Trichloroethylene aerobic cometabolism by suspended and immobilized butane-growing microbial consortia: A kinetic study

Bioresource Technology 144 (2013) 529–538

Contents lists available at SciVerse ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Trichloroethylene aerobic cometabolism by suspended and immobilized butane-growing microbial consortia: A kinetic study Dario Frascari ⇑, Giulio Zanaroli, Giacomo Bucchi, Antonella Rosato, Nasrin Tavanaie, Serena Fraraccio, Davide Pinelli, Fabio Fava Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna, Via Terracini 28, Bologna, Italy

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Butane and TCE kinetics by

suspended and immobilized cells at 15 and 30 °C.  Cell adhesion increases butane (15 and 30 °C) and TCE (15 °C) biodegradation rate.  Butane/TCE inhibition: competitive for free cells, mainly mixed for attached cells.  Cell immobilization significantly reduces the mutual butane/TCE inhibition.  Simulated continuous-flow biofilm PFR: 99.96% TCE conversion with 0.4–0.5 day HRT.

a r t i c l e

i n f o

Article history: Received 15 May 2013 Received in revised form 28 June 2013 Accepted 2 July 2013 Available online 8 July 2013 Keywords: Kinetic study Biofilm Aerobic cometabolism Trichloroethylene Inhibition

a b s t r a c t A kinetic study of butane uptake and trichloroethylene (TCE) aerobic cometabolism was conducted by two suspended-cell (15 and 30 °C) and two attached-cell (15 and 30 °C) consortia obtained from the indigenous biomass of a TCE-contaminated aquifer. The shift from suspended to attached cells resulted in an increase of butane (15 and 30 °C) and TCE (15 °C) biodegradation rates, and a significant decrease of butane inhibition on TCE biodegradation. The TCE 15 °C maximum specific biodegradation rate was 1 1 with suspended cells and 0:021 mgTCE mg1 with attached cells. equal to 0:011 mgTCE mg1 protein d protein d The type of mutual butane/TCE inhibition depended on temperature and biomass conditions. On the basis of a continuous-flow simulation, a packed-bed PFR inoculated with the 15 or 30 °C attached-cell consortium could attain a 99.96% conversion of the studied site’s average TCE concentration with a 0.4–0.5-day hydraulic residence time, with a low effect of temperature on the TCE degradation performances. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Aerobic cometabolism (AC) by bacteria grown on methane, propane, butane, n-pentane, n-hexane, toluene, ammonia and vinyl chloride can lead to the rapid and complete dechlorination of a wide range of chlorinated aliphatic hydrocarbons (CAHs) (Verce et al., 2002; Hori et al., 2005; Alpaslan Kocamemi and Çeçen, ⇑ Corresponding author. Tel.: +39 051 2090416; fax: +39 051 6347788. E-mail address: [email protected] (D. Frascari). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.006

2010a; Cappelletti et al., 2012a; Frascari et al., 2013). The kinetic modeling of CAH AC processes is of primary importance for the rational design of in situ and on-site technologies for the bioremediation of CAH-contaminated sites. Among the aspects to be investigated in a AC kinetic study, the type and entity of the mutual substrate–CAH inhibition plays a crucial role, as the application of different inhibition models can lead to significantly different results in terms of the predicted effluent concentrations in a given treatment system (Kim et al., 2002a). While numerous kinetic studies of CAH AC by suspended-cell pure strains or consortia at

530

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

room temperature or 30 °C were published (e.g., Hori et al., 2005; Kim et al., 2002b; Alpaslan Kocamemi and Çeçen, 2010b), there is a general lack in the literature of AC kinetics performed at real-site temperatures and with immobilized cells. On-site AC in bioreactors represents an interesting alternative to in situ applications, in particular when the necessity to implement a hydraulic barrier leads to the choice of a pump and treat remediation. Immobilized-cell bioreactors present specific advantages over suspended-cell reactors, such as higher cell retention times, a partial cell protection against toxic compounds and the elimination of the biomass settler (Flemming and Wingender, 2010). The general goal of this work was to perform a kinetic study of butane uptake and trichloroethylene (TCE) AC by four butaneutilizing mixed consortia obtained from the indigenous biomass of a CAH-contaminated aquifer, so as to perform a model-based preliminary design of an on-site bioreactor treatment of the studied groundwater. More specifically, the work was aimed at: (i) studying the kinetics of butane uptake and TCE AC by a butanegrown suspended-cell consortium developed at 30 °C from the indigenous biomass of the studied aquifer; (ii) evaluating the effect on the TCE/butane kinetics of a shift from 30 to 15 °C; (iii) evaluating at both 15 and 30 °C the effect on the TCE/butane kinetics of microbial immobilization on a porous biofilm carrier; (iv) identifying the most suitable model to describe the mutual butane–TCE inhibition for each of the four above-listed microbial consortia; (v) performing a simulation of a continuous-flow TCE AC process, aimed at evaluating the optimal bioreactor configuration for the on-site bioremediation of the studied groundwater. The comparison of the 15 and 30 °C kinetics was aimed at allowing the evaluation of the effect of temperature on the proposed on-site AC process. 2. Methods 2.1. Assay description The experimental work was divided into 5 assays. Assays 1–4, schematically represented in the left part of Table 1, were aimed at investigating the kinetics of butane uptake and TCE AC by the following 4 consortia obtained from the indigenous biomass of the studied aquifer: suspended cells, 30 °C (S30); suspended cells, 15 °C (S15); attached cells, 30 °C (A30); attached cells, 15 °C (A15). The porous ceramic carrier utilized for cell immobilization, named Biomax, was selected in a previous study aimed at identifying the best-performing biofilm carrier for the development of a packed-bed reactor process (Cappelletti et al., 2012b). The main characteristics of the cell carrier are: specific surface area 64 m2 g1, porosity 58%, average pore diameter 58 lm, bulk density 670 kg m3, specific density 3330 kg m3. The procedure for the development of the 4 consortia is described in Section 2.2. Each assay was divided into 4 sub-assays, aimed at investigating the

