty acids (weight %) Saturated Total cyclopropane 1238 1538 1465 1725 1921 1498 1669 ND 1648 ND Cyclopropane 17:0 cy 1238 1538 051 066 017 099 000 ND 357 ND 19:0 cy x10c 000 000 1414 1659 1904 1399 1669 ND 1291 ND Unsaturated 4880 1980 3778 1345 3045 1932 3148 ND 4032 ND
Time Day 1 Day 4 Day 8 Day 16 Day 24
FC soil FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B
Straight-chain 3305 3796 3649 3923 3807 3878 3748 ND 3466 ND
Branched 348 2247 803 2853 894 2551 1067 ND 615 ND
Hydroxylated 229 439 305 154 333 141 368 ND 239 ND
Values are the means of three replicates (standard errors <5%). FCP, contaminated and nonbioaugmented soil; FCP+B, contaminated and bioaugmented soil; ND, not determined.
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1068% on day 16 in FC17. Similarly, in FC50+B, the highest increase in percentages of branched fatty acids (2994%) was noticed during the rst 4 days of the incubation and then maintained at the similar level. In contrast, in FC50, content of branched fatty acids increased about four times from day 1 to day 24 and then gradually declined (Table 6). Phenol contamination caused also the appearance of new fatty acids that were not present in untreated soil. They were mainly represented by branched fatty acids such as 13:0 iso, 13:0 anteiso, 14:0 iso, 16:0 anteiso, 17:0 anteiso and hydroxylated 16:0 2OH and 16:0 3OH; however, their contribution in FAME proles was low and did not exceed 1% of total fatty acids (data not shown). The another signicant changes in FAME proles of tested soils were related to cyclopropane fatty acid abundance. In FC soil, independently of phenol concentrations used the content of 17:0 cy strongly declined during the rst 4 days of the experiment. In the following days, its contents in FAME patterns still decreased, even to 0%. Interestingly, the other cyclic fatty acid 19:0 cy x10c appeared only from day 4. In general, its abundance depended on the degree of phenol utilization. In bioaugmented FC soils, the highest contents of 19:0 cy x10c were detected between 4 and 8 days when about 60% of phenol added was degraded. However, in nonbioaugmented FC soil polluted with lower phenol doses (17 and 33 mg g)1), the highest content of this fatty acid was
found when about 50% of phenol was removed (day 8), whereas in soil with phenol at the concentration of 50 mg of g)1, it was found when above 70% of this pollutant was degraded (day 32) (Tables 46). Similar responses of bacterial communities to phenol were found in both augmented and nonbioaugmented FS soil. Observed changes included alterations in the amount of branched and cyclopropane fatty acids. In augmented FS soil, phenol treatment caused the increase in branched fatty acid content from day 1 to day 8 (17 mg g)1) and day 24 (33 and 50 mg g)1) (Tables 79). In nonbioaugmented soil, the abundance of branched fatty acids also increased at the rst days of incubation; however, it was about four times lower as compared to bioaugmented FS soil. During the following days till the end of the experiment in both soils, the amount of branched fatty acids in FAME proles decreased (Tables 79). Changes in cyclopropane fatty acids were related to a decrease in 17:0 cy content and appearance of 19:0 cy x10c over the experimental period. In phenol-polluted soil with the dosages of 17 and 33 mg g)1 and bioaugmented FS soil, the highest abundance of 19:0 cy x10c in FAME proles was observed on day 16 when 6080% of contaminant was degraded, whereas in FS50+B, it was observed on day 56 when phenol was almost completely degraded (Tables 79). No such effect was observed in phenol-contaminated but nonbioaugmented soils. In contrast to contaminated FC soils, in FS soils under phenol
