Water Air Soil Pollut (2014) 225:1980 DOI 10.1007/s11270-014-1980-3

Potential Use of Newly Isolated Bacterial Strain Ochrobactrum anthropi in Bioremediation of Polychlorinated Biphenyls Slavomíra Murínová & Katarína Dercová

Received: 16 January 2014 / Accepted: 28 April 2014 / Published online: 24 May 2014 # Springer International Publishing Switzerland 2014

Abstract The degradation ability of newly isolated bacterial strain Ochrobactrum anthropi toward polychlorinated biphenyls (PCBs) was examined under aerobic conditions. The strain was isolated from historically PCB-contaminated sediments from Strážsky canal in eastern Slovakia, surrounding of the former PCB producer. The degradation ability of the strain was enhanced by addition of other substrates and degradation inducers—biphenyl, glucose, both biphenyl and glucose, ivy leaves, and pine needles. The adaptation of cells membrane toward PCBs in the presence of abovementioned substrates was evaluated with the changes in fatty acid composition (membrane saturation, cis–trans isomerization, and changes in branched fatty acids synthesis). The highest induction of PCB degradation and lowest cell adaptation in liquid medium was achieved using ivy leaves. On the other hand, lowest degradation was achieved when PCBs were added alone. Similar low degradation was observed in the presence of glucose addition together with biphenyl. Contrary, highest growth stimulation under the applied condition was observed. Obtained results indicated that addition of glucose together with biphenyl induced PCB S. Murínová : K. Dercová Department of Biochemical Technology, Institute of Biotechnology and Food Science, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia S. Murínová (*) Water Research Institute, Nábrežie arm. gen. L. Svobodu 5, 812 49 Bratislava, Slovakia e-mail: [email protected]

degradation via bacterial growth stimulation, not via the induction of activity of degradation enzymes. Cut ivy leaves (containing terpenoic compounds serving as degradation inducer and structural analog of biphenyl) increased PCB removal from contaminated sediment by O. anthropi. Results indicate the degradation ability of O. anthropi toward penta-, hexa-, and hepta-chlorinated PCB congeners. The degradation of congeners with more than five chlorine atoms per molecule was detected in higher extent compared to dichlorinated congeners. Keywords Bioaugmentation . Biodegradation . Bioremediation . Membraneadaptation . Polychlorinated biphenyls

1 Introduction Long-term industrial production and use of polychlorinated biphenyls (PCBs) have led to a massive contamination of the environment. Due to their physicochemical properties as thermal and chemical stability, low solubility in water, and bioaccumulation in living organisms, they had spread to long distances from contamination sources worldwide (Hiller et al. 2010; Brázová et al. 2012; Langer et al. 2012). Slovakia belonged to top ten world PCB producers with total production of 21,500 t. The leakage of produced mixtures from the production plant in Strážske through Strážsky canal and untreated liquid waste into the water reservoir Zemplínska Šírava caused dangerous

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Water Air Soil Pollut (2014) 225:1980

contamination of Laborec River and Zemplínska Šírava lake in Eastern part of Slovakia (Dercová et al. 2008). PCBs constitute a class of 209 congeners with biphenyl core and varying numbers of chlorine atoms per molecule that are structurally related (Tříska et al. 2004; Field and Sierra-Alvarez 2008; Furukawa and Fujihara 2008). PCBs represent potential health risks for living organisms due to their lipophilicity, toxicity, and possible carcinogenic properties (Sylvestre 1995; Komancová et al. 2003). PCBs can stimulate various metabolic responses in human body because of their similarities with endocrine hormones. Moreover, PCBs belong to endocrine disruptors (Langer et al. 2012). Physical and chemical decontamination processes have limitations due to their costing and PCB-concentrationdependent effectiveness. Bioremediation has long been seen as a cost-effective way to eliminate diffusive contamination of PCBs in various environmental matrices, e.g., soils, sediments, and sludge (Hickey 1999; Tandlich et al. 2011). It is a set of techniques that improve the degradation capacity of contaminated areas. They use bioaugmentation strategy (introduction of specific degradable strains or consortia of microorganisms) or biostimulation strategy (introduction of nutrients, inducers, and oxygen) (Mrozik and Piotrowska-Seget 2010). Important factor for successful bioaugmentation is the selection of proper bacteria that can not only degrade contaminants but can also adapt to adverse environment, usually higher toxicity of the contaminated area (Zorádová-Murínová et al. 2012). The stimulation of PCB removal can be achieved by the addition of structurally related compounds as biphenyl or terpenoic compounds. These chemicals can stimulate the activity of enzymes of aerobic upper degradation pathway (Fig. 1).

Other mechanism of stimulation of PCB removal with easily utilized carbon sources is based on the enhancement of bacterial growth. Glucose may possibly have a catabolic repression on PCB degradation; however, it stimulates bacterial growth. Therefore, glucose was added to mineral media to evaluate whether the stimulation of bacterial growth (more biomass, higher degradation) can overcome their possible inhibition of degradation enzymes. Natural plant material as ivy leaves or tangerine peel can probably stimulate both degradation ability (terpenes) and bacterial growth (plant oils) (Gilbert and Crowley 1997; Hernandez et al. 1997; Dzantor et al. 2002; Kwon et al. 2009; Tortella et al. 2013). These materials can possibly affect the adaptation responses of bacterial cells. Hydrophobic compounds as PCBs can incorporate into cytoplasmic membrane and increase the membrane fluidity. Bacterial cells must counteract this effect to provide cell survival. Several adaptation mechanisms have been evolved to diminish membrane fluidity and dispose hydrophobic pollutants out of the cell structures. The most important adaptation is realized through an increase in membrane saturation, isomerization of cis-unsaturated fatty acids (UFA) into their trans isomers, and stimulation of the synthesis of iso-branched fatty acids at the expense of anteiso ones (Weber and de Bont 1996; Heipieper et al. 2004; Mrozik et al. 2005). The purpose of this study was to evaluate the degradation ability and adaptation responses of a newly isolated strain Ochrobactrum anthropi in defined liquid mineral medium and in the contaminated sediment on laboratory scale. For the stimulation of PCB removal, various degradation inducers with probably different stimulation mechanisms were added. The best degradation inducer observed using liquid mineral medium was