kinetics of (a) butane uptake in the absence of TCE, (b) TCE AC in the absence of butane, (c) TCE inhibition on butane uptake, and (d) butane inhibition on TCE AC. Each sub-assay consisted of a set of 6–11 experimental conditions tested in parallel and characterized by different initial concentrations of butane or TCE, without inhibitor for sub-assays (a) and (b), or with a fixed inhibitor concentration (reported in the legend of Fig. 1) for sub-assays (c) and (d). The fixed inhibitor concentration was calculated, on the basis of preliminary suspended-cell tests, so as to attain a roughly 50% decrease of the butane or TCE specific biodegradation rate. The inhibitor concentration was maintained roughly constant during each inhibition test by sequential additions of butane or TCE calculated on the basis of the GC measurements. The initial butane or TCE concentrations of each sub-assay, that can be read in the X-axis of Fig. 1, were attained by adding the required amount of pure butane or of a TCE-saturated aqueous solution, maintained at 4 °C. In the experimental conditions characterized by initial butane concentrations above 25 mg L1, the headspace was enriched with pure O2 in order to avoid the attainment of oxygen-limited conditions during the tests. Previous tests of butane uptake with different initial headspace O2 levels (20%, 60% and 100%) indicated the absence of any effect of the O2 concentrations on the butane uptake rate (data not shown). Each experimental condition was studied in triplicate 120-mL vials containing 50 mL of cell suspension for the suspended-cell assays (1 and 2), or 60 mL (bulk volume) of biofilm-containing Biomax carriers and 50 mL of water for the attached-cell assays (3 and 4). To evaluate possible phenomena of butane or TCE abiotic depletion, for each experimental condition an additional control vial sterilized with NaN3 (3.5 g L1) and containing 50 mL of water, plus 60 mL of non-biofilm-containing carriers for assays 3 and 4, was set up. After the butane and/or TCE additions, the vials were placed in agitation (150 rpm) at 15 °C (assays 2 and 4) or 30 °C (assays 1 and 3) and subjected to the initial measurement of butane and TCE headspace concentration after 20 min. The subsequent analysis were made at intervals of about 30 min in the 30 °C tests and 60 min in the 15 °C tests. The initial 4–5 measurements (corresponding to about 2 h of biodegradation at 30 °C and 4 h at 15 °C) were utilized to calculate the initial biodegradation rate by linear regression, as explained in Section 2.4. In order to determine the initial specific rates, the cell concentration was measured in each vial at the onset of each sub-assay by sampling 1 mL of suspension for assays 1 and 2, or 3 Biomax carriers for assays 3 and 4. The resulting cell concentrations varied from 60 mgprotein L1 (15 °C) to 150 mgprotein L1 (30 °C) for suspended cells, and from 12 mgprotein L1 (15 °C) to 70 mgprotein L1 (30 °C) for attached cells. Attached-cell concentrations were referred to the reaction volume (volume occupied by the carriers + interstitial water volume). Assay 5 was aimed at evaluating the endogenous decay constant kd of the 15 and 30 °C suspended-cell consortia (S15 and S30). For each consortium, 3 replicate 120-mL vials filled with

Table 1 Best estimates of the kinetic parameters relative to the assays of butane uptake and TCE biodegradation by the four studied consortia, with 95% confidence intervals. Assay Consortium Cell No. condition

1 2 3 4 a b c d e

S30 S15 A30 A15

Suspended Suspended Attached Attached

Temp. Butane (°C) qmax,Ba 30 15 30 15

TCE KS,Bb

4.5 ± 0.5 4 ± 2 2.0 ± 0.5 7 ± 2 29 ± 2 8±2 13 ± 1 12 ± 3

mg mgprotein1 d1. mg L1. Type of inhibition: C = competitive; M = mixed. L mgprotein1 d1. Not applicable.

Inhib. typec

KI,C,TCE-Bb

C C M M

0.05 ± 0.01 0.09 ± 0.01 0.4 ± 0.2 1.0 ± 0.5

KI,UC, TCE-B

k1,Bd

qmax,TCEa

KS,TCEb

b

n.a.e n.a.e 1.5 ± 1.1 2.1 ± 1.3

1.1 ± 0.5 0.082 ± 0.002 0.3 ± 0.1 0.0109 ± 0.0004 3.5 ± 0.9 0.06 ± 0.02 1.1 ± 0.3 0.021 ± 0.001

0.80 ± 0.07 0.75 ± 0.05 2.7 ± 1.1 1.10 ± 0.07

Inhib. typec

KI,C,B-TCEb

C C M C

0.07 ± 0.02 0.40 ± 0.06 1.4 ± 0.7 0.9 ± 0.2

KI,UC,BTCE

k1,TCEd

b

n.a.e n.a.e 2.2 ± 1.5 n.a.e

0.102 ± 0.01 0.015 ± 0.001 0.021 ± 0.01 0.019 ± 0.001

531

Initial net specific biodegr. rate (mg mg protein-1 d-1)

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

16

A

o

Butane - Attached cells - 15 C

0.030

o

o

Butane - Attached cells - 15 C - with TCE 0.43 mg/L o Butane - Susp. cells - 15 C

14 12

B

o

TCE - Attached cells - 15 C TCE - Attached cells - 15 C with butane 1.4 mg/L o TCE - Susp. cells - 15 C

0.025

o

o

Butane - Susp. cells - 15 C - with TCE 0.48 mg/L

TCE - Susp. cells - 15 C - with butane 1.3 mg/L

0.020

10 8

0.015

6

0.010 4

0.005 2

0.000

0 0

10

20

30

0

2

4

6

8

Initial concentration (mg L-1) Fig. 1. Butane and TCE initial net specific biodegradation rate versus initial concentration in the 15 °C assays (2 and 4): experimental data ± 95% confidence intervals and best fitting simulations. For the inhibition tests, the simulations were performed with the inhibition models indicated in Table 1. The best-fitting values of the kinetic parameters are reported in Table 1. A: butane, 15 °C; B: TCE, 15 °C.

60 mL of cell suspension were maintained in agitation for 30 days without any substrate supply, and the concentration of viable cells was measured every 4–5 days as CFU mL1, as described previously (Frascari et al., 2005). 2.2. TCE-degrading microbial consortia Four butane-grown, TCE-degrading microbial consortia obtained from the indigenous biomass of a TCE (0.04–5.8 mg L1) and 1,1,2,2-tetrachloroethane (TeCA) (0.2–4.0 mg L1) contaminated aquifer located in Northern Italy were utilized for the kinetic tests. Consortium S30 consisted of suspended cells enriched at 30 °C by exposing 60 mL of aquifer’s groundwater to consecutive pulses of butane (2 mg L1 in the aqueous phase), O2 (8 mg L1), TCE (1–7 mg L1) and TeCA (1–5 mg L1) for 60 days in a 120-mL microcosm. Given the poor TeCA degradation capacity of S30, the kinetic study was not extended to TeCA. To produce the amount of biomass required for the butane/TCE kinetic study, S30 was first grown in 2-L bottles containing 1.1 L of sterilized water and then in a 15-L BiostatÒ Cplus fermentor (Sartorius, Goettingen, Germany) containing 7 L of sterilized water and operated at 30 °C with consecutive pulses of butane, O2 and TCE. Upon the attainment of a 150 mgprotein L1 cell concentration, a 1.05-L aliquote of S30 suspension was sampled every 3–4 days for 4 times, in order to set-up the kinetic assays. Consortium S15 consisted of suspended cells enriched at 15 °C, i.e., the yearly average temperature of the studied aquifer. S15 was obtained from consortium S30 by transferring it (10% v/v) in a vial containing 60 mL of site groundwater, maintained at 15 °C and exposed for 90 days to consecutive pulses of butane, O2 and TCE. Given the impossibility to operate the fermentor at 15 °C, consortium S15 was re-inoculated in four 2-L bottles each containing 1.1 L of sterilized water exposed to further butane/O2/TCE pulses at 15 °C until the attainment of a 60 mgprotein L1 cell concentration, in order to obtain the amount of biomass required to set up the kinetic assays. To evaluate the effect on the kinetic parameters of bacterial immobilization, the suspended-cell consortia S30 and S15 were grown as attached cells on the selected biofilm carrier (Biomax), to obtain the corresponding attached-cell consortia A30 and A15.

