1 Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a class of hazardous contaminants that are widely distributed and persistent in terrestrial and aquatic environments (Haritash and Kaushik 2009; Vila et al. 2015; Wilcke 2000; Nam et al. 2003). The environmental fate of PAHs is of great research interest owing to their clear or potential toxic, carcinogenic and mutagenic properties, and their high recalcitrance and tendency to accumulate in food chains (Samanta et al. 2002). The stability and hydrophobicity of PAHs increase with increase in number of aromatic rings (Kanaly and Harayama 2000); thus, high molecular weight (HMW) PAHs which have greater than three aromatic rings are more persistent in the environment. The PAHs pollution is very serious in China, and the total PAHs emission in 2003 was reported to be 25,300 t, a high portion of which was HMW PAHs due to large contributions of domestic coal and coking industry (Xu et al. 2006; Yang et al. 2012).

Compared with other remediation techniques such as photodegradation, chemical oxidation, and plant uptake, biodegradation of HMW PAHs is accepted as the most cost-effective and sustainable method (Haritash and Kaushik 2009; Tyagi et al. 2011). There are extensive studies on the biodegradation of low-molecular-weight PAHs, while only a few bacteria have been reported to use HMW PAHs as the sole growth substrate due to the lower bioavailability and higher resistance (Peng et al. 2008). Recently, considerable effort has been made on the isolation of microorganisms that can biodegrade HMW PAHs (Ma et al. 2013; Luo et al. 2014; Liao et al. 2015; Ghosh et al. 2014; Xu et al. 2011).

Pyrene (PYR), a tetracyclic PAH, is one of the 16 PAHs listed as priority pollutants by the Environmental Protection Agency (EPA) of the US and has often been used as a model compound in studies on HMW PAHs biodegradation (Yuan et al. 2014; Liao et al. 2015; Li et al. 2005). Many of the pyrene-degrading bacteria described are mainly Gram-positive such as Rhodococcus sp. (Kanaly and Harayama 2000), Mycobacterium sp. (Kim et al. 2005; Samanta et al. 2002), and Brevibacillus brevis (Liao et al. 2015). Ho et al. (2000) isolated 21 pyrene-degrading strains from 16 environmental samples from the US, Germany, and Norway, all of which were be Gram-positive (Ho et al. 2000). In comparison with Gram-positive bacteria, the cell wall of Gram-negative bacteria is thinner, which might advantage the mass transfer and thereby facilitate the PAHs biodegradation (Ma et al. 2013; Ghosh et al. 2014). Besides, the shorter growth cycle of Gram-negative bacteria may also help to accelerate the degradation process (Ma et al. 2013). Although there have been few studies on the PYR utilization by Gram-negative bacteria, such as Pseudomonas (Ping et al. 2011; Ghosh et al. 2014), biodegradation of pyrene using Gram-negative bacterial is rarely found in literature. Knowledge gathered from the actions of pure cultures towards sole substrate is the first and fundamental step in the progressive approach for comprehending the fate of PAHs in environment (Vila et al. 2015). Therefore, isolation and characterization of Gram-negative bacteria that could effectively biodegrade pyrene is necessary and may help to improve the available bank of microbial resources and information.

Moreover, it is important to address certain issues such as substrate inhibition, cell protection, and reusability to develop a successful biodegradation process (Neralla and Weaver 1997; Jorgensen et al. 2000; Tam and Wong 2008). With regard to these concerns, immobilization technology of microorganisms has been exploited and proved to be more advantageous than free cells (Neelakanteshwar K. Patil et al. 2003; Sanjay and Sugunan 2006; Li et al. 2005). Immobilized cells offer much better reusability, high operational stability, and ensures continuous biotreatment by maintaining high cell density and activity. There are numerous matrices available for immobilization, such as polyvinyl alcohol (PVA), polyurethane foam (PUF), and sodium alginate (SA) (Idris et al. 2008; N. K. Patil et al. 2006). Among synthetic polymers, PVA and SA are the most commonly investigated carriers for bioremediation applications, and a great many studies have been carried out to exploit efficient PVA/SA based carrier for the bioremediation of contaminants like phenol (Liu et al. 2009), petroleum (Karamalidis et al. 2010), and naphthalene (Lin et al. 2014). To improve the porosity and mechanical strength of PVA/SA beads, supplements such as powdered activated carbon (Li et al. 2005), lignin (Zhang et al. 2008), and bentonite and kaolin (Lin et al. 2014) were tested, and positive results were obtained. However, attempts to produce PVA-SA beads with the addition of diatomite, a common clay mineral, was scarcely reported in literature, especially in the biotreatment of HMW PAHs like pyrene.