Fig. 1 Upper biphenyl/PCB degradation pathway of aerobic bacteria encoded by the bph locus. Metabolites: 1 biphenyl, 2 2,3dihydro-2,3-dihydroxybiphenyl, 3 2,3-dihydroxybiphenyl, 4 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, 5 benzoic acid. Enzymes: A biphenyl-2,3 dioxygenase (BphA), B 2,3-dihydro-2,3-

dihydroxybiphenyl-2,3 dehydrogenase (BphB), C 2,3dihydroxybiphenyl-1,2 dioxygenase (BphC), D 2-hydroxy-6oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD) (modified according to Seeger et al. 1995; Furukawa and Fujihara 2008)

Water Air Soil Pollut (2014) 225:1980

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tested for the stimulation of bioremediation of PCBcontaminated sediments in a microcosm (under laboratory conditions) by O. anthropi.

2 Materials and Methods 2.1 Chemicals and Media Growth medium—culture broth no. 2 (Imuna, Slovakia), minimal mineral medium containing 1 g l−1 (NH4)2SO4, 2.7 g l−1 KH2PO4, 5.2 g l−1 NaHPO4 ·2 H2O, 0.2 g l−1 MgSO4 ·7 H2O, 0.01 g l−1 FeSO4 ·7 H2O and 0.03 g l−1 Ca(NO3)2 ·4 H2O (Lachema Brno, Czech Republic), a commercial mixture of PCBs— DELOR 103, containing 40–42 % (w/v) of bound chlorine (Chemko Strážske, Slovakia), Florisil (VWR), ivy leaves (Hedera helix), pine needles (Pinus sylvestris) standard mixture of PCB congeners (Dr. Ehrenstorfer, Germany), n-hexane, biphenyl, glucose, NaCl, and dimethylsulfoxide (DMSO) (Mikrochem, Slovakia) were used.

2.2 Microorganism Used for Bioaugmentation O. anthropi was isolated from contaminated sediment from Strážsky canal with the same method as described in Dudášová et al. (2014). The strain was identified and has been maintained at the Czech Collection of Microorganisms (CCM), Masaryk University, Czech Republic, for safety storage without numbering. Bacterial strain was cultivated in 200 ml of growth medium on a rotary shaker 180 rpm at 28 °C in the dark. Overnight culture was centrifuged and washed three times with saline solution. Twenty milliliters of saline solution was added into the sediment and mixed. A total of 0.1, 0.2, 0.4, 0.8, 1, 1.4, and 2 ml of solution were diluted with saline solution into 10ml volume. The absorbance of each sample was measured spectrophotometrically. Subsequently, every solution was purred into Petri plate and dried for 6 h at 60 °C. Dry plates were weighed, and the correlation between the biomass concentration and absorbance (A620nm) was measured with a final equation: Abs ¼ 1:886⋅c þ 0:0683

ð1Þ

2.3 Biodegradation of PCBs in Defined Liquid Mineral Medium Bacterial inoculum was prepared in 200 ml of growth medium for 48 h on a rotary shaker 180 rpm at 28 °C in the dark. Biomass was harvested by centrifugation and washed three times with saline solution. The concentration of biomass in 20 ml of saline solution was measured according to Eq. 1. Biodegradation of PCBs was carried out in 500-ml Erlenmeyer flasks equipped with a glass columns filled with SILIPOR C18 sorbent and closed with a cotton wool stopper to achieve sterile environment. Each degradation flask contained 50 ml of minimal mineral medium, 1 g l−1 of bacterial strain, and 100 mg l−1 of DELOR 103. Biphenyl, glucose, or ivy leaves were added into a flask to observe the stimulation of PCB removal. Biphenyl in a final concentration 10 mg l−1, glucose in a final concentration 5 g l−1, and 3 g of cut ivy leaves (H. helix) or cut pine needles (P. sylvestris) were added into appropriate flask. The ivy leaves mainly consist of bidesmosidic triterpene saponins with hederagenin, oleanolic acid, and bayogenin (2β-hydroxyhederagenin) as aglycones and flavonoids such as quercetin and kaempferol (Wichtl 2004). The oil of pine needles consists of monoterpenes α-pinene (20.7–69.1 %), camphene (0.1–1.7 %), β-pinene (0.17–11.6), and αphellandrene (0.1–31.4 %) (Berta et al. 1997). Fresh ivy leaves and pine needles were washed with distilled water, dried at room temperature, cut into small pieces (diameter 1 mm), and subsequently added into liquid medium. Five different sets of experiments were used as follows: PCBs alone, glucose with PCBs, biphenyl with PCBs, ivy leaves with PCBs, and pine needles with PCBs. Three parallel sets for each experiment ran to enable statistical data evaluation. The flasks were incubated in the dark at 28 °C on a rotary shaker for 7 days. Abiotic control with PCBs and without biomass ran in parallel. Biodegradation amount was calculated as the initial amount minus the amount eliminated by abiotic degradation. Elimination of PCB congeners was evaluated and expressed as percentage of the final amount minus the abiotic decay and evaporation according to equation: D¼

