Evaluation of scale-up effect on cold-active enzyme production and biodegradation tests using pilot-scale bioreactors and a 3D soil tank.

Despite recent attention being paid to the biodegradation of petroleum hydrocarbons in cold environments, scale-up studies of biodegradation are lacking. Herein, the effect of scale-up on the enzymatic biodegradation of highly contaminated soil at low temperatures was studied. A novel cold-adapted bacteria (Arthrobacter sp. S2TR-06) was isolated that could produce cold-active degradative enzymes (xylene monooxygenase (XMO) and catechol 2,3-dioxygenase (C2,3D)). Enzyme production was investigated on 4 different scales (lab to pilot scale). The results showed a shorter fermentation time, and the highest production of enzymes and biomass (107 g/L for biomass, 109 U/mL, and 203 U/mL for XMO and C2,3D after 24 h) was achieved in the 150-L bioreactor due to enhanced oxygenation. Multi-pulse injection of p-xylene into the production medium was needed every 6 h. The stability of membrane-bound enzymes can be increased up to 3-fold by adding FeSO4 at 0.1% (w/v) before extraction. Soil tests also showed that biodegradation is scale-dependent. The maximum biodegradation rate decreased from 100% at lab-scale to 36% in the 300-L sand tank tests due to limited access of enzymes to trapped p-xylene in soil pores, low dissolved oxygen in the water-saturated zone, soil heterogeneity, and the presence of the free phase of p-xylene. The result demonstrated that formulation of enzyme mixture with FeSO4 and direct injection of enzyme mixture (third scenario) can increase the efficiency of bioremediation in heterogeneous soil. In this study, it was demonstrated that cold-active degradative enzyme production can be scaled up to an industrial scale and enzymatic treatment can be used to effectively bioremediate p-xylene contaminated sites. This study could provide key scale-up guidance for the enzymatic bioremediation of mono-aromatic pollutants in water-saturated soil under cold conditions.

Pilot-scale production and in-situ application of petroleum-degrading enzyme cocktail from Alcanivorax borkumensis.

Petroleum degrading enzymes can be used as an alternative way to improve petroleum bioremediation approaches. Alcanivorax borkumensis is an alkane-degrading bacteria that can produce petroleum degrading enzymes such as alkane hydroxylase and lipase. In this study, pilot-scale Alcanivorax borkumensis fermentation was developed for producing large volumes of petroleum degrading enzymes cocktail (∼900 L). Different process conditions, such as inoculum age 72 h and size 4% v/v, temperature 30 ± 1 °C, agitation speed at 150 rpm and, fermentation period 3 days were determined as the optimum for producing alkane hydroxylase and lipase activity. The oxygen transfer capacity was studied for obtaining better bacterial growth and higher enzyme activities in bioreactor process optimization as well as scale-up. Results showed that the maximum values of oxygen mass transfer coefficient (kLa), oxygen uptake rate (OUR), oxygen transfer rate (OTR), alkane hydroxylase, lipase, and cell count were 196.95 h-1, 0.92 mmol O2/L/h, 1.8 mmol O2/L/h, 222.49 U/mL, 325 U/mL, and 8.6 × 1010 CFU/mL, respectively. Compared with the bench-scale bioreactors, the 150 L fermenter showed a better oxygen transfer rate which affected the cell growth that doubled the number and enzymes production that increased. Then, the enzyme cocktail was used for a field test in a diesel source zone using a 5-spot well pattern. The results showed a significant reduction in concentrations of C10 - C50 (from 36% to > 99%) after one injection of enzyme cocktail, mainly for the contaminated soils located in the saturated zone of the unconfined aquifer. This study confirmed the scaling-up ofalkane-degrading enzyme production to an industrial-scale and its application for effective bioremediation of petroleum contaminated sites.

Column tests for evaluation of the enzymatic biodegradation capacity of hydrocarbons (C10-C50) contaminated soil.