In our previous study on the biodegradation of HMW PAH (fluoranthene), a Gram-negative bacterium, Herbaspirillum chlorophenolicum strain FA1 (H. chlorophenolicum FA1), was isolated from activated sludge which contained various kinds of organic pollutants, including 4.51 ± 0.37 mg g−1 pyrene. To our knowledge, biodegradation and kinetics of pyrene by free and immobilized cells of H. chlorophenolicum FA1 has not been reported so far. Thus, the objectives of this study were (1) to evaluate the pyrene-degrading capacity of strain FA1, (2) to examine the performance of PVA/SA based carrier supplemented with diatomite, and (3) to compare the pyrene degradation kinetics by free and immobilized cells of strain FA1.

2 Materials and Methods

2.1 Microorganism and Medium

H. chlorophenolicum FA1 (CGMCC 3797), a potential pyrene biodegradation strain used in this study, was isolated from the activated sludge of wastewater treatment plant, Yangzi Petrochemical Company in Nanjing, China. This strain has been identified based on 16S rDNA gene sequence analysis in our previous work (Xu et al. 2011).

The mineral salt medium (MSM) used in this paper contained (l−1) (100 ml phosphate buffer (pH 7.0), 0.0675 g MgSO4, 0.0364 g CaCl2, 2.5 × 10−4 g FeCl3, 1.0 ml microelement solution (Mn2SO4 39.9 mg, ZnSO4 · H2O 42.8 mg, (NH4)Mo7O24 · 4H2O 34.7 mg, per liter)), and the pH was adjusted to 7.0. All glassware and solutions were sterilized before experiments by autoclaving at 121 °C for 30 min.

2.2 Microbial Culture

Strain FA1 was cultured in MSM supplemented with pyrene (10 mg l−1) as the sole carbon source, then incubated at 120 rpm, 30 °C and subcultured once in a week for a month. The cells were then transferred to liquid Luria-Bertani (LB) medium and incubated at 200 rpm, 30 °C until the late exponential growth phase was reached. Subsequently, the cell suspension was prepared as described before (Xu et al. 2011). Fresh cells were cultured in solutions containing 2.5 % glutaraldehyde (Liao et al. 2015) to gain inactivate cells of strain FA1.

2.3 Biodegradation and Biosorption of Pyrene

PYR degradation was carried out in 50-ml flasks containing 20 ml MSM with an initial PYR concentration of 10 mg l−1, and strain FA1 was inoculated at 7.2 × 107 CFU ml−1. The flasks were then enclosed with eight sheets of sterile gauze and cultured in rotary shaker at 120 rpm, 30 °C in the dark. At regular time intervals, flasks were harvested to determine the residual PYR concentrations, and the cell growth was determined based on absorbance (OD600 nm). Flasks without inoculation were used as controls, showing that the abiotic losses in PYR concentration were negligible.

To determine the influence of temperature and pH on PYR degradation, the flasks containing the same reaction solution (pH 7.0) as described above were first cultured at different temperatures of 15, 20, 25, 30, and 35 °C, respectively. Thereafter, experiments were performed under the optimum temperature with the pH at 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. As a result, 30 °C and pH 7.5 (see Fig. S1 and S2 in Online Resource) were proved to be high-effective for pyrene biodegradation and were used in the following experiments.