A − ðX þ Y Þ ⋅ 100% A

ð2Þ

where D stands for the degradation of the particular PCB congener, A is the congener amount determined under

1980, Page 4 of 16

abiotic condition at the end of experiment (μg), X is the amount of congener in the cultivation medium at the end of exposure (μg), and Y is the amount of PCB congener evaporated during the experiment (μg). 2.4 Biodegradation of PCBs in the Contaminated Sediment Sediment samples originated from Strážsky canal from the site downstream the junction with the effluent canal from sewage treatment plant (Fig. 2). Sediment had been historically contaminated with PCBs because of the production of commercial mixtures of PCBs in factory Chemko situated in town Strážske located upstream. Initial amount of PCB contamination in sediment and estimate of accessible fraction of selected congeners in sediment are shown in Table 1. Accessibility of PCB congeners was measured using mild supercritical fluid extraction according to Hallgren et al. (2006). Three parallel samples were measured to achieve statistic evaluation. Sediment contained 1.74 % of organic matter, 4.40 g kg−1 of total nitrogen, and 1.82 g kg−1 of total phosphorus. Before the bioremediation experiment, whole wet sediment was sieved through the sieve with 60-μm mesh size and dried for 4 days at laboratory

Fig. 2 Sediment sampling site situated in the Eastern Slovakia

Water Air Soil Pollut (2014) 225:1980

temperature. Five grams of sediment sample was transported into 50-ml Erlenmeyer flask and poured with 15-ml of minimal mineral medium. Three different sets of experiments ran in parallel as follows: control, sediment bioaugmented with O. anthropi (10 mg kg−1 sedim ent), and sediment bioaugmented and biostimulated with O. anthropi (10 mg kg−1 sediment) and ivy leaves (75 mg equally 15 g kg−1 sediment). To prevent bacterial and fungal growth, 2 ml of 2.5 % sodium azide was added into the control experiment. Cultivation flasks were closed with glass columns with 1 g of SILIPOR C18 to measure PCB evaporation. Columns were covered with foil and incubated stationary in the dark at 28 °C. Water evaporation was controlled every week with subsequent immixture. Whole cultivation flask of each set of experiment was analyzed immediately after the preparation and after 7, 28, 42, 56, 70, and 85 days. Three sets of each experiment ran in parallel. 2.5 PCB Extraction The degradation experiment in the defined liquid mineral medium prolonged 7 days. After this time, whole flasks were taken from rotary shaker and enriched with 5 ml of n-hexane. Ten minutes of ultrasonic bath was

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Table 1 Total concentration of PCB congeners in the sediment and accessibility of selected congeners, determined by supercritical fluid extraction according to Hallgren et al. (2006) Congener no. according to IUPAC

PCB8 2,4′-di-CB

PCB28 2,4,4′tri-CB

PCB5 2 2,2′,5,5′tetra-CB

PCB101 2,2′,4,5,5′penta-CB

PCB118 2,3′,4,4′, 5-penta-CB

Concentration (mg PCB kg sediment−1) Accessibility (%)

0.84±0.05

4.18±0.88

6.77±0.38

3.46±0.55

1.35±0.05 68±21

Concentration (mg PCB kg sediment−1) Accessibility (%)

88±23

92±13

81±13

65±17

PCB138 2,2′,3,4, 4′,5-hexa-CB 4.03±0.14

PCB153 2,2′,4,4′, 5,5′-hexa-CB 1.36±0.35

PCB180 2,2′,3,4, 4′,5,5′-hepta-CB 3.80±0.65

PCB203 2,2′,3,4,4′, 5,5′,6-octa-CB 1.37±0.35

65±25

67±21

63±29

60±20

used to release PCBs from bacterial cells. The extraction was performed in separatory funnel with 2-min shaking. After extraction, anhydrous K2CO3 was added to reduce the foam originated from biomass. The n-hexane layer was collected, and the aqueous layer was returned into separatory funnel and extracted again with 15 ml of nhexane. n-Hexane layers were dried with anhydrous Na2SO4 and filled with n-hexane to a volume 25 ml. PCBs were analyzed by gas chromatography (GC) with electron capture detection (ECD). The effectiveness of PCB extraction was determined by the addition of PCB209 congener as internal standard into MM medium prior to ultrasonic treatment. Evaporated PCB congeners captured into SILIPOR C18 sorbent were extracted with 8 ml of n-hexane and analyzed by GC-ECD. Samples of used contaminated sediment were transferred into Petri plate and dried for 4 days at room temperature after remediation. Two grams of homogenous sediment sample of each flask was extracted with Soxhlet apparatus with 60 ml of n-hexane for 4 h. Danube sediment with incorporation of 100 ng of standard mixture of PCB congeners was extracted to determine extraction recovery. Hexane was evaporated on a vacuum rotary vaporizer to a volume of 1–2 ml. The cleanup process included 45-min ultrasonic bath with copper powder. Subsequently, the extract was filtrated through the Florisil (30–60 US mesh) column. Extract volume was adjusted to 1 ml and analyzed by GC. 2.6 PCB Analysis Gas chromatograph (7890A Agilent Technologies, USA) with ECD (300 °C, make up gas N 2 at