For both S30 and S15, 105 mL of suspension were utilized to inoculate (10% v/v) 21 120-mL vials, each containing 60 mL (bulk volume) of sterilized biofilm carriers and 50 mL of sterilized water. The vials, maintained at 30 or 15 °C depending on the inoculum utilized, were spiked with 3 consecutive pulses of butane (2 mg L1), TCE (1 mg L1) and O2 (8 mg L1). To remove the suspended biomass and promote growth in the biofilm, starting from the 4th butane/TCE/O2 pulse, before each re-spike the carriers were washed in physiological solution (NaCl 9 g L1) and re-supplied with 50 mL of fresh sterilized water. After 10 butane/TCE/oxygen spikes the vials were utilized for the 30 or 15 °C attached-cell kinetic tests (assays 3 and 4, respectively). To this goal, at each temperature the 4 sub-assays were applied to the 21 vials in the following order: butane uptake in the absence of TCE; TCE AC in the absence of butane; TCE inhibition on butane uptake; butane inhibition on TCE AC. Before each sub-assay, the vials were exposed to a butane/O2 pulse and to water replacement with fresh sterilized water, in order to minimize the suspended cell concentration. Based on PCR–DGGE analysis, performed according to the procedures described previously (Zanaroli et al., 2012; Valentino et al., 2013), the suspended cell consortia S30 and S15 were highly similar to each other (69.2% similarity) and consisted mainly of Bacteroidetes, Betaproteobacteria and Alphaproteobacteria not related to known butane oxidizing or chlorinated solvent co-metabolizing bacteria. Conversely, attached cells consortia A30 and A15 remarkably differed from the corresponding free cells consortia (40.2% and 38.5% similarity, respectively) and included phylotypes related to the degradation of chloroaromatics and CAH, such as Cupriavidus necator, Bacillus sp., Sphingobacterium sp. and Dechloromonas sp. strains. 2.3. Chemicals and analysis Butane (99.95%) and TCE (99.5%) were purchased from Aldrich (Gillingham, UK). Gas-phase butane, O2 and TCE concentrations were measured with a HP6890 GC connected to a 7694E headspace sampler, utilizing a flame ionization detector for butane, and an electron capture detector for O2 and TCE. Cl was measured by Ion Chromatography. Details on the chromatographic methods are reported by Frascari et al. (2005, 2006a). Protein concentrations

532

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

in the soil-free tests were measured as described by Frascari et al. (2006b). The quantification of attached cells was performed with the same protocol on carriers, after their incubation in sterile physiological solution for 1 h at 30 °C under shaking (90 rpm) and centrifugation at 8000 rpm for 20 min followed by removal of the supernatant. 2.4. Data elaboration and modeling In each kinetic test, total amounts in the vial and liquid-phase concentrations of butane and TCE were calculated assuming liquid–gas equilibrium. To validate the latter assumption, the gas/ liquid transport characteristic time (tT, evaluated as 1/kLa) was compared, for each compound, to the observed reaction characteristic time (tR, evaluated as ci/ri) following the procedure described by Frascari et al. (2008). The ranges of variation of the tR/tT ratio were 19–26 for butane, and 240–4400 for TCE. Gas–liquid mass transfer limitations on the calculated biodegradation rates and kinetic parameters were therefore considered negligible. To validate this analysis, the 30 °C suspended-cell butane kinetic test characterized by the highest butane concentration was repeated at higher shaking rates (185 and 220 rpm) than that utilized throughout the kinetic study (150 rpm), without any significant effect on the calculated butane uptake rate. The absence of gas–liquid mass transfer resistances justifies the comparison of the kinetic parameters estimated from the suspended- and attached-cell tests, characterized by different liquid/gas ratios. The following gas–water partition coefficients were utilized: butane, 24.6 at 15 °C and 44.0 at 30 °C; TCE, 0.251 at 15 °C and 0.497 at 30 °C (Sander, 1999). Preliminary tests showed that no butane or TCE adsorption on the Biomax carriers occurred. In each test, the butane or TCE initial depletion rate (ri) was calculated by dividing the slope of the linear regression of the initial 4–5 points of the plot of total amount in the vial versus time by the reaction volume, defined as the volume of the liquid phase in the suspended cell tests, or the bulk volume of the biofilm carriers in the immobilized-cell tests. The initial biodegradation rate (ri,bio) was calculated as ri  ri,abio, where ri,abio indicates the depletion rate obtained in the corresponding abiotic test. The TCE abiotic depletion rates, satisfactorily fitted with a 1st order model (1st order constant = 4.9  109 d1 at 30 °C and 2.5  1010 d1 at 15 °C), always resulted <1% of the corresponding values of rTCE. The initial specific biodegradation rate (qi) was finally obtained by dividing ri,bio by the cell concentration measured at the onset of each test (X0). The variation of active cell concentration DX occurring during the time interval associated to the headspace measurements utilized to calculate ri was evaluated as YX/S DmB/Vreaction in the butane kinetic tests and DmTCE/(Tc,TCE  Vreaction) in the TCE kinetic tests (all symbols are defined in Appendix A). As |DX|/X0 resulted <0.15 in all the tests, the approximation of constant biomass concentration during the time interval associated to the evaluation of each degradation rate was considered acceptable. The specific rates of both butane uptake and TCE biodegradation were interpreted by means of a Michaelis–Menten-type kinetic equation:

qi ¼

qobs max;i c i

ð1Þ

K obs S;i þ c i

In the single-compound kinetics (sub-assays (a) and (b)) the obobs served kinetic parameters qobs max;i and K S;i coincide – for each temperature and cell condition – with the ‘‘true’’ kinetic parameters qmax;i and K S;i . Conversely in sub-assays (c) and (d), characterized obs by the contemporary presence of butane and TCE, qobs max;i and K S;i were expressed according to the four inhibition models reported in Table 2 (competitive, noncompetitive, uncompetitive and mixed inhibition). Thus the apixobs refers to the effect of inhibition, and not to the influence of the diffusional resistances that occur in the attached-cell tests. In the single-compound kinetics, kinetic parameters qmax;i and K S;i were estimated by non-linear least squares regression (NLSR) of the simulated specific biodegradation rates qi (Eq. (1)) to the corresponding values obtained from the experimental data. To minimize the risk of attaining a local rather than a global minimum, reliable first-guess values of qmax;i and K S;i were obtained using the method of the direct linear plot (Kim et al., 2002b). In the double-compound kinetics, inhibition constants K I;C and K I;UC were obs estimated by NLSR of Eq. (1) (with the expressions of qobs max;i and K S;i reported in Table 2) to the corresponding experimental values, using the best-estimates of qmax;i and K S;i obtained in the corresponding single-compound kinetics as input parameters. In each inhibition sub-assay, to identify the most suitable inhibition model the best fits obtained with the four models listed in Table 2 were compared by means of the coefficient of determination R2, defined so as to allow the comparison of models with different numbers of parameters (Holman, 2001):

R2 ¼ 1 

PN

 qcalc;i Þ2 NP1

i¼1 ðqexp;i

!, P N

 qexp;m Þ2 N1

i¼1 ðqexp;i

! ð2Þ

The model characterized by the highest R2 was then compared to the other 3 models by means of F tests, and was considered the right model for describing the studied inhibition if, in each comparison, the probability that the two models are not statistically different resulted <5%. In the case of F tests resulting in probabilities >5%, the correct inhibition model was identified as follows. The observed obs parameters qobs max;i and K S;i were estimated directly by NLSR of Eq. obs (1) on the experimental values of qi. qobs max;i and K S;i were then represented in a (KS, qmax) plot, together with the estimates of qmax;i and K S;i obtained in the corresponding non-inhibited test. The inhibition type was then identified on the basis of the direction of the shift of the ðK S;i ; qmax;i Þ point due to inhibition: for competitive inhibition the shift is to the right, for uncompetitive inhibition it is toward the origin, for mixed inhibition it is intermediate between these extremes, and for noncompetitive inhibition (a mixed inhibition with K I;C ¼ K I;UC ) the point shifts down vertically (Kim et al., 2002b). In assay 5, the endogenous decay constants kd of the 15 and 30 °C suspended-cell consortia (S15 and S30) were estimated by interpolating the experimental concentrations of viable cells with