To elucidate the contribution of biosorption of PYR during biodegradation, the degradation experiment was performed at optimum temperature and pH with an initial PYR concentration of 0.135 mg l−1. The systems were inoculated with strain FA1 at 7.2 × 107 CFU ml−1 and incubated at 120 rpm in the dark. Samples were harvested for PYR analysis at 2, 4, 6, 12, 18, 24, and 48 h, separately. The collected samples were first centrifuged, and the residual PYR in the supernatant was detected to calculate the removal amount. To determine the pyrene inside cells, the cell deposits were resuspended in PBS and extracted by ultrasonic for 20 min with dichloromethane as the solvent for three successive extractions (Liao et al. 2015). The PYR biodegradation was calculated by subtracting the PYR inside cells from the whole removal amount. Experiments were also conducted using inactivated cells of strain FA1 to examine the passive biosorption of pyrene.

2.4 Effect of Carboxylated Aromatic Metabolite

2.4.1 Biodegradability of Carboxylated Aromatic Metabolite by Strain FA1

A carboxylated aromatic compound, phthalic acid (PA), which has often been detected as one of the model intermediates in PYR biodegradation (Kanaly and Harayama 2000; Ghosh et al. 2014) was added into MSM as the sole substrate. Biodegradation experiments of phthalic acid at different initial concentrations (10, 20, 30, 50 mg l−1) by strain FA1 were carried out in 500-ml Erlenmeyer flasks containing 200 ml of MSM. The flasks were cultured in rotary shaker at 120 rpm at 30 °C, and the residual PA was determined after 10 days.

2.4.2 Influence of Phthalic Acid on PYR Biodegradation

Experiments were performed at initial PYR concentration of 10 mg l−1, and PA was added as a model carboxylated aromatic intermediate to the culture at initial concentrations of 0–50 mg l−1. The residual PYR and PA were determined after 10 days, as well as the cell growth.

2.5 Carbon Source Utilization

The potential of strain FA1 to utilize low-molecular-weight (LMW) PAHs, HMW PAHs and some other aromatic compounds including naphthalene, fluorene, phenanthrene, anthracene, chrysene, benzo[b]fluoranthene, benzo[a]pyrene, benzene, toluene, and phenol were investigated, respectively. The MSM was supplied with one of the abovementioned chemicals at 10 mg l−1 and then incubated at 30 °C, 120 rpm in the dark. Growth of the strain was measured by the increase of OD600 of the culture after 10 days.

2.6 PYR Biodegradation by Immobilized Cells

2.6.1 Cell Immobilization

H. chlorophenolicum FA1 was immobilized using two widely used matrices, SA and PVA. During the immobilization process, the suspension cells were added at 10 % (v/v).

SA-diatomite carrier: sodium alginate (4 %, w/v) solution was prepared, mixed with cells of strain FA1 and diatomite (1.75 %, w/v), then the mixture was gently dropped into 4 °C CaCl2 solution (4 %, w/v) (Prabu and Thatheyus 2007). The mixture was placed at room temperature for 1 h and cured for 20 h at 4 °C. Then, the formed beads were washed with sterile saline thrice and stored at 4 °C.

PVA-diatomite carrier (chemical method): suspended cells were added to the solution containing (w/v) 11 % PVA, 1 % SA, and 2.5 % diatomite. The solution was thoroughly mixed and placed at room temperature for 1 h before being gently dropped into saturated boric acid (pH 6.5–7.0, containing 2 % CaCl2, w/v) (Idris et al. 2008). During the crosslinking process, the boric acid was stirred with a magnetic stirrer to prevent adhesion of beads. After being cured for 20 h at 4 °C, the beads were stirred for 30 min in 0.5 mol l−1 Na2SO4 solution and washed with sterile saline thrice.

PVA-diatomite carrier (physical method): PVA solution (11 %, w/v) containing 1 % SA and 2.5 % diatomite (w/v) was added with suspension cells and mixed thoroughly to be uniform. After being placed at room temperature for 1 h, 20 μl of this mixture was transferred to each well of a 60-well microplate, frozen at −18 °C for 12 h and thawed at 4 °C for 24 h in two cycles (Partovinia and Naeimpoor 2013; Liu et al. 2009) and washed with sterile saline thrice.