25 ml min−1) and fused-silica capillary column (Agilent 19091 J-413, USA) with HP-5 stationary phase (30 m× 0.32 mm×0.25 μm) was used for analyzing PCBs. Helium was used as a carrier gas (68 kPa, 1.8 ml min−1, split–splitless inlet mode). The column was held at 80 °C for 1 min and then raised to 160 °C at 30 °C min−1, held for 1 min, and then raised to 260 °C at 4 °C min−1, held for 3 min. The injector temperature was 250 °C. Peak identification and calibration were measured on Agilent ChemStation A.10.02 [1757] according to the standard mixture of PCB congeners. 2.7 Bacterial Cell Enumeration The number of vital cell of O. anthropi in liquid media and total heterotrophic bacteria in sediment samples were quantified using the drop plate method. Solid minimal mineral medium supplemented with 15 g l−1 Nobel agar (Difco) was used for bacterial growth. One milliliter of wet sediment after degradation or 1 ml of liquid media after degradation was diluted with particular volume of sterile minimal mineral medium and applied to Petri plates. Plates were incubated at 28 °C for 48 h in the dark with subsequent colonies counting. 2.8 Design of the Adaptation Response Experiment Bacterial inoculum was prepared equally to that used for biodegradation experiment. The experiment for adaptation responses was carried out in 500-ml Erlenmeyer flasks closed with a cotton wool stopper to achieve sterile environment. Each cultivation flask contained 200 ml of minimal mineral medium and 1 g l−1 of bacterial strain and 100 mg l−1 of DELOR 103. A control set of experiments containing 5 g l−1 glucose

1980, Page 6 of 16

as carbon source was run in parallel. Biphenyl, glucose, ivy leaves, and pine needles were added equally as in biodegradation experiment. Five experimental sets equal to those run in degradation experiment were used in triplicate to provide statistic evaluation. After 7-day cultivation, bacterial biomass was harvested by centrifugation (1,500×g for 5 min), subsequently dried at 65 °C for 10 h, and weighted. Lipids from the homogenized dry biomass were isolated, fractionated by thin-layer chromatography according to Zorádová-Murínová et al. (2012). 2.9 Fatty Acid Analyses Fatty acid components of total lipids and membrane lipids—phosphatidylcholine (PC) and phosphatidylethanolamine (PE)—were analyzed by GC as their methyl esters using an Agilent model 6890 gas chromatograph (Agilent Technologies, USA) equipped with a 60 m× 0.25 mm capillary column DB-23 (film thickness of 0.25 μm, Agilent Technologies, USA) and a flame ionization detector (FID). The conditions for the analyses were defaulted according to Zorádová-Murínová et al. (2012). The fatty acid methylester peaks were identified by comparison to the authentic standards of C4 −C24 fatty acid methylester mixture (Supelco, USA) and evaluated by ChemStation B.01.03 (Agilent Technologies, USA). 2.10 Statistical Analysis All experiments were performed in triplicate. Data were processed using standard software packages Microsoft Excel and Kruskal–Wallis one-way analysis of variance on ranks (Sigma plot) for statistical evaluation. The differences in the mean values among the treatment groups are greater than would be expected by chance (there is a statistically significant difference), if the p<0.05.

3 Results 3.1 Adaptation Responses to PCBs The bacterial cell adaptation responses toward PCBs were compared with those of cells grown on glucose carbon source in the absence of PCBs. The initial

Water Air Soil Pollut (2014) 225:1980

concentration of O. anthropi was 1 g l−1 (colonyforming units (CFU)×108 × ml−1 ± SD 18.13 ±2.65). The concentration of bacterial strain after 7-day degradation changed depending on substrate added at the beginning of the experiment (Fig. 3a). Some aerobic bacteria are able to grow on PCBs as a sole carbon source (Kim and Picardal 2000, 2001; Parnell et al. 2010). However, according to Fig. 3a, it seems that O. anthropi can only co-metabolize PCBs using another organic compound in energy acquisition and cofactor recovery. Bacterial growth after the 7-day cultivation reached 1.8 g l−1 (CFU×108 ×ml−1 ±SD 54.56±8.72). This growth level was similar with the level obtained when bacterial strain grown in the presence of PCBs together with glucose and biphenyl. The main adaptation responses, increase saturation, cis–trans isomerization, and changes in branched fatty acids were evaluated in total lipids (TLs), main membrane fraction PE, and minor membrane fraction PC at the end of experiment. An increase in TL saturation compared to control was observed in the presence of PCBs in all experimental sets (Fig. 4a) (p<0.05). The highest saturation in TL of bacterial cells was measured when PCBs were present alone, together with biphenyl, or together with both glucose and biphenyl. The saturation in membrane PE was the topmost under these conditions as well (p <0.05). The minor membrane fraction PC revealed the highest saturation in the presence of natural matrices (ivy leaves or pine needles). The trans/cis ratio expresses the level of cis–trans isomerization of bacterial fatty acids. The ratio in TL exceeded control value only when cells were grown in the presence of PCBs together with biphenyl (Fig. 4b). Membrane adaptation by cis–trans isomerization was present in cells grown on PCBs alone or together with synthetic compounds (glucose, biphenyls, or both). The presence of both natural materials decreases this isomerization in both membrane fractions compared to control. The changes in synthesis of branched fatty acids could be also found under the adverse conditions. The lowest the anteiso/iso ratio is the higher effort cells must provide to adapt to toxic compounds. The lowest anteiso/iso ratio in TL was observed when PCBs were added alone or together with glucose or biphenyl or both (p<0.05) (Fig. 4c). Contrary, ivy leaves together with PCBs increased this ratio in all lipid fractions. The highest increase was observed in the main membrane fraction PE (p<0.05).