Table 2 obs Expressions of qobs utilized for the simulation of the butane and TCE specific rates in the sub-assays dedicated to the analysis of the mutual butane–TCE inhibition. max and K S Type of inhibition

qobs max;B

K obs S;B

qobs max;TCE

K obs S;TCE

Competitive Non competitive

qmax;B

K S;B  ð1 þ cTCE =K I;C;TCE-B Þ K S;B

qmax;TCE

K S;TCE  ð1 þ cB =K I;C;B-TCE Þ K S;TCE

Uncompetitive Mixed

qmax;B ð1þcTCE =K I;UC;TCE-B Þ qmax;B ð1þcTCE =K I;UC;TCE-B Þ qmax;B ð1þcTCE =K I;UC;TCEB Þ

K S;B ð1þcTCE =K I;UC;TCE-B Þ

K S;B 

ð1þcTCE =K I;C;TCEB Þ ð1þcTCE =K I;UC;TCEB Þ

qmax;TCE ð1þcB =K I;UC;B-TCE Þ qmax;TCE ð1þcB =K I;UC;B-TCE Þ qmax;TCE ð1þcB =K I;UC;B-TCE Þ

K S;TCE ð1þcB =K I;UC;B-TCE Þ ð1þc =K

Þ

I;C;B-TCE K S;TCE  ð1þcBB=K I;UC;B-TCE Þ

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

a first-order model. For all parameters, 95% confidence intervals were determined according to Smith et al. (1997). 3. Results and discussion The butane and TCE initial specific biodegradation rates and the corresponding best fitting simulations are represented in Fig. 1 for the 15 °C consortia (S15, A15), and in Fig. S1 in the Supplementary data for the 30 °C consortia (S30, A30). The best estimates of the kinetic parameters ± 95% confidence intervals, as well as the inhibition type that resulted more appropriate in each double-compound test, are reported in Table 1. 3.1. Kinetics of butane uptake and TCE cometabolism in the absence of inhibition As shown in Fig. 1 and Fig. S1, the Michaelis–Menten model allowed a satisfactory fit of the single-compound specific rates expressed for both butane and TCE by the four studied consortia (R2 = 0.955–0.994). Although no substrate inhibition phenomena were observed in the studied concentration ranges (butane 0–50 mg L1, TCE 0–9 mg L1), other authors report a significant substrate inhibition in studies of TCE biodegradation even at lower TCE concentrations (e.g., Shukla et al., 2011). Cell attachment had remarkable effects on the kinetic parameters (Table 1). Cell adhesion led at both temperatures to a marked increase of the butane and TCE KS, that can be ascribed to the masstransfer resistances across the biofilm. In the case of butane, cell adhesion also led, at both 30 °C and 15 °C, to a notable increase of qmax. As a result, the attached-cell average butane specific rate was 5–6 times higher than the corresponding suspended-cell average specific rate (Fig. 1A). The fact that, despite the mass-transfer resistances across the biofilm, cell attachment determined a marked increase of qB can be ascribed to the remarkable differences in composition between the attached cell consortia A30 and A15 and the corresponding suspended cell consortia S30 and S15 (38–40% similarity). Such a difference probably results from the different growth procedures applied to the suspended- and attached-cell consortia. Indeed, during the extended fed-batch growth of suspended-cell consortia cross-feeding phenomena associated to the non-negligible concentration of cell debris might have led to the growth of non-butane utilizers. Conversely, the periodic liquid replacement with fresh sterile water performed during the fed-batch growth of the attached-cell consortia reduced cross-feeding, thus allowing the attainment of a higher fraction of butane utilizers, and favored the wash-out of fast-growing species from the bulk liquid, thus promoting the prevalence in the biofilm of slowly-growing microorganisms. As for TCE, bacterial adhesion to the carrier determined a marked increase of qmax;TCE at 15 °C (Fig. 1B), whereas a decrease of qmax;TCE was observed at 30 °C (Fig. S1 in the Supplementary data). In the evaluation of the different effects of biomass immobilization on the TCE specific rates at 15 °C and 30 °C, it should be noted that TCE degraders are likely to be a small fraction within each microbial consortium, and that therefore changes in the concentration of 1 or 2 TCE degraders as a result of cell adhesion could determine drastic variations of the TCE specific rates. For both butane and TCE, the shift from 30 to 15 °C markedly reduced the estimates of qmax and consequently the average specific rates under both cell conditions. Since, for both suspended- and attached-cells, the consortia developed at the two temperatures were highly similar, the lower rates observed are likely due to lower metabolic activity of the biomass at 15 °C. Assuming 1 mgprotein = 2 mgdw (Alvarez-Cohen and Speitel, 2001), the estimates of qmax,TCE,30 obtained in this study are in

533

agreement with those obtained at 25–30 °C both for suspended cells (e.g.: Keenan et al., 1994: 0.080 L mgprotein1 d1 with a mixed propane-utilizing consortium) and for biofilm processes (e.g.: Zhang and Tay, 2012: 0.041 mg mgprotein1 d1 with phenolgrown granules; Segar et al., 1995: 0.076 mg mgprotein1 d1 with a phenol-grown biofilm). 3.2. Kinetics of butane uptake and TCE cometabolism in the presence of inhibition In all the double-compound assays (TCE ? butane and butane ? TCE inhibition, at both temperatures and for both biomass conditions), the competitive and mixed inhibition models resulted in high R2 values (0.91–0.995) and were not statistically different from each other (p > 0.05), whereas the uncompetitive inhibition model gave rather low fits (R2 = 0.12–0.89). The noncompetitive model generally yielded intermediate R2 values, with the exception of the 30 °C attached-cell inhibition tests where noncompetitive inhibition was characterized by R2 values similar to those of competitive and mixed inhibition, and the three models resulted to be not statistically different. The poor fits provided by the uncompetitive inhibition model are in agreement with the observation that an uncompetitive inhibitor only binds to the enzyme–substrate complex, whereas in AC processes CAHs are oxidized even in the absence of the growth substrate, and vice versa. For each double-compound sub-assay, to identify the most suitable inhibition model, the observed kinetic parameters qobs max;i and K obs S;i – estimated directly by NLSR of Eq. (1) on the experimental qi values – were compared to the best estimates of the corresponding values obtained in the single-compound sub-assays. For all the suspended-cell double-compound sub-assays inhibition resulted to be competitive, as it determined minor variations of qmax – comparable with the uncertainties on the estimate of this parameter – and 2–17-fold increases of KS. Conversely, in the 15 °C and 30 °C attached-cell sub-assays of TCE inhibition on butane the attainment of comparable decreases of qmax and KS led to the choice of the mixed inhibition model. Lastly, in the attached-cell tests of butane inhibition on TCE the latter resulted competitive at 15 °C and mixed at 30 °C. To explain this peculiar finding, it should be considered that the inhibition type observed in these tests is a combination of the inhibition phenomena occurring in each TCE-degrading strain, and that the 15 and 30 °C consortia present not negligible differences (36% similarity, on the basis of the DGGE profiles). The best estimates of K I;C and K I;UC are reported in Table 1. As an example, the best fits obtained with the four tested models in the sub-assay of butane inhibition on TCE AC at 15 °C are shown in Fig. 2. The analysis of the literature on the mutual CAH–substrate inhibition provides a complex scenario, the type and entity of inhibition being strongly dependent on the enzyme involved and on the studied CAH. Numerous studies of AC found or assumed competitive inhibition (e.g.: Anderson and McCarty, 1996; Chang and Criddle, 1997; Hori et al., 2005; Alpaslan Kocamemi and Ferhan, 2007; Frascari et al., 2008; Balasubramanian et al., 2011). While the theory of enzyme kinetics with competitive inhibition indicates that K I;C should be equal to the KS of the inhibitor, most of the above-listed studies report a K I;C –K S of the inhibitor. In agreement with these findings, in this study we obtained for the two suspended-cell consortia (S15 and S30) that the mutual butane–TCE inhibition was best described by a competitive inhibition model with a KI,C/KS ratio in the 0.02–0.12 range. Conversely, the identification of mixed inhibition as the best model to describe the mutual butane–TCE inhibition for consortium A30 and the TCE ? butane inhibition for A15 is in agreement with Kim et al. (2002a,b), who found mixed inhibition of butane on the AC of 1,1,1-trichloroethane, 1,1,-dichloroethylene and