The diameter of the obtained beads was approximate 4 mm.

2.6.2 Biodegradation and Kinetics

Experiments were carried out in 50-ml Erlenmeyer flasks containing 20 ml MSM. Immobilized cells of FA1 were inoculated to give an initial bacterial population of 7.8 × 107 CFU l−1. The initial concentration of PYR was set to be 10 mg l−1, and the pH of the system was adjusted to 7.5. Flasks were capped with eight sheets of sterile gauze to allow air circulation and cultured at 30 °C in the dark. For comparison, a set of experiments was conducted with free cells under identical reaction conditions. Flasks without inoculation were used as control. Samples were taken periodically for residual PYR analysis.

Using the obtained most efficient carrier, PYR degradation kinetics by both free and immobilized cells were examined at initial PYR concentrations of 10, 20, and 50 mg l−1. In order to distinguish between the decrease of PYR as a result of biodegradation or of adsorption, experiments were also conducted using beads without biomass. Samples were collected periodically and analyzed for fluoranthene remaining.

2.6.3 Storage Stability and Reusability of Immobilized Cells

To test the storage stability of PYR degradation, immobilized cells of strain FA1 were stored in distilled phosphate buffer for 0, 5, 10, 15, 30, and 45 days at 4 °C. The beads were washed with distilled water before use and added to the MSM (pH 7.5) containing 50 mg l−1 pyrene. System was cultured for 15 days at 30 °C, 120 rpm, and the biodegradation of pyrene was detected. Besides, in order to investigate the reusability of immobilized cells, the beads were consecutively reused for 8 cycles of pyrene degradation with the same reaction conditions as in the storage stability tests. The reaction system was cultured for 15 days as one cycle.

2.7 Analysis Methods

The whole residual pyrene in samples was extracted by dichloromethane and collected using a former established procedure (Xu et al. 2011). The obtained pyrene was filtered through a 0.22-μm syringe filter for HPLC analysis. The HPLC conditions were C18 column (5 μm, 4.6 mm × 250 mm), methanol-water (90:10, v/v), flow rate of 1.0 ml min−1, detection wavelength of 254 nm, injection volume 20 μl, and column temperature of 40 °C. The concentration of PYR was calculated by external standard method. The mean value of extraction efficiency for PYR in solution was (95.6 ± 1.59) %.

The PA concentration in solution samples was determined with the absorbance at 280 nm (Khan et al. 2015) by dual beam UV/vis spectrometer (Thermo Scientific, EVO 60, Waltham, MA).

All of the experiments in this paper were conducted in triplicate, and the data were expressed as arithmetic means ± standard deviations.

3 Results and Discussion

H. chlorophenolicum FA1, which was used in this study, was isolated from activated sludge using fluoranthene as the sole carbon and energy source (Xu et al. 2011). The activated sludge was found to contain various kinds of organic pollutants, including pyrene (4.51 ± 0.37 mg g−1), indicating possible degradability of PYR and other organic compounds by strain FA1.

3.1 PYR Removal, Biodegradation, and Biosorption by Free Cells

3.1.1 Removal of Pyrene

Time course of PYR removal as well as the cell growth during biodegradation at initial PYR concentration of 10 mg l−1 was monitored over 20 days (Fig. 1). The results demonstrated that fluoranthene-degrading bacteria strain FA1 can grow well on PYR. Rapid biodegradation of PYR was observed in the first few days, 38.9 % of PYR was removed in 6 days, and the 10-d removal reached over 50 %, after which the degradation continued while the rate slowed down. This could be due to the decrease of nutrients and microelement supply and the accumulation of toxic intermediates (Wu et al. 2012; Ghosh et al. 2014), which would cause reduction in activity of the cells and enzyme. It was further confirmed by the cell growth curve which was in good consistent with the corresponding removal curve of Pyrene (Fig. 1). Microbial growth exhibited significant acceleration at day 4, and the increased removal efficiency of pyrene was observed. As growth rate decreased, the degradation rate of PYR decreased accordingly.