Total fatty acids (weight %) Saturated Total cyclopropane 1233 1549 1702 1663 2085 2006 1962 1492 1626 1435 1333 ND 1257 ND Cyclopropane 17:0 cy 1233 1549 127 041 080 000 101 158 177 351 312 ND 766 ND 19:0 cy x10c 000 000 1575 1622 2005 2006 1861 1334 1449 1084 1021 ND 491 ND Unsaturated 4845 1792 3465 1160 2913 1026 2850 1794 3697 1902 4189 ND 4457 ND
Time Day 1 Day 4 Day 8 Day 16 Day 24 Day 32 Day 36
FC soil FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B
Straight-chain 3355 3811 3686 3994 3709 3811 3764 3650 3518 3702 3488 ND 3411 ND
Branched 341 2411 828 3016 967 3027 1107 2880 847 2767 706 ND 624 ND
Hydroxylated 226 437 319 167 326 130 317 184 312 194 284 ND 251 ND
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Total fatty acids (weight %) Saturated Total cyclopropane 1227 1536 1678 1836 2133 2344 2229 2241 2299 2166 2471 1844 2261 1622 1944 1518 1721 1256 1501 ND 1301 ND Cyclopropane 17:0 cy 1227 1536 056 000 000 000 000 000 000 000 000 000 000 000 000 047 000 226 000 ND 097 ND 19:0 cy x10c 000 000 1622 1836 2133 2344 2229 2241 2299 2166 2471 1844 2261 1622 1944 1471 1721 1040 1501 ND 1204 ND Unsaturated 4840 1881 3618 986 2795 785 2375 805 2263 857 2138 1037 2418 1528 2743 1650 3129 1990 3651 ND 4000 ND
Time Day 1 Day 4 Day 8 Day 16 Day 24 Day 32 Day 40 Day 48 Day 56 Day 64 Day 68
FC soil FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B FCP FCP+B
Straight-chain 3356 3876 3572 4119 3713 4010 3876 4031 3888 4005 3851 4116 3801 3955 3787 3904 3774 3899 3684 ND 3601 ND
Branched 339 2296 835 2994 1093 2809 1240 2866 1273 2889 1266 2919 1257 2802 1241 2816 1110 2701 915 ND 845 ND
Hydroxylated 238 411 297 065 266 052 280 057 277 083 274 084 263 093 285 112 266 154 249 ND 253 ND
exposure, any new fatty acids as compared to untreated FS soil were detected. Discussion In this study, we demonstrated that soil inoculation with Pseudomonas sp. JS150 characterized by high catabolic potential towards many aromatic compounds is an effective way to enhance the rate of phenol removal and soil restoration. Its capability to degrade a wide range of contaminants was achieved by genetic modication through mutagenesis (Haigler et al. 1992). Bacteria from genus Pseudomonas are known to have versatile metabolic capabilities, and therefore, they are often used to increase the efciency of aromatic compounds mineralization in contaminated sites (Stalwood et al. 2005; Das and Mukherjee 2007; Juhanson et al. 2009; Karamalidis et al. 2010; Afzal et al. 2011). Degradation studies revealed that soil bioaugmentation signicantly accelerated the rate of phenol degradation as compared to nonbioaugmented soils. While in FC50 soil
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this process lasted 68 days, in FC50+B, it proceeded 2 weeks shorter. In FS50+B, phenol was completely degraded 24 days faster than in nonbioaugmented soil. The enhanced catabolic potential by soil inoculation with specialized bacterial single strain was well documented in many studies. For example, Teng et al. (2010) reported that soil bioaugmentation by Paracoccus sp. strain HPD-2 decreased total polycyclic aromatic hydrocarbons (PAHs) concentrations from 9942 to 7638 lg kg)1 dry soil after 28 days, whereas it decreased only to 9601 lg kg)1 in noninoculated control soil. In other study, Wang et al. (2004) showed that after introduction of Burkholderia picketti into soil, quinoline at the concentration of 1 mg g)1 soil was completely removed within 6 and 8 h with and without combined effect of indigenous bacteria. The nal effect of soil bioaugmentation depends on the ability of inoculant to colonize soil niches and compete with autochthonous micro-organisms. Data on the survival of introduced cell are contradictory. In most studies, a sharp decrease in inoculant number was observed immediately after inoculation (between 4 and 7 days after