534

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

Uncompetitive inhibition

Initial net specific rate (mg mg protein-1 d-1)

0.010

Non-competitive inhibition Mixed inhibition 0.008 Competitive inhibition

0.006

0.004

0.002

0.000 0

1

2

3

4

5

6

Initial concentration (mg L-1) Fig. 2. TCE initial net specific biodegradation rate versus initial concentration in sub-assay 2(d) (suspended cells, 15 °C, butane inhibition on TCE): experimental data ± 95% confidence intervals and best fitting simulations performed with the 4 models illustrated in Table 2. Competitive inhibition was selected as the bestfitting model.

1,1-dichloroethane, and Alpaslan Kocamemi and Çeçen (2010b), who found mixed inhibition of ammonia on the AC of 1,2dichloroethane. In order to compare, for both butane and TCE, the effects of inhibition on the suspended and attached cells specific rates, for each temperature and cell conditions we calculated the qwith inhibition =qwithout inhibition ratio, using a fixed concentration of inhibitor at each temperature. For butane inhibition on TCE, the inhibitor concentration was set at half of the solubility of butane in water at each temperature (at 1 atm). The resulting values (29 mg L1 at 30 °C and 50 mg L1 at 15 °C) roughly correspond to the average butane concentration in a hypothetical continuous-flow tubular reactor fed at the solubility of butane in water, in the case of complete butane conversion at the end of the bioreactor. For TCE inhibition on butane, following an analogous criterion, the inhibitor concentration was set at half of the average TCE concentration of the studied aquifer (3.4/2 = 1.7 mg L1). For each compound, the qwith inhibition =qwithout inhibition ratio was calculated at regular intervals over the concentration range attainable in a tubular reactor under the above-reported assumptions (TCE: 0–3.4 mg L1; butane: 0–58 mg L1 at 30 °C, 0–100 mg L1 at 15 °C). The plots of qwith inhibition =qwithout inhibition over the above-reported concentration ranges are shown in Fig. 3. This analysis indicates that, for both compounds, the biofilm process is significantly less affected by inhibition than the suspended cell process. This finding might be ascribed to the fact that the average inhibitor concentration within the biofilm is lower than that measured in the liquid phase, as a result of the inhibitor concentration profile in the biofilm. The data reported in Fig. 3 also show that, under the above-listed assumptions, a continuous-flow tubular-reactor process would be characterized by very strong inhibition effects, with a 50–82% decrease of the butane rate due to TCE inhibition, and a 95–99% decrease of the TCE rate due to butane inhibition. 3.3. Estimates of decay constant, growth yield and TCE transformation capacity The estimates of endogenous decay constant, growth yield on butane and TCE transformation capacity of the studied consortia

were aimed at completing the set of kinetic parameters required for the simulation of the continuous-flow TCE AC process under various bioreactor configurations that will be presented in Section 3.4. The constants of endogenous decay of the suspended-cell consortia, evaluated from the data of assay 5, were equal to 0.035 ± 0.005 d1 at 30 °C, and 0.011 ± 0.002 d1 at 15 °C. An analogous evaluation could not be performed for the attached-cell consortia, given the unavailability of a method to plate the cells immobilized on a porous biofilm carrier such as the one used in this study. The growth yield on butane of the 30 °C suspended-cell consortium (S30), determined by dividing the mass of cells produced by the amount of butane consumed during the growth of S30 in the fermentor, resulted equal to 0.23 ± 0.03 gprotein gbutane1. This parameter was assumed to be independent of temperature (Tchobanoglous et al., 2003). A selection of the experimental data relative to the sub-assays of TCE biodegradation by suspended-cells (consortia S30 and S15) were utilized to estimate the TCE transformation capacity, defined as the ratio of the mass of TCE transformed to the mass of cells inactivated (Alvarez-Cohen and McCarty, 1991). This ratio was evaluated, in sub-assays 1(b) and 2(b), for the experimental conditions that did not lead to the complete biodegradation of the amount of TCE initially supplied, assuming that the incomplete TCE transformation was due to the complete inactivation of the amount of cells initially present in each vial. A specific simulation indicated that endogenous decay had a negligible effect on the concentration of active cells during the biodegradation tests object of this evaluation. The suspended-cell transformation capacity resulted equal to 32 ± 3 mgTCE gprotein1 at 30 °C and 28 ± 3 mgTCE gprotein1 at 15 °C. This method to estimate the transformation capacity provides an overall evaluation of the effects of the inactivation of cellular functions due to transformation product toxicity and of the exhaustion of the reducing power (NADH) accumulated during the previous phase of cell growth on butane. An analogous evaluation for the attached-cell consortia was not possible, as sub-assays 3(b) and 4(b) were not monitored until the complete halt of the TCE transformation process. The above-reported estimates of TCE transformation capacity are in good agreement with those reported in previous studies (e.g.: Zhang and Tay, 2012: 22 mgTCE gprotein1 with phenol-grown granules; Chen et al., 2008: 8 mgTCE gprotein1 with a phenol-utilizing strain).

3.4. Simulation of a continuous-flow bioreactor process of TCE biodegradation by the studied consortia The last section of this work was aimed at evaluating the feasibility of an on-site bioreactor treatment of the studied TCEcontaminated groundwater, operated either at the aquifer’s average temperature (15 °C) or at 30 °C and inoculated with the suspended- or attached-cell consortia object of this study. The inlet TCE concentration (cTCE,0) was set equal to the average value in the studied aquifer (3.4 mg L1), whereas the outlet TCE concentration was set equal to the limit imposed by the Italian law for the remediation of aquifers (0.0015 mg L1). In each equation, the specific rates qB and qTCE were expressed by Eq. (1), combined for each consortium with the previously determined inhibition model (Table 2) and with the kinetic parameters reported in Table 1. The biomass mass balances were written following the approach proposed by Alvarez-Cohen and McCarty (1991). The main results of the simulations are reported in Table 3, further model hypothesis and details are described in Table S1 in the Supplementary data and the explanation of each symbol is reported in Appendix A.