Fig. 1
figure 1

Time course of pyrene removal by free cells of strain FA1

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3.1.2 Influence of Temperature and pH

A series of PYR degradation experiments at initial concentration of 10 mg l−1 were performed at various temperatures (15–35 °C) and pH (6.0–8.0). As shown in Fig. S1 and S2 (Online Resource), PYR removal was apparently affected by the initial temperature and pH, since the variation of which affected the essential groups in activate center of enzyme and the membrane permeability (Żyłka et al. 2009; Ye et al. 2014). The optimal temperature and pH for strain FA1 to degrade PYR were determined to be 30 °C and 7.5, respectively. Strain FA1 showed effective removal of PYR under lower temperature of 15 °C, though the 10-d PYR biodegradation of 18.7 % was much less than that under 30 °C (54.3 %). As present in Fig. S2, higher PYR removal was achieved within alkaline system than that within acidic system. Under the optimum conditions, the 10-d degradation of PYR increased from 54.3 to 66.3 %. The optimum temperature and pH for contaminants biodegradation depended on the specific bacteria species and the target substrate. Thirty degrees celsius and pH 8.0 were found to be the most high-effective in fluoranthene-degrading using the same strain H. chlorophenolicum FA1 (Xu et al. 2011). Same results were reported when Acinetobacter strain USTB-X was used to biodegrade pyrene (Yuan et al. 2014), while the most appropriate temperature and pH for degradation of diesel oil by Achromobacter sp. HZ01 were at 28 °C and pH 7.5 (Deng et al. 2014).

3.1.3 Biodegradation and Biosorption of Pyrene

To illustrate the individual contribution of biosorption and biodegradation to the whole removal of PYR, biosorption and biodegradation of PYR by strain FA1 were determined separately. The experiments were conducted with an initial PYR concentration at its water solubility (0.135 mg l−1) which would allow the biosorption and biodegradation process to be evaluated independently (Desai et al. 2008). At the beginning, PYR removal was much higher than the biodegradation due to the contribution of biosorption (Fig. 2). The gap between removal and biodegradation reached the highest of 41.2 % at 6 h, and the gap decreased gradually after that. The 48-h biodegradation of PYR was 93.2 ± 5.3 %, very close to the PYR removal at 48 h (98.4 ± 7.8 %). It can also been seen in Fig. 2 that the biosorption of PYR was relatively high in comparison with biodegradation at 0–18 h. These results indicated that the biosorption was a major contribution to the PYR removal at early stage. Similarly, Liao et al. (2015) reported that the fast rising of intracellular pyrene in cells of Brevibacillus brevis led to higher pyrene removal than biodegradation initially. However, the biosorption of phenanthrene by microbial consortium was not significant at all (Partovinia and Naeimpoor 2013). The PYR accumulated in cells gradually decreased along with the degradation process (Fig. 2), implying that the biodegradation was the predominant process.

Fig. 2
figure 2

Pyrene removal, biodegradation, and biosorption by strain FA1

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The PYR biosorption experiment was also conducted using inactivated cells of strain FA1 for better understanding of biosorption when it was metabolism-independent. The inactivated cells showed rapid sorption ability for PYR and nearly half the initial PYR was sorbed within 4 h (Fig. 2), indicating quick interaction between PYR and the active groups of H. chlorophenolicum FA1. There was a slight decrease in biosorption amount after 48 h, which might be caused by the release of adsorbed PYR as a result of cell breakdown. The adsorbed amount of PYR by inactivated cells was higher than that by live cells throughout the experiment (Fig. 2), providing additional evidence of intracellular degradation of PYR.

It must be mentioned that the extraction of PYR in the following experiments was performed using the whole culture in each flask, and the extraction solvent was added directly to the culture. Thus, we assumed that the reduction in PYR concentration was a result of biodegradation.