Water Air Soil Pollut (2014) 225:1980

Page 7 of 16, 1980

biomass concentration (g l

−1

)

2 1.8 1.6

A

1.4 1.2 1 0.8 0.6 0.4 0.2 0 PCBs

PCBs+glc

PCBs+bip

PCBs+bip+glc

PCBs+ivy leaves

PCBs+pine needles

PCBs+glc

PCBs+bip

PCBs+bip+glc

PCBs+ivy leaves

PCBs+pine needles

100

degradation efficiency (%)

90 80

B

70 60 50 40 30 20 10 0 PCBs

Fig. 3 Bacterial growth of O. anthropi after 7 days of cultivation in mineral medium (a) and the degradation efficiency (degradation of PCBs normalized to 1 g l−1 biomass) (b) in the presence of glucose (PCBs + glc) and biphenyl (PCBs + bip), the combination

of glucose and biphenyl (PCBs + bip + glc), plant terpenes as inducers (in the form of cut ivy leaves (PCBs + ivy leaves) or cut pine needles (PCBs + pine needle)), and in the control experiment (contained PCBs alone, PCBs)

3.2 PCB Degradation in the Defined Liquid Mineral Medium

and degradation ability were observed in the presence of ivy leaves as well as pine needle addition. Bacterial growth in the presence of ivy leaves was almost three times higher compared to that in control (addition of PCBs alone). Moreover, the degradation range was the highest among all experimental sets and exceeded 53 % (p<0.05) of the initial amount of PCBs. Other natural material, pine needles, stimulated bacterial growth similarly as ivy leaves (2.4 higher biomass in comparison with control). The degradation efficiency with pine needle addition reached 45 %.

The degradation of PCBs was examined in the minimal mineral medium at pH 7.5 and 28 °C. The amount of PCBs degraded by O. anthropi without any other carbon source (glucose) or degradation inducer (biphenyl) slightly crossed 20 %. Lower degradation ability was observed only in the presence of both glucose and biphenyl addition (p<0.05). Synergic effect of both substrates seems to be appropriate for bacterial growth (Fig. 3a), however, did not stimulate the activity of degradation enzymes, as the degradation efficiency (PCBs normalized to biomass unit) reached only 17 % (Fig. 3b). The total amount of PCB degraded under abovementioned conditions was higher (30.4 %) probably due to a higher biomass amount. Biphenyl induced probably the activity of degradation enzymes, as the degradation efficiency reached 42.5 %, although the bacterial growth was not positively influenced at all. Positive results for the stimulation of bacterial growth

3.3 The Congener Specification of PCB Degradation Figure 5a represents the stimulation of decrease of PCB congeners of different groups with addition of glucose, biphenyl, both glucose and biphenyl, ivy leaves, and pine needles. This stimulation was compared with control containing DELOR 103 as a sole carbon and energy source. Figure 5a shows the degradation efficiency and the induction ability of various primary substrates

1980, Page 8 of 16

Water Air Soil Pollut (2014) 225:1980 3.5

A

TL PE

saturated/unsaturated FA ratio

3

PC 2.5 2 1.5 1 0.5 0 cont

0.7

PCBs

PCBs+glc

PCBs+bip

PCBs+bip+glc

PCBs+ivy leaves

TL

B

PE

0.6

trans/cis UFA ratio

PCBs+pine needles

PC

0.5 0.4 0.3 0.2 0.1 0 cont

3

PCBs

PCBs+glc

PCBs+bip

PCBs+bip+glc

PCBs+ivy leaves

C

TL PE

2.5

anteiso/iso FA ratio

PCBs+pine needles

PC

2

1.5

1

0.5

0 cont

PCBs

PCBs+glc

PCBs+bip

PCBs+bip+glc

PCBs+ivy leaves

PCBs+pine needles

Water Air Soil Pollut (2014) 225:1980

Page 9 of 16, 1980

ƒFig. 4

The ratio of saturated to unsaturated fatty acids (FA) (a), trans to cis unsaturated fatty acids (UFA) (b), and anteiso to isobranched FA (c) were measured in total lipids (TL) and membrane lipids—phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The cells were cultivated in the presence of PCBs (PCBs), PCBs and glucose (PCBs + glc), PCBs and biphenyl (PCBs + bip), PCBs and both biphenyl and glucose (PCBs + bip + glc), PCBs and ivy leaves (PCBs + ivy leaves), or PCBs and pine needles (PCBs + pine needles). Control experiment contains only glucose as carbon source

toward the PCB degradation enzymes. Figure 4b shows the overall degradation of PCBs measured in the experiment. This figure could indicate the stimulation of PCB

PCB

PCB+glc

PCB+bip

degradation through the growth stimulation. The comparison of Fig. 5a, b suggests the mechanism of degradation induction. According to Fig. 5a, none of the primary substrates used in this study revealed stimulation effect to degradation of hepta-chlorobiphenyls (hepta-CBs) via the induction of activity of degradation enzymes. However, this stimulation mechanism was achieved with biphenyl or natural material (ivy leaves and pine needles) addition in all other congener groups (except for hepta-CB). Biphenyl revealed the highest induction of tri-chlorobiphenyls (3-CBs) degradation via the induction of the activity of degradation enzymes

PCB+glc+bip

PCB+ivy leaves

PCB+pine needles

100

degradation efficiency (%)

90

A

80 70 60 50 40 30 20 10 0 di-CB

PCB

tri-CB

PCB+glc

PCB+bip

tetra-CB

penta-CB

PCB+glc+bip

hexa-CB

PCB+ivy leaves

hepta-CB

PCB+pine needles

100

PCBs congener degradation (%)