535

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

A

0.8

0.10

o

Attached cells 30 C

0.7

o

B

o

Suspended cells 30 C

Suspended cells 30 C o

Attached cells 30 C o

Suspended cells 15 C

o

qwith inhibition / qwithout inhibition

Suspended cells 15 C 0.6

o

Attached cells 15 C

0.08

o

Attached cells 15 C

0.5 0.06

0.4 0.04

0.3 0.2

0.02

0.1 0.00

0.0 0

20

40

Butane concentration (mg

0

60

1

2

3

TCE concentration (mg L-1)

L-1)

Fig. 3. Plots of the ratio (specific rate with inhibition/specific rate without inhibition) versus butane or TCE concentration for the 4 consortia object of the study: A, butane specific rates; B, TCE specific rates. The ratios were evaluated under the following assumptions: for qB,with inhibition/qB,without inhibition, TCE fixed concentration = 1.7 mg L1, butane concentration range = 0–58 mg L1 at 30 °C, 0–100 mg L1 at 15 °C. For qTCE,with inhibition/qTCE,without inhibition, butane fixed concentration = 29 mg L1 at 30 °C and 50 mg L1 at 15 °C, TCE concentration range = 0–3.4 mg L1.

Table 3 Main results of the continuous-flow simulations of TCE aerobic cometabolic treatment of the groundwater of the studied site (cTCE = 3.4 mg L1) by the four consortia object of this study in CSTR and PFR reactors. Details on the simulations are provided in Section 3.4 and in Table S1 in the Supplementary data.

a b

Reactor type

Biomass condition

Temp. (°C)

HRT (d)

mB/mTCE (g g1)a

1 1 b _ 000 d ) m B;G!L (mg L

CSTR with recycle of settled biomass

Suspended

15 30

75.5 24.0

3435 3376

0.16 0.48

Packed-bed CSTR

Attached (+suspended)

15 30

40.6 37.2

1984 5269

0.17 0.48

PFR with recycle of settled biomass

Suspended

15 30

0.90 0.20

206 171

1.5 5.7

Packed-bed PFR

Attached (+suspended)

15 30

0.50 0.38

235 136

1.3 1.9

Ratio of butane mass fed to TCE mass biodegraded. Mass flow rate of butane transferred from the gas to the liquid phase, referred to the reactor volume.

As a first step, we considered a suspended-cell continuous stirred-tank reactor (CSTR) with biomass settling and recycle of concentrated biomass. This system is described by the butane, TCE and biomass steady-state mass balance referred to the (bioreactor + settler) system, reported in Eqs. (3)–(5) for the general case of a mixed suspended-cell/attached-cell CSTR, and by the biomass steady-state mass balance in the bioreactor (Eq. (6), referred only to the suspended-cell case):

_ 000 0¼m B;G!L  0¼

cB;CSTR  qB;attached  X CSTR;attached  qB;susp:  X CSTR;susp: HRT

cTCE;0  cTCE;CSTR  qTCE;attached  X CSTR;attached  qTCE;susp: HRT  X CSTR;susp: qTCE;susp: X OUT;susp:  ð1  f Þ þ X R  f þ ðqB;susp:  Y X=S  kd  Þ HRT T C;TCE qTCE;attached  X CSTR;susp: þ ðqB;attached  Y X=S  kd  Þ  X CSTR;attached T C;TCE

ð3Þ

ð4Þ

0¼

0¼

ð5Þ

qTCE;susp: R  X R  ð1 þ RÞ  X CSTR;susp: þ ðqB;susp:  Y X=S  kd  Þ HRT T C;TCE  X CSTR;susp:

ð6Þ

The calculation was performed by assigning reasonable values to XR (6 gprotein L1), XOUT (5 mgprotein L1), f (0.01) and R (1). As shown in Table 3, the solution relative to the suspended-cell CSTR ðX CSTR;attached ¼ 0Þ resulted at both 15 and 30 °C in excessively high values of both HRT and ratio of butane fed to TCE biodegraded (mB/mTCE). A second feasibility evaluation focused on a Biomax-filled packed-bed CSTR, where cell retention in the carriers allows the elimination of the biomass settler. As a result of the co-existence of attached- and suspended-cells, this system is described by Eqs. (3)–(5), with f = 0 as the absence of the settler implies the absence of waste concentrated sludge. On the basis of the results previously obtained in a chloroform-degrading packed reactor continuously fed with butane (Ciavarelli et al., 2012), in the absence of specific information on the rate of cell detachment from the cell carrier X CSTR;attached was prudentially assumed equal to the cell concentration of the suspended-cell CSTR (3 gprotein L1), and X CSTR;susp: was set at 1/100 of X CSTR;attached . Also the packed-bed CSTR resulted at both temperatures in very high values of HRT and mB/mTCE (Table 3). The excessively high CSTR HRTs obtained for both biomass conditions are due to the fact that, as a result of the particularly low cTCE imposed in the effluent, both CSTRs work at extremely low TCE biodegradation rates. The third evaluation focused on a suspended-cell plug-flow reactor (PFR) with biomass settling and recycle of concentrated

536

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

biomass. This system is described by the butane, TCE and biomass steady-state differential mass balances in the bioreactor:

dcB ¼ qB;susp:  X susp: d HRT0

ð7Þ

dcTCE ¼ qTCE;susp:  X susp: d HRT0

ð8Þ

qTCE;susp: dX ¼ ðqB;susp:  Y X=S  kd  Þ  X susp: TC d HRT0

ð9Þ

Eqs. (7)–(9) were solved with the boundary conditions described in Table S1. As a single butane injection in the PFR, even with a cB,0 equal to the solubility of butane in water at 15 or 30 °C and 1 atm, could not satisfy the condition of stationary global cell mass balance, the simulation was modified by introducing several points of injection of gaseous butane. As shown in Fig. S2 in the Supplementary data, that reports the TCE, butane and biomass concentration profiles along the PFR at 15 and 30 °C, a partially (but not completely) optimized configuration consists in attaining in the first reactor portion a nearly complete TCE biodegradation in the absence of butane, so as to eliminate the marked effect of butane inhibition on TCE. The second reaction portion is thus dedicated to regenerating – through the addition of multiple butane pulses – the amount of biomass killed by the toxic degradation products in the first portion, while completing the degradation of TCE until the attainment of the assigned effluent concentration (0.0015 mg L1). This configuration has the additional advantage of drastically reducing TCE inhibition on butane uptake. As shown in Table 3, the suspended-cell PFR was characterized by reasonable HRTs for a wastewater treatment process (0.2–0.9 day, depending on temperature), and by a decrease of over one order of magnitude of mB/mTCE in comparison with the suspended- or attached-cell CSTR. The last simulation focused on a packed-bed PFR, characterized by a prevailing presence of attached cells. Due to the very low biomass convective term, the continuous substrate supply would determine in this configuration an excessively high cell concentration in the first bioreactor portion, with the risk of clogging the cell carrier porosity. The attached-cell PFR should therefore be fed with a pulsed substrate supply, a solution that allows a strong reduction of substrate inhibition on TCE AC, as well as the attainment of a nearly homogeneous distribution of cell concentration along the bioreactor (Goltz et al., 2001; Frascari et al., 2012). A rigorous simulation of the attached-cell PFR with pulsed butane feed goes beyond the scope of this work, as it requires a non-stationary expression of the PFR equations and an optimization of the pulsed substrate feed. To obtain an approximate evaluation of the packedbed PFR HRT, we simulated the TCE concentration profile (Eq. (8)) with a constant X CSTR;attached equal to the average X CSTR;susp: obtained in the suspended-cell PFR with recycle, and an X CSTR;susp: equal to X CSTR;attached =100. Assuming that butane is fed to the PFR for 1/4 of the duration of each cycle of butane/oxygen pulsed feed (Ciavarelli et al., 2012), the HRT resulting from the approximate PFR solution was multiplied by 4/3, under the prudential hypothesis that no TCE AC occurs in the reactor portion occupied at each instant by butane. As shown in Table 3, the 15 °C packed-bed PFR resulted in a HRT equal to about half the HRT of the suspended-cell PFR, thanks to the increase in qmax;TCE associated to cell adhesion at 15 °C (Table 1). Conversely, at 30 °C cell attachment determined an increase in HRT. An overall evaluation of the different bioreactor scenarios indicates that all the proposed PFR configurations – designed so as to minimize butane inhibition on TCE AC – allow a drastic HRT