3.2 Effect of Carboxylated Aromatic Metabolite on Pyrene Biodegradation

The metabolites formed via the PAHs pathway have been reported to either facilitate or inhibit the PAHs biodegradation (Vila et al. 2015; Haritash and Kaushik 2009). Phthalic acid (PA), a signature carboxylated aromatic metabolite, has been identified in pyrene biodegradation by various bacteria such as Mycobacterium flavescens (Dean-Ross and Cerniglia 1996), Mycobacterium sp. (Rehmann et al. 1998; Kanaly and Harayama 2000), Pseudomonas aeruginosa strain RS1 (Ghosh et al. 2014). In this study, PA was selected to investigate the influence of metabolites on the pyrene biodegradation.

Experiments were first conducted to examine the capability of stain FA1 utilizing PA. The residual PA after 10 days is presented in Table 1. Results showed that PAHs-degrading strain FA1 could use PA as the sole substrate and achieve almost complete removal of PA at all tested concentrations (10–50 mg l−1). At higher initial PA concentration up to 50 mg l−1, the 10-day removal reached 96.18 %. Further experiments were performed to investigate the influence of phthalic acid on PYR degradation, and the results are present in Fig. 3. The introduction of PA at low concentrations (10–20 mg l−1) exerted slight inhibition on PYR degradation, but the decrease was not significant (p < 0.05). Similarly, no negative effect on phenanthrene biodegradation was observed when culture medium was added with hydroxylated aromatic metabolite (Partovinia and Naeimpoor 2014). Significant promotion in PYR degradation was observed when PA was added at high concentrations (30–50 mg l−1). This could be explained that higher amount of available carbon sources enhance the biomass production and hence facilitate the biodegradation (Partovinia and Naeimpoor 2013). This was further confirmed by the measured cell density in PYR-PA ternary biodegradation system (Fig. 3). The 10-d cell growth increased with the increase of PA concentration and was more than three times higher with 50 mg l−1 PA than without PA. The residual PA in PYR-PA binary system was also monitored for comparison (Table 1). PA biodegradation was lower in the binary mixture than in sole substrate system except when PA was added at 50 mg l−1. The inhibitory effect could be due to the interactions existing in multi-substrate systems (Dimitriou-Christidis and Autenrieth 2007), while the exception at PA concentration of 50 mg l−1 could be contributed to the high biomass increased biodegradation surpassing the inhibitory effect.

Table 1 Residual phthalic acid in biodegradation by strain FA1 after 10 days
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Fig. 3
figure 3

10-day biodegradation of Pyrene with the presence of phthalic acid under different concentrations

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3.3 Utilization of Carbon Source

There has been considerable debate over the efficiency of bioaugmentation (TrzesickaMlynarz and Ward 1996; Yu et al. 2005; Tam and Wong 2008; Pathak et al. 2009) because the biodegradation conditions in natural environment were much more complicated than that in laboratory. The targeted strain FA1 has already proved its survival and colonizing capacities with the challenges from indigenous flora (Xu et al. 2011). Here in this work, the comprehensive degrading-capacity of strain FA1 was studied for PAHs in the environment are often found as complex mixtures. Ten carbon sources were tested, including LMW PAHs, HMW PAHs, and several other typical aromatic pollutants. These compounds were selected due to their wide spread in environment, especially in the oil-contaminated sites (Kanaly and Harayama 2000; Wu et al. 2012; Vila et al. 2015). Strain FA1 was found to have a relative broad substrate profile (Table 2) and could utilize LMW PAHs of naphthalene, fluorene, phenanthrene and anthracene, HMW PAH of benzo[b]fluoranthene, and other aromatic compounds of benzene and toluene as sole carbon and energy source. Besides, strain FA1 could also grow well when Tween 80 served as the sole carbon source (Xu et al. 2011).

Table 2 Ability of fluoranthene-degrading strain FA1 utilizing other carbon sources
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3.4 Biodegradation of Pyrene by Immobilized Cells

3.4.1 Pyrene Biodegradation with Different Carriers

H. chlorophenolicum FA1 was immobilized for the purpose of improving the biodegradation capacity and reusability. Due to low-cost, easy fixation of bacteria, durability, and high mechanical strength, clay minerals has been frequently utilized for bacterial immobilization (Lin et al. 2014), while immobilization using a clay mineral of diatomite was scarcely reported. In this study, diatomite was utilized to produce modified SA and PVA beads, and their PYR-degrading ability was tested and compared with free cells.