90

B

80 70 60 50 40 30 20 10 0 di-CB

tri-CB

tetra-CB

Fig. 5 The efficiency of PCB congener degradation (a) and overall PCB congener degradation (b) in liquid MM medium by O. anthropi. The induction of degradation was studied with

penta-CB

hexa-CB

hepta-CB

addition of various primary substrates: glc glucose, bip biphenyl, glc + bip glucose together with biphenyl, PCB + ivy leaves ivy leaves, and PCB + pine needles pine needles

1980, Page 10 of 16

among all studied primary substrates (p<0.05). Ivy leaves likely stimulated the activity of degradation enzymes toward di- and tetra-chlorobiphenyls (di-CBs and tetra-CBs) in highest extent (p<0.05). The stimulation effect of biphenyl, pine needles, and ivy leaves toward penta- and hexa-chlorobiphenyls (penta-CBs and hexaCBs) degradation was comparable. The addition of glucose with biphenyl did not stimulate the degradation via abovementioned mechanisms at all. The degradation efficiency with both biphenyl and glucose addition revealed similar results as were observed in the case of glucose addition. This observation is interesting because much higher degradation efficiency was observed in the presence of just biphenyl for all congener groups except for heptaCBs. Figure 5b shows the predominant degradation of di-, tri-, and tetra-CBs with ivy leave addition and predominant degradation of penta-, hexa-, and hepta-CBs with ivy leaves or pine needle addition. Because of the highest bacterial growth as well as the highest PCB degradation efficiency, we can conclude that natural materials (ivy leaves and pine needles) induce both bacterial growth as well as the activity of degradation enzymes. These stimulators and inducers seem to be appropriate for further bioremediation study in contaminated sediments. It is necessary to mention that the stimulation effect of ivy leaves on bacterial growth and degradation ability was higher in comparison with that of pine needles. The addition of glucose and addition of glucose together with biphenyl revealed 40 to 50 % removal of penta- and hexa-CBs. The overall removal of tetra-, penta-, hexa-, and heptaCBs was lower when biphenyl alone was used compared to other primary substrates. However, the removal of di- and tri-CBs with biphenyl addition was higher than with glucose addition or glucose with biphenyl. Generally, it revealed that glucose is not a convenient inducer of PCB degradation enzymes of O. anthropi, moreover, because of its growth stimulation effects (addition of glucose to mineral medium can lead into higher PCB congener removal). Similar results were observed with combination of glucose and biphenyl addition. Biphenyl in used concentration did not affect bacterial growth positively (comparable growth with the control sample was observed); however, it induced degradation efficiency of biomass because of the stimulation of degradation

Water Air Soil Pollut (2014) 225:1980 Table 2 Number of colony-forming units (CFU) in 1 ml of wet sediment throughout bioremediation CFU

Experimental set

Time of experiment (day)

Control

0 7

7.14×10

2.17×10

8.92×109

28

3.82×103

4.12×109

4.98×109

42

3

9

2.75×109

9

4.82×109

8

6.06×109

9

9.57×109

56 70 85

Bioaugmentation

Bioaugmentation + biostimulation

4.35×103

3.21×1010

3.19×1010

3

10

2.35×10

2

5.06×10

3

1.71×10

3

3.22×10

3.53×10 1.41×10 9.53×10 1.12×10

Relative standard deviation did not exceed 10 % of particular value

enzyme activity (increase of the bacterial growth did not correspond with an increase of biodegradation). 3.4 Bioremediation of PCB-Contaminated Sediment Ivy leave addition revealed the highest induction of PCB degradation in liquid media among all tested amendments. They also decreased the adaptation responses caused with PCBs and stimulated bacterial growth. These findings were the reasons for its further use in bioremediation experiments. Bioaugmentation strategy ran with using bacterial strain O. anthropi with PCB degradation ability. Combination of biostimulation and bioaugmentation strategy used O. anthropi with 15 g of cut ivy leaves per kilogram of dry sediment. Both types of bioremediation experiments were compared with the abiotic control experiment when bacterial activity was inhibited with the addition of 2.5 % sodium azide. The number of CFU in control experiment is described in Table 2. Biomass amount in bioaugmentation experiment decreased 1.5 times within first week. Biomass reduction of 3.6 times in bioaugmentation and biostimulation experiments was observed in the first week. This could be explained with an adaptation of O. anthropi and its colonization of sediment. The amount of organic matter in sediment limited bacterial strain as well Fig. 6 Concentration of PCB congeners during the cultivation in„ the control experiment (a), bioaugmentation experiment with O. anthropi (b), and in bioaugmentation and biostimulation experiments with O. anthropi and ivy leave addition (c)

PC

PC

PC

PC

PC

PC

B

B

B

B

B

52

28

15

18

20 3

18 0

13 8

15 3

11 8

10 1

B

B

B

B

B

PC

PC

PC

PC

8

4

16

B

B

PC

PC

PC

PC

PC

PC

B5 2

B2 8

B1 5

B1 8

B8

B4

B2 03

B1 80

B1 38

B1 53

B1 18

B1 01

PC

PC

PC

PC

PC

PC

concentration of PCB congeners (mg kg−1) 16

PC

PC

concentration of PCB congeners (mg kg−1)

PC

PC

PC

PC

PC

PC

B

B

B

B

B

52

28

15

20 3

18 0

13 8

15 3

11 8

10 1

B

B

B

8

4

18

B

B

B

B

PC

PC

PC

PC

PC

PC

concentration of PCB congeners (mg kg−1)