reduction in comparison with the CSTRs, because the higher average value of cTCE along the bioreactor determines a correspondingly higher value of qTCE. The HRT decrease determines a corresponding decrease of butane consumption (mB/mTCE) as a result of the lower contribution of endogenous decay in the cell mass balance. The minimum HRT (0.20 day) was obtained with the 30 °C suspended-cell PFR. However, considering that the packed-bed PFR allows the elimination of the biomass settler, the relatively low HRTs obtained with the latter configuration (0.38–0.50 day) are of particular interest. Furthermore, the weak effect of temperature – in the 15–30 °C range – on the packedbed PFR HRT indicates that this reactor solution might be implemented without temperature control (or with a moderate temperature control during the winter) without a marked effect on the outlet TCE concentration. 4. Conclusions The increase of the 30 and 15 °C butane rate and of the 15 °C TCE rate associated to the shift from suspended to attached cells, together with lower mutual butane/TCE inhibition observed for attached cells, indicate the potential utilization of the 15 °C attached-cell consortium for the on-site bioremediation of TCEcontaminated aquifers. The continuous-flow simulation indicated that a packed-bed PFR with a 0.4–0.5 day HRT can lead to a 99.96% conversion of the studied aquifer’s average TCE concentration, with a weak effect of temperature on the TCE degradation performances in the 15–30 °C range. Acknowledgements Project co-funding by the European Commission under Grant Agreement No. 265946 (Minotaurus project, 7th FP) is acknowledged. The authors greatly thank Askoll Due for supplying the Biomax carriers. Appendix A. Notation

ci ci,CSTR f

HRT HRT0

kd KI,C,TCE-B KI,C,B-TCE KI,UC,TCE-B KI,UC,B-TCE kLa KS,i

concentration of compound i (mg L1) concentration of compound i in the CSTR reactor (mg L1) ratio of the waste sludge flow rate to the flow rate of treated groundwater, in the simulation of the CSTR with settler (–) bioreactor hydraulic residence time, referred to the flow rate of treated groundwater (d). bioreactor hydraulic residence time referred to the flow rate actually entering the bioreactor, in case of CSTR or PFR with recycle of settled biomass (HRT0 = HRT/(1 + R)) (d) constant of endogenous decay (d1) constant expressing the competitive inhibition of TCE on butane uptake (mginhibitor L1) constant expressing the competitive inhibition of butane on TCE biodegradation (mginhibitor L1) constant expressing the uncompetitive inhibition of TCE on butane uptake (mginhibitorr L1) constant expressing the uncompetitive inhibition of butane on TCE biodegradation (mginhibitorr L1) gas/liquid volumetric mass transfer coefficient (d1) true half-saturation constant of compound i

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

K obs S;i k1,i mB/mTCE _ 000 m B;G!L

N p P qi qmax,i qobs max;i R

R2 ri ri,abio ri,bio TC,TCE tR tT Vreaction

X1 XR XCSTR XOUT YX/S

Dm B

DmTCE

DX

Subscripts 0 B calc exp m O2 susp. TCE total

(mgr L1) observed half-saturated constant of compound i (mgr L1) pseudo first order constant of compound i (k1 = qmax/KS) (d1 mgprotein1 L) ratio of butane mass fed to the bioreactor to TCE mass biodegraded mass flow rate of butane transferred from the gas to the liquid phase, referred to the reactor volume (mgbutane d1 L1) number of experimental specific rates in a given kinetic test (–) probability level (–) number of model parameters (–) specific biodegradation rate of compound i (mgi mgprotein1 d1) true maximum specific biodegradation rate of compound i (mg mgprotein1 d1) observed maximum specific biodegradation rate of compound i (mg mgprotein1 d1) recycle ratio (R = volumetric flow rate of the recycle/volumetric flow rate of treated groundwater) (–) coefficient of determination (–) initial depletion rate of compound i (ri = ri,bio + ri,abio) (mg L1 d1) initial abiotic degradation rate of compound i (mg L1 d1) initial biodegradation rate of compound i (mg L1 d1) TCE transformation capacity (mgTCE gprotein1) reaction characteristic time (d) gas/liquid transport characteristic time (tT = 1/kLa) (d) liquid volume in the suspended-cell tests, or bulk volume occupied by the carriers in the attachedcell tests (L) biomass concentration in the outlet of the PFR (mgprot L1) biomass concentration in the recycle (mgprot L1) biomass concentration in the CSTR (mgprot L1) biomass concentration in the system outlet (mgprot L1) cell/butane yield ðmgprotein mg1 butane Þ butane mass consumed during the time interval associated to the evaluation of each initial biodegradation rate (g) TCE mass consumed during the time interval associated to the evaluation of each initial biodegradation rate (g) variation of active cell concentration DX occurring during the time interval associated to the evaluation of each initial biodegradation rate (mgprot L1) referring to the inlet stream referring to Butane calculated value experimental value average value referring to molecular oxygen referring to suspended cells referring to TCE referring to the whole system