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Table 7 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FS soil during phenol degradation at the concentration 17 mg g)1
Total fatty acids (weight %) Saturated Total cyclopropane 1100 1241 1195 1121 1078 1115 1116 1225 1163 1021 1190 ND 1228 ND 1200 ND 1089 ND Cyclopropane 17:0 cy 1100 1241 1195 1121 577 394 617 166 712 268 916 ND 1001 ND 1010 ND 1015 ND 19:0 cy x10c 000 000 000 000 501 721 499 1059 451 753 274 ND 227 ND 190 ND 074 ND Unsaturated 4353 2024 4281 2051 4220 1538 3843 1172 3803 1771 3875 ND 3908 ND 3905 ND 4141 ND
Time Day 1 Day 4 Day 8 Day 16 Day 24 Day 32 Day 40 Day 48 Day 56
FS soil FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B
Straight-chain 3926 3866 3870 4006 4013 4406 4264 4738 4244 4533 4176 ND 4154 ND 4162 ND 4065 ND
Branched 621 2681 654 2721 689 2813 777 2672 790 2559 759 ND 710 ND 733 ND 705 ND
Hydroxylated 000 148 000 101 000 128 000 193 000 116 000 ND 000 ND 000 ND 000 ND
Values are the means of three replicates (standard errors <5%). FSP, contaminated and nonbioaugmented soil; FSP+B, contaminated and bioaugmented soil; ND, not determined.
inoculation) and then maintained at the similar level for a long time, while others found that number of introduced cell slightly decreased or even increased over time. In our studies, the number of Pseudomonas sp. JS150 instantly decreased during the rst 4 days in contaminated FC and FS soils. The observed decrease depended on the concentrations of phenol added. The higher phenol doses were applied, and the higher cell count decline was observed. Similarly, initial decreasing of CFU of Pseudomonas aeruginosa was found by Nasseri et al. (2010), who studied the effect of bioaugmentation on phenanthrene degradation. However, in contrast to our study, the decline in introduced bacteria was followed by the 4- to 6-fold increase in bacterial counts during 2 months. As showed by Juhanson et al. (2009), introduced Pseudomonas strains could survive and demonstrate their catabolic traits at phenol-contaminated soil even 40 months after inoculation. The correlation between CFU numbers of inoculants and contamination level was observed by Sejakova et al. (2009) studying the effect of Comamonas testosteroni CCM7530 inoculation on pentachlorophenol (PCP) biotransformation. In bioaugmented Fluvisol soil containing 10 mg PCP kg)1, number of CFUs decreased over 7 days and then increased till day 17, whereas in soil with 100 mg PCP kg)1, number of CFUs rapidly
increased from day 1 to 17. The observed decrease in introduced cells may be explained by the fact that bacterial inoculants cultured in laboratory optimum conditions undergo stress when enter natural soil. The fate of introduced strains depends on several abiotic and biotic factors such as uctuations in temperature, water content, pH, lack of nutrients as well a level of contaminants and interactions with indigenous organisms (Mrozik and Piotrowska-Seget 2010; Tyagi et al. 2011). What is important, stress because of drastic changes in environmental conditions may lead to loss of microbial viability and even death of inoculated cells (Goldstein et al. 1985; van Veen et al. 1997; Liu et al. 2009). Sometimes unfavourable environmental circumstances, especially during the rst days after inoculation, may be a reason that bioaugmentation does not enhance the degradative potential of contaminated soil (Mariano et al. 2009; Silva et al. 2009; Ruberto et al. 2010). A success of microbial survival, phenols removal and subsequently the efciency of their degradation were strongly correlated with soil organic matter content. In our study, we revealed that phenol biodegradation rate was signicantly faster in soil with higher organic matter content what had especially seen in soils polluted with the highest phenol dose. The organic matter, especially