As shown in Fig. 4, cells immobilized in SA-diatomite carrier exhibited the lowest degradation of PYR. SA-diatomite was found to be very fragile with the beads tending to disintegrate around 5 days. PVA-diatomite carrier by both chemical and physical methods showed higher pyrene removal after 20 days in comparison with free cells. The carrier could provide better survival rate for microbes by acting as a protective shelter under stressed environmental conditions such as the toxicity of pyrene and intermediates (Liu et al. 2009), as well as better mass transfer (Lin et al. 2014; Tyagi et al. 2011) and therefore resulted in increased biodegradation. The most efficient biodegradation of PYR occurred with PVA-diatomite immobilized cells adopting chemical method, by which 92.8 % of initial PYR was removed within 10 days, much higher than that with PVA-diatomite beads by physical method (77.4 %). This was in accord with the degradation of phenanthrene and pyrene in soil slurry by PVA-PAC immobilized bacteria Zoogloea sp. (Li et al. 2005), in which chemical chemically produced PVA-PAC carriers showed better performance. However, PVA beads prepared by physical method of freezing-thawing were considered to be advantageous as reported by Partovinia and Naeimpoor (2013). Though comparison has been made, it is noteworthy that the specific matrices, immobilizing-procedure, and bacteria cells used for producing PVA-based carriers were not the same.

Fig. 4
figure 4

Pyrene biodegradation by free and immobilized cells of strain FA1

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3.4.2 Kinetics of Pyrene Biodegradation

PVA-diatomite carrier (chemical method) was selected due to its high efficiency to study the kinetics of PYR biodegradation by immobilized cells of strain FA1. Kinetic investigation was also performed with free cells for comparing. Experiments were carried out with different initial concentrations of pyrene from 10 to 50 mg l−1, and the results are shown in Fig. 5. Both free and immobilized cells could degrade PYR without any lag phase, and the biodegradation rate was much higher in system with immobilized cells. At initial concentration of PYR up to 50 mg l−1, immobilized cells maintained high biodegradation efficiency and removed 47.75 mg l−1 of PYR within 20 days, while only 24.10 mg l−1 of PYR was degraded in free cell system. Immobilized cells could enable a faster and more efficient biodegradation as compared to free cells via minimizing cell membrane damage and lowering the concentration of toxic compounds in the microenvironment of cells (Tyagi et al. 2011). Additionally, experiments were conducted using beads without biomass at initial pyrene concentration of 50 mg l−1, where an insignificant PYR decrease about 2 % was observed.

Fig. 5
figure 5

Effect of initial pyrene concentration on the biodegradation kinetics by free and immobilized cells of strain FA1

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Application of experiment data to different kinetic models was performed, and it was found that the biodegradation kinetics by the immobilized and free cells were fitted well to the exponential first-order reaction kinetics model (Shen 2002):

$$ -\frac{dS}{dt}=k{S}^1=kS $$
(1)

where S is the concentration of pyrene (mg l−1); k is the first-order reaction rate constant for the biodegradation of pyrene (d−1).

The kinetic results of PYR biodegradation were summarized in Table 3. In free cell system, when the initial PYR concentration increased from 10 to 50 mg l−1, the biodegradation rate of PYR by free cells of FA1 decreased substantially with the k value decreased from 0.135 to 0.0353 d−1. In immobilized cell system, the stresses exerted on the cells by the increasing PYR concentration could be reduced due to the protection from carrier (Tyagi et al. 2011). Thus, when initial PYR concentration increased from 10 to 20 mg l−1, the first-order rate constant (k) increased with the increasing amount of carbon source. Nonetheless, biodegradation rate by immobilized cells decreased at higher PYR concentration of 50 mg l−1 due to the inhibitory effect of PYR and possible soluble toxic intermediates (Ping et al. 2014), which might cause reduction of the activities of enzymes responsible for degrading PYR. Similar constrained biodegradation at high concentrations of pyrene were observed in previous studies employing different microorganisms (Partovinia and Naeimpoor 2013; Lu et al. 2014). However, the uptake rate of PYR by Pseudomonas aeruginosa strain RS1 increased as PYR concentrations increased from 25 to 400 mg l−1 (Ghosh et al. 2014).