537

Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 07.006. References Alpaslan Kocamemi, B., Ferhan, C., 2007. Kinetic analysis of the inhibitory effect of trichloroethylene (TCE) on nitrification in cometabolic degradation. Biodegradation 18, 71–81. Alpaslan Kocamemi, B., Çeçen, F., 2010a. Biological removal of the xenobiotic trichloroethylene (TCE) through cometabolism in nitrifying systems. Bioresour. Technol. 101, 430–433. Alpaslan Kocamemi, B., Çeçen, F., 2010b. Cometabolic degradation and inhibition kinetics of 1,2-dichloroethane (1,2-DCA) in suspended-growth nitrifying systems. Environ. Technol. 31, 295–305. Alvarez-Cohen, L., McCarty, 1991. A cometabolic transformation model for halogenated aliphatic compounds exhibiting product toxicity. Environ. Sci. Technol. 25, 1381–1387. Alvarez-Cohen, L., Speitel Jr., G.E., 2001. Kinetics of aerobic cometabolism of chlorinated solvents. Biodegradation 12, 105–126. Anderson, J.E., McCarty, P.L., 1996. Effect of three chlorinated ethenes on growth rates for a methanotrophic mixed culture. Environ. Sci. Technol. 30, 3517–3524. Balasubramanian, P., Philip, L., Bhallamudi, S.M., 2011. Biodegradation of chlorinated and non-chlorinated VOCs from pharmaceutical industries. Appl. Biochem. Biotechnol. 163, 497–518. Cappelletti, M., Frascari, D., Zannoni, D., Fedi, S., 2012a. Microbial degradation of chloroform. Appl. Microbiol. Biotechnol. 96, 1395–1409. Cappelletti, M., Bucchi, G., De Sousa Mendes, J., Alberini, A., Fedi, S., Bertin, L., Frascari, D., 2012b. Biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of four hyperthermophilic Thermotoga strains. J. Chem. Technol. Biotechnol. 87, 1291–1301. Chang, W.K., Criddle, C.S., 1997. Experimental evaluation of a model for cometabolism: prediction of simultaneous degradation of trichloroethylene and methane by a methanotrophic mixed culture. Biotechnol. Bioeng. 54, 491– 501. Chen, Y.M., Lin, T.F., Huang, C., Lin, J.C., 2008. Cometabolic degradation kinetics of TCE and phenol by Pseudomonas putida. Chemosphere 72, 1671–1680. Ciavarelli, R., Cappelletti, M., Fedi, S., Pinelli, D., Frascari, D., 2012. Chloroform aerobic cometabolism by butane-growing Rhodococcus aetherovorans BCP1 in continuous-flow biofilm reactors. Bioprocess Biosyst. Eng. 35, 667–681. Flemming, H.C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623– 633. Frascari, D., Zannoni, A., Fedi, S., Pii, Y., Zannoni, D., Pinelli, D., Nocentini, M., 2005. Aerobic cometabolism of chloroform by butane-grown microorganisms: longterm monitoring of depletion rates and isolation of a high-performing strain. Biodegradation 16, 147–158. Frascari, D., Zannoni, A., Pinelli, D., Nocentini, M., Baleani, E., Fedi, S., Zannoni, D., Farneti, A., Battistelli, A., 2006a. Long-term aerobic cometabolism of a chlorinated solvent mixture by vinyl chloride-, methane- and propaneutilizing biomasses. J. Hazard. Mater. 138, 29–39. Frascari, D., Pinelli, D., Nocentini, M., Pii, Y., Fedi, S., Zannoni, D., 2006b. Chloroform degradation by butane-grown cells of Rhodococcus aetherovorans BCP1. Appl. Microbiol. Biotechnol. 73, 421–428. Frascari, D., Pinelli, D., Nocentini, M., Baleani, E., Cappelletti, M., Fedi, S., 2008. A kinetic study of chlorinated solvent cometabolic biodegradation by propanegrown Rhodococcus sp. PB1. Biochem. Eng. J. 42, 139–147. Frascari, D., Cappelletti, M., Fedi, S., Verboschi, A., Ciavarelli, R., Nocentini, M., Pinelli, D., 2012. Application of the growth substrate pulsed feeding technique to a process of chloroform aerobic cometabolism in a continuous-flow sand-filled reactor. Process Biochem. 47, 1656–1664. Frascari, D., Fraraccio, S., Nocentini, M., Pinelli, D., 2013. Aerobic/anaerobic/aerobic sequenced biodegradation of a mixture of chlorinated ethenes, ethanes and methanes in batch bioreactors. Bioresour. Technol. 128, 479–486. Goltz, M.N., Bouwer, E.J., Huang, J., 2001. Transport issues and bioremediation modeling for the in situ aerobic co-metabolism of chlorinated solvents. Biodegradation 12, 127–140. Holman, J.P., 2001. Experimental Methods for Engineers, seventh ed. McGraw Hill, Boston. Hori, K., Mii, J., Morono, Y., Tanji, Y., Unno, H., 2005. Kinetic analyses of trichloroethylene cometabolism by toluene-degrading bacteria harboring a tod homologous gene. Biochem. Eng. J. 26, 59–64. Keenan, J.E., Strand, S.E., Stensel, H.D., 1994. Degradation kinetics of chlorinated solvents by a propane oxidizing enrichment culture. Bioremediation of Chlorinated and Polycyclic Aromatic Hydrocarbon Compounds. Lewis Publishers, Boca Raton, FL, pp. 1–13. Kim, Y., Arp, D., Semprini, L., 2002a. Kinetic and inhibition studies for the aerobic cometabolism of 1,1,1-trichloroethane, 1,1-dichloroethylene, and 1,1dichloroethane by a butane-grown mixed culture. Biotechnol. Bioeng. 80, 498–508. Kim, Y., Arp, D., Semprini, L., 2002b. A combined method for determining inhibition type, kinetic parameters and inhibition coefficients for aerobic cometabolism of

538

D. Frascari et al. / Bioresource Technology 144 (2013) 529–538

1,1,1-trichloroethane by a butane-grown mixed culture. Biotechnol. Bioeng. 77, 564–576. Sander, R., 1999. Compilation of Henry’s Law Constants for Inorganic and Organic Species of Potential Importance in Environmental Chemistry. Max-Planck Institute of Chemistry, Mainz. Segar Jr., R.L., DeWys, S.L., Speitel Jr., G.E., 1995. Sustained trichloroethylene cometabolism by phenol-degrading bacteria in sequencing biofilm reactors. Water Environ. Res. 67, 764–774. Shukla, A.K., Singh, R.S., Upadhyay, S.N., Dubey, S.K., 2011. Substrate inhibition during bio-filtration of TCE using diazotrophic bacterial community. Bioresour. Technol. 102, 3561–3563. Smith, L.H., Kitanidis, P.K., McCarty, P.L., 1997. Numerical modeling and uncertainties in rate coefficients for methane utilization and TCE cometabolism by a methane-oxidizing mixed culture. Biotechnol. Bioeng. 53, 320–331.

Tchobanoglous, G., Franklin, L.B., Stense, H.D., 2003. Wastewater Engineering: Treatment and Reuse, fourth ed. Metcalf & Eddy Inc., McGraw-Hill, Boston. Valentino, F., Brusca, A.A., Beccari, M., Nuzzo, A., Zanaroli, G., Majone, M., 2013. Start up of biological sequencing batch reactor (SBR) and short-term biomass acclimation for polyhydroxyalkanoates production. J. Chem. Technol. Biotechnol. 88, 261–270. Verce, M.F., Gunsch, C.K., Danko, A.S., Freedman, D.L., 2002. Cometabolism of cis1,2-dichloroethene by aerobic cultures grown on vinyl chloride as the primary substrate. Environ. Sci. Technol. 36, 2171–2177. Zanaroli, G., Balloi, A., Negroni, A., Borruso, L., Daffonchio, D., Fava, F., 2012. A Chloroflexi bacterium dechlorinates polychlorinated biphenyls in marine sediments under in situ-like biogeochemical conditions. J. Hazard. Mater. 209–210, 449–457. Zhang, Y., Tay, J.H., 2012. Co-metabolic degradation activities of trichloroethylene by phenol-grown aerobic granules. J. Biotechnol. 162, 274–282.