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Total fatty acids (weight %) Saturated Total cyclopropane 1141 1236 1201 1241 1063 1123 1216 1255 1263 1206 1270 1227 1142 1202 1009 1198 1090 ND 1040 ND Cyclopropane 17:0 cy 1141 1236 1201 1241 419 382 172 098 077 155 000 411 094 513 194 577 515 ND 416 ND 19:0 cy x10c 000 000 000 000 644 741 1044 1157 1186 1051 1270 816 1048 689 815 621 575 ND 624 ND Unsaturated 4269 1862 4013 1162 3694 904 3130 776 2938 961 2755 1290 3047 1379 3130 1581 3015 ND 3605 ND
Time Day 1 Day 4 Day 8 Day 16 Day 24 Day 32 Day 40 Day 48 Day 56 Day 64
FS soil FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B
Straight-chain 3966 3947 4074 4584 4277 4694 4455 4699 4499 4691 4671 4411 4511 4455 4670 4216 4680 ND 4411 ND
Branched 624 2801 712 2911 966 3101 1199 3104 1300 3142 1304 2916 1300 2805 1191 2844 1115 ND 944 ND
Hydroxylated 000 154 000 102 000 178 000 166 000 000 000 156 000 159 000 161 000 ND 000 ND
humic substances, are considered to be growth-promoting and protective factors against harmful organic compounds for soil micro-organisms. The protective character of humic acids is connected with their ability to bind recalcitrant contaminants, reduce their bioavailability and limit the toxicity for soil microbiota (Nam and Kim 2002). It is known that phenolic carbon, which is enzymatically incorporated into humic acids, is much more stable against biodegradation in soil than the carbon of free phenols (Vinken et al. 2005). In this study, the content of humic carbon did not change during biodegradation experiments suggesting that aromatic carbon did not strongly incorporate into humic substances. Phenol could reversibly associate with humic substances and after releasing phenolic particles were subjected to biodegradation. Similarly, we did not observe signicant changes in the humic material content during phenol dissipation in contaminated sterile MF and WM soils inoculated with Pseudomonas stutzeri (Mrozik et al. 2008). In many environmental studies, analysis of FAME proles has been used to determine changes in microbial populations and their activity in soil (Kozdroj and van Elsas 2001), wastewater treatment (Quezada et al. 2007)
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and sediments (Dunn et al. 2008). Moreover, alterations in FAME patterns obtained directly from soil may indicate the response of microbial communities to natural and antropogenic stress (Kozdroj 2000; Islam et al. 2009). In this study, we successfully applied FAME analysis to assess the progress in phenol degradation in both nonbioaugmented FC and FS soils and bioaugmented with Pseudomonas sp. JS150. Phenol contamination as well as soil inoculation shifted microbial communities FAME proles, and the most signicant alterations were connected with the distribution of branched and cyclopropane fatty acids. Studying phenol biodegradation in sterile soils inoculated with P. stutzeri and Pseudomonas sp. CF600, we found that cyclopropane fatty acid 19:0 cy x8c apparent at substantial amount when more than 50% of phenol added was degraded (Mrozik et al. 2008, 2010). It was interesting to check whether the similar effect can occur in nonsterile soil bioaugmented with Pseudomonas sp. JS150. Results of our study conrmed that appearance of a new cyclopropane fatty acid 19:0 cy x10c is connected with the degree of phenol removal. Depending on soil type and phenol contamination in both augmented and nonbioaugmented
A. Mrozik et al.