Water Air Soil Pollut (2014) 225:1980 Page 11 of 16, 1980

18

16

A 0

7

14 28

12 42

56

10 70

8 85

6

4

2

0

18

B 0

7

14 28

12 42

10 56

70

8 85

6

4

2

0

18

C 0

14 7

28

12 42

10 56

70

8

85

6

4

2

0

1980, Page 12 of 16

(1.74 % of organic matter). After 42-day adaptation in the presence of ivy leaves, the decrease of O. anthropi stopped and an increase in its population was observed, although it did not reach the initial amount (Table 2). Control experiment with suppressed bacterial growth revealed none or just very low PCB congener transformation (Fig. 6a). The evaporation of PCB congeners was highest in the control experiment and lowest in the experiment with addition of bacterial strain together with ivy leaves. Evaporated PCB congeners were captured with SILIPOR C18 and subtracted from degradation. Results of analyses of specific congeners revealed the degradation ability of O. anthropi toward wide spectrum of chlorinated biphenyls. Both lower (di-, tri-, and tetra-CBs) and higher (penta-, hexa-, and hepta-CBs) chlorinated congeners were reduced during the bioremediation experiment in contaminated sediment similarly as in liquid mineral medium. Higher degradation was achieved with the addition of ivy leaves as degradation inducer (Fig. 6c). Linearity for the removal of PCB 101 (2,2′,4,5,5′-penta-CB) and PCB 118 (2,3′,4, 4′,5-penta-CB) by O. anthropi in bioaugmentation experiment was observed (Fig. 5b). The highest degradation contribution in the experiment with ivy leaves was observed within the first 7 days when 5 % (PCB 8) to 34 % (PCB 180) degradation was achieved. Biomass amount in bioaugmentation experiment decreased by the beginning of the third month (70 days), and then, it remained constant. The presence of ivy leaves in sediment led to higher biomass decrease within the first 7 days; however, after 42 days, the number of viable cells increased. Interestingly, the decrease of biomass within first 7 days of cultivation with ivy leaves was accompanied with the highest degradation rate (Fig. 6c). Ivy leaves could probably induce the activity of PCB degradation enzymes first and, after, the utilization of other carbon substrates present in sediment that they served as energy source. The degradation rate of PCB congeners accelerated after first 28 days in bioaugmentation experiment (Fig. 7b). Ivy leave addition stimulated PCB biodegradation which led into higher removal of PCB congeners. The highest degradation rate in the presence of ivy leaves was observed within the first 28 days. The removal of overall PCBs was significantly higher when the combination of bioaugmentation and biostimulation strategy was used (p<0.05). Total degradation of PCB congeners in the used sediment is present in Fig. 7. Lower chlorinated congeners (PCB 4 and PCB 8) underwent the transformation

Water Air Soil Pollut (2014) 225:1980

in smaller extent. Contrarily, higher chlorinated congeners (PCB 118, PCB 138, and PCB 153) were transformed more easily. PCB 118 represents 14 % toxicity of DELOR 103 (Larsen et al. 1993). Interestingly, the degradation of this toxic congener achieved 56 % in bioaugmentation experiment and 61 % in experiment with O. anthropi and ivy leaves. This phenomenon could be explained probably with the availability of 2,3-biphenyl position for the degradation enzymes. Ivy leaves increased mostly the degradation of PCB203, PCB8, PCB101, and PCB28 (18.6, 17, 15.2, and 14.3 % above bioaugmentation PCB removal) in comparison with bioaugmentation experiment. At the end of experiment, the highest degradation of PCB 18 (2,2′,5-tri-CB) in both remediation experiments was observed (Fig. 7a). Figure 6b shows the overall PCB remaining in sediment within the time of experiments. After 85 days, 27 % of initial PCB amount remained in sediment amended with O. anthropi and ivy leaves. Sediment amended with just O. anthropi revealed 1.5 times higher PCB residue.

4 Discussion PCBs are generally subjected to both aerobic and anaerobic metabolism of different microorganisms. Under aerobic conditions, biphenyl dioxygenase attacks biphenyl core and transforms PCB congeners into the particular chlorobenzoate and a pentanoic acid derivative (Seeger et al. 1995). Under anaerobic conditions, PCB congeners are subjected to reductive dechlorination resulted in the intact biphenyl or lower chlorinated biphenyls. In sediments, in slow flowing rivers, both types of metabolism can be observed because of the limitation of the distribution of oxygen (Dudková et al. 2012). Present study deals with the degradation ability of newly isolated bacterial strain O. anthropi and its ability to adapt to the presence of PCBs in the presence of various substrates. The membrane adaptation toward PCBs in the presence of ivy leaves decreased. Lower saturation, cis–trans isomerization, and lower synthesis of iso-branched FA at the expense of anteiso ones were observed. These results corresponded with those observed by Zorádová-Murínová et al. (2012). It seems that natural matrices containing high amount of terpenoic compounds could help bacterial strains to survive in the environment polluted with hydrophobic compounds and degrade them (Zorádová-Murínová et al. 2012; Dudášová et al. 2014).

Water Air Soil Pollut (2014) 225:1980

Page 13 of 16, 1980 bioaugmentation

bioaugmentation + biostimulation

biodegradation of PCB congeners (%)

100 90

A

80 70 60 50 40 30 20 10 0

PCB4

PCB8

PCB18

PCB15

PCB28

bioaugmentation

120

PCB52 PCB101 PCB118 PCB153 PCB138 PCB180 PCB203

bioaugmentation+biostimulation

control

PCBs remaining (%)

100

B

80

60

40

20

0 0

7

28

42

56

70

85

time (days)

Fig. 7 SBiodegradation of PCB congeners at the end of the bioremediation experiment, after 85 days (a) and the progress of overall PCB remaining throughout the experiment (b).