Table 3 Parameters of first-order biodegradation kinetics of pyrene by free and immobilized cells of H. chlorophenolicum strain FA1
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With an initial PYR concentration of 20 mg l−1, the strain Klebsiella pneumonia PL1 degraded 63.4 % of PYR in 10 days (Ping et al. 2014). According to Liao et al. (2015), the biodegradation of pyrene (1 mg l−1) by Brevibacillus brevis reached 69 % within 7 days. With initial PYR concentrations of 10, 20, and 50 mg l−1, the 15-day biodegradation of pyrene by free cells of H. chlorophenolicum FA1 were 89.1, 67.3, and 42.3 %, respectively (Fig. 5), indicating that strain FA1 owns comparable pyrene-degrading capacity. Despite the lower bioavailability of pyrene, there were a few reports on bacteria with extraordinary high biodegrading efficiency such as Leclercia adecarboxylata PS4040 (61.5 % within 20 days at 200 mg l−1 PYR) (Sarma et al. 2010), Acinetobacter strain USTB-X (63 % within 16 days at 100 mg l−1 PYR) (Yuan et al. 2014).

3.5 Reusability and Storage Stability of PVA-diatomite Carrier

For the purpose of practical application, the reusability and stability during long-term storage of PVA-diatomite beads (chemical method) were examined. PVA-diatomite immobilized cells were tested in eight consecutive degradation processes with an initial PYR concentration of 50 mg l−1. The time courses of PYR biodegradation for the first 4 cycles were given in Fig. S3 (Online Resource). Enhanced degradation efficiency was observed after being consecutively used for 2 cycles, up to 98.2 % PYR was removed after 15 days in the third cycle comparing to 91.3 % of the first one. The 15-day removal of PYR only decreased 2.7 % after the PVA-diatomite beads (chemical method) were used for 8 cycles (data not shown), and no physical damage in bead structure was observed. This indicated that the chemically produced PVA-diatomite beads could be reused more than eight times, the reusability of which was comparable to PVA bead added with different clay, i.e., bentonite (Lin et al. 2014). As shown in Fig. S4 (Online Resource), the pyrene degradation by immobilized cells remained high with the extension of storage time, and 82.4 % of PYR was biodegraded after the beads being stored for 45 days at 4 °C. Moreover, no breakage or morphological change in the structure of beads was observed after the long-term storage.

4 Conclusion

H. chlorophenolicum FA1, isolated from active sludge using fluoranthene as the sole carbon source, was capable of biodegrading both pyrene and a model carboxylated aromatic metabolite (phthalic acid) of pyrene biodegradation. The presence of phthalic acid at higher concentration significantly enhanced the PYR biodegradation. The contribution of biosorption and biodegradation to the whole removal of pyrene was illustrated separately, showing that the removal of pyrene by strain FA1 was mainly due to biodegradation. Immobilized cells in PVA-diatomite carrier adopting chemical method significantly showed enhanced biodegradation capacity of PYR, with which over 90 % of pyrene (50 mg l−1) was biodegraded within 15 days. Further studies on kinetics revealed that biodegradation of pyrene by free and immobilized cells of strain FA1 followed the first-order kinetic model. Furthermore, the high levels of reusability and storage stability of PVA-diatomite immobilized cells (chemical method) for biodegrading PYR were proved. Besides, strain FA1 exhibited a relative broad substrate profile, including LMW PAHs, HMW PAHs, and other typical aromatic pollutants. These results indicate that immobilized H. chlorophenolicum FA1 possesses a good application potential in the bioremediation of pyrene contamination. Moreover, although we assessed the influence of a carboxylated aromatic metabolite (phthalic acid) on pyrene biodegradation, further work is needed to analyze the formation of intermediate metabolites and their concentrations to provide more insight to PYR biodegradation by H. chlorophenolicum FA1.