Total fatty acids (weight %) Saturated Total cyclopropane 1114 1273 1217 1189 1227 1189 1366 1368 1399 1511 1419 1521 1472 1533 1677 1544 1717 1562 1377 1311 1185 1191 1155 ND 1101 ND 1205 ND Cyclopropane 17:0 cy 1114 1273 1217 1189 411 000 209 000 000 000 000 000 000 000 000 000 000 000 000 000 157 000 244 ND 288 ND 457 ND 19:0 cy x10c 000 000 000 000 816 1189 1157 1368 1399 1511 1419 1521 1472 1533 1677 1544 1717 1562 1377 1311 1028 1191 911 ND 813 ND 748 ND Unsaturated 4395 1932 3753 1243 3113 957 2664 766 2655 774 2525 655 2481 725 2148 850 2147 893 2388 1188 2999 1395 3229 ND 3285 ND 3508 ND
Time Day 1 Day 4 Day 8 Day 16 Day 24 Day 32 Day 40 Day 48 Day 56 Day 64 Day 72 Day 80 Day 88 Day 96
FS soil FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B FSP FSP+B
Straight-chain 3878 3877 4164 4520 4497 4640 4590 4624 4525 4502 4598 4694 4563 4656 4631 4517 4559 4512 4629 4477 4401 4405 4400 ND 4414 ND 4276 ND
Branched 613 2764 866 2951 1163 3124 1370 3166 1421 3121 1468 3043 1484 2995 1544 2991 1577 2951 1606 2912 1415 2888 1216 ND 1200 ND 1011 ND
Hydroxylated 000 154 000 097 000 090 000 076 000 092 000 084 000 091 000 098 000 082 000 112 000 121 000 ND 000 ND 000 ND
soils, the highest content of this fatty acid in FAME proles was detected when phenol was biodegraded in 60 80%. Our earlier results (Mrozik et al. 2008, 2010) showed that bacteria from genus Pseudomonas used cyclopropane ring formation in response to high phenol concentration. Therefore, we indicate that it is an important adaptive mechanism of bacteria to chemical stress although the protective role of cyclopropane fatty acids in cytoplasmic membrane is not understood in details. Moreover, cyclopropane fatty acids are very useful markers for monitoring phenol degradation in soil. Essential changes in FAME patterns of sterile and nonsterile soils inoculated with different strains from genus Pseudomonas included also the alterations in branched fatty acid contents (Mrozik et al. 2008, 2010). In this study immediately after inoculation, the abundance of
this fatty acid group increased and then gradually declined in parallel with the overall decrease in phenol concentrations. The active remodelling of fatty acid composition including alterations in cyclopropane and branched fatty acids in the membrane structure and regulation of membrane functions allows bacteria to adapt and survive in unfavourable conditions. In conclusion, Pseudomonas sp. JS150 did not lose capability of phenol degradation after soil inoculation, well adapted to stress conditions and survived over the experimental time. For these reasons, it seems to be an attractive micro-organism for bioremediation technology to enhance the capability of soil towards phenol degradation. Moreover, cyclopropane and branched fatty acid contents are sensitive probes for monitoring the progress of phenol removal from soil.
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Acknowledgements This work was supported by grant no. N N305 049536 from the Polish Ministry of Science and Higher Education. References
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Enhanced of Phenol Degradation by Soil Bioaugmentacion With Pseudomonas SP JS150

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Enhancement of phenol degradation by soil bioaugmentation with Pseudomonas sp. JS150

Table 1 Characteristics of soils

*Total humic and fulvic acids.

Phenol (mg g1)

Values are the means of three replicates (standard errors <5%).

Values are the means of three replicates (standard errors <5%).

3 4 Phenol (mg g1)

109 107 105 103

109 107 105 103 109 107 105 103 0

Time Day 1 Day 4 Day 8 Day 16 Day 24

Straight-chain 3305 3796 3649 3923 3807 3878 3748 ND 3466 ND

Branched 348 2247 803 2853 894 2551 1067 ND 615 ND

Hydroxylated 229 439 305 154 333 141 368 ND 239 ND

Time Day 1 Day 4 Day 8 Day 16 Day 24 Day 32 Day 36

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