Bioaugmentation with O. anthropi, bioaugmentation + biostimulation with addition of O. anthropi and ivy leaves

O. anthropi grows in the aerobic environment. Prevalence of biphenyl ring attack is therefore expected in bioremediation experiments in the river sediment as well as in the experiments performed in the defined liquid mineral medium. The degradation ability of abovementioned strain in mineral liquid medium and in contaminated sediment originated from Strážsky canal was confirmed. Highly chlorinated biphenyls (penta- and hexa-CBs) were degraded in higher extent compared to lower chlorinated (di-CB). Researchers generally described under aerobic conditions only the degradation of lower chlorinated

congeners (Sondossi et al. 1992; Mondello et al. 1997; Potrawfke et al. 1998; Field and Sierra-Alvarez 2008; Luo et al. 2008). However, first studies mentioning degradation ability toward tetra-, penta-, and hexa-CBs appeared in last decades (Commandeur et al. 1995; Komancová et al. 2003; Liz et al. 2009). Low degradation of di-CB compared to higher chlorinated congeners could be explained with the higher evaporation of di-CB compared to penta- and hexa-CBs that can diminish the amount of di-CB accessible to microorganisms. Results obtained from experiments with the contaminated sediment followed the results from the liquid media besides

1980, Page 14 of 16

one exception; PCB18 in the sediment was degraded in the highest extent. The addition of biphenyl increased the PCB degradation efficiency. However, the presence of glucose and biphenyl led into similar low PCB degradation as that of glucose alone. Other early publications dealt with the effect of glucose and biphenyl on PCB degradation (Billingsley et al. 1997; Parnell et al 2010). The enhancing effect of biphenyl on PCB degradation enzymes (biphenyl-2,3-dioxygenase) has been described in details (Sylvestre 1995; Singer et al. 2000; Dzantor et al. 2002; Luo et al. 2008). Walia et al. (1990) described inhibitory effect of glucose on the efficiency of PCB degradation. However, the effect of high amount of glucose on biphenyl induction ability toward PCB degradation is reported by us for the first time. Our results suggest that glucose could hamper induction effect of biphenyl. As the results, the degradation of PCB congeners in the presence of glucose addition reached similar levels independently on the presence of biphenyl. Natural degradation processes in the environment (sediments, soil) include aerobic and anaerobic degradation. Both types of PCB transformation occurs simultaneously that leads into PCB mineralization (Abramowicz 1990; Dudková et al. 2012). Sediments naturally contain indigenous microflora (mostly anaerobic) capable of PCB dehalogenation. These anaerobic microorganisms colonize mainly deeper part of sediment, where none or only low oxygen content could be present (Natarajan et al. 1998). The results obtained from bioremediation experiments in unsterile sediments could indicate the dehalogenation activity of anaerobic species during the experiment. This conclusion would explain massive degradation of higher chlorinated congeners and contrast poor removal of lower chlorinated congeners. However, the results obtained from liquid media with pure bacterial culture contradict the theory of PCB dehalogenation, because a significant degradation of higher chlorinated biphenyls was observed under aerobic conditions in the artificial medium. Penta- and hexa-CBs were degraded in the same or higher extent than di- and tri-CBs. Therefore, the degradation ability of the bacterial strain toward higher chlorinated biphenyls strain is highly probable. The mechanism of such PCB transformation of highly chlorinated congeners could possibly include the exchange of chlorine atom with hydroxyl group as was described by Komancová et al. (2003). Similar degradation of highly chlorinated biphenyls was described in Egorova et al. (2013). The

Water Air Soil Pollut (2014) 225:1980

mechanism of transformation of highly chlorinated congeners should be verified in future experiments. Contribution of ivy leave and pine needle addition in stimulation of degradation ability of abovementioned strain is an interesting finding. Previous results described the biphenyl and ivy leave induction potential toward PCB-degradation enzymes (Sierra et al. 2003; Field and Sierra-Alvarez 2008); however, the information of induction effect of pine needles is rarely accessible. Our results indicate that ivy leaves stimulated bacterial PCBdegradation in higher extent in comparison with pine needles. The results of biodegradation of PCBs in liquid media indicated that ivy leaves and pine needles can served as growth substrate as well as PCB degradation inducer. However, the positive effect of ivy leaves on growth ability of O. anthropi in contaminated sediment was rather low. These findings could be explained with the presence of other microorganisms in the sediment. Microorganisms living in sediments with poor organic fraction compete for substrate (Luo et al. 2007). Other explanation for low growth stimulation could be the lower availability of ivy leaves to bacteria living in sediment compared to bacteria present exclusively in liquid media. Since the sediment contained only 1.74 % of organic matter, ivy leaves present the best available source of utilizable carbon. Limited contact of sediment bound bacteria with ivy leaves would explain lower bacterial growth in sediment.

5 Conclusions Our experiments confirmed the positive effects of ivy leaves and pine needles toward the degradation ability of O. anthropi and its adaptation to PCBs. Ivy leaves stimulated bacterial growth as well as the activity of degradation enzymes. Addition of pine needles revealed lower PCB degradation efficiency compared to ivy leaves. The total degradation of di-, tri-, and tetra-CBs was also lower for strain grown on pine needles compared to that grown on ivy leaves. The ability of O. anthropi to transform higher chlorinated biphenyls in various contaminated matrices (liquid media and sediments) was observed. Our findings could be profitable for bioremediation technologies used in the cleaning of polluted environment. Acknowledgment The financial support from Slovak Grant Agency (grant No. 1/0734/12) and grant APVV-0656-12 from Ministry of Education, Science, and Sports are gratefully

Water Air Soil Pollut (2014) 225:1980 acknowledged. Authors are thankful to Branislav Vrana, PhD., from the National Reference Laboratory in Water Research Institute Bratislava for the critical comments.

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