Though many studies pertaining to soil bioremediation have been performed to study the microbial kinetics in shake flasks, the process efficiency in column tests is seldom. In the present study, soil columns tests were carried out to study the biodegradation of soil contaminated with a high concentration of diesel (≈19.5 g/kg) petroleum hydrocarbons expressed as C10-C50. Experiments were done with crude enzymatic cocktail produced by the hydrocarbonoclastic bacterium, Alcanivorax borkumensis. A. borkumensis was grown on a media with 3% (v/v) motor oil as the sole carbon and energy source. The effects of the enzyme concentration, treatment time and oxidant on the bioremediation efficiency of C10-C50 were investigated. A batch test was also carried out in parallel to investigate the stability of the enzymes and the effect of the biosurfactants on the desorption and the bioconversion of C10-C50. Batch tests indicated that the biosurfactants significantly affected the desorption and alkane hydroxylase and lipase enzymes, maintained their catalytic activity during the 20-day test, with a half-life of 7.44 days and 8.84 days, respectively. The crude enzyme cocktail, with 40 U/mL of lipase and 10 U/mL of alkane hydroxylase, showed the highest conversion of 57.36% after 12 weeks of treatment with a degradation rate of 0.0218 day-1. The results show that the soil column tests can be used to optimize operating conditions for hydrocarbon degradation and to assess the performance of the overall bioremediation process.

Bench-scale production of enzymes from the hydrocarbonoclastic bacteria Alcanivorax borkumensis and biodegradation tests.

This study investigates motor oil (3, 5, 7.5 and 10% (v v-1)) as a sole carbon source for the production of Alcanivorax borkumensis in shake flasks and a 5 L bench-scale fermenter in comparison to the standard media. Shake flask studies showed a significant and higher cell growth (p=0.000038), lipase (p = 0.006900) and alkane hydroxylase production (p = 0.000921) by Alcanivorax borkumensis when motor oil was used as the substrate. Based on Tukey post-hoc tests, 5% motor oil concentration was selected as the optimal substrate concentration. The 5 L fermenter experiments conducted using motor oil at 5% (v v-1) concentration, under controlled conditions exhibited significant and higher alkane hydroxylase and lipase activities (55.6 U mL-1 (p = 0.018418) and 208.30 U mL-1 (p = 0.020087), respectively) as compared with those of motor oil at 3% (v v-1) and n-hexadecane at 3% (v v-1) concentration which was used as control. Cell growth was significantly higher when motor oil (3 or 5%) was used as a substrate (p = 0.024705). Enzymatic degradation tested on two different polycyclic aromatic hydrocarbons (PAHs) contaminated groundwaters showed 37.4% removal after 5 days with a degradation rate of 196.6 ppb day-1 and 82.8% removal after 10 days with a degradation rate of 217.54 ppb day-1 for the 1st site and an almost complete biodegradation with 95% removal and 499.02 ppb day-1 removal rate after only 5 days for the 2nd site.

Production and characterization of novel hydrocarbon degrading enzymes from Alcanivorax borkumensis.

This study investigates the production of alkane hydroxylase, lipase and esterase by the marine hydrocarbon degrading bacteria Alcanivorax borkumensis. The focus of this study is the remediation of petroleum hydrocarbons, hexane, hexadecane and motor oil as model substrates. A. borkumensis showed an incremental growth on these substrates with a high cell count. Growth on motor oil showed highest alkane hydroxylase and lipase production of 2.62 U/ml and 71 U/ml, respectively, while growth on hexadecane showed the highest esterase production of 57.5 U/ml. The percentage of hexane, hexadecane, and motor oil degradation during A. borkumensis growth after 72 h, was around 80%, 81.5% and 75%, respectively. Zymogram showed two different bands with a molecular weight of approx. 52 and 40 kDa, respectively with lipase and esterase activity. Alkane hydroxylase reached optimum activity at pH 8.0 and 70 ±â€¯1 °C for hexane and hexadecane and 75 ±â€¯1 °C for motor oil. Lipase and esterase showed optimum activity at 35 ±â€¯1 °C and 40 ±â€¯1 °C, respectively and pH 7.0. The crude enzymes showed higher stability in a wide range of pH, but they were not thermostable at higher temperatures.

Impact of heterogeneous properties of soil and LNAPL on surfactant-enhanced capillary desaturation.

This paper investigates low-concentration (<1wt%) surfactant flushing when used as a follow-up technology for multiphase vacuum extraction on heterogeneous sites. Challenges posed by soil permeability, pore-size distribution, mineralogy, light non-aqueous phase liquid (LNAPL) weathering and groundwater hardness were quantified through batch and soil column tests. Compatibility issues between the mixed mineralogy soils, hard groundwater, mixed LNAPL and usual anionic surfactants were observed. The selected solution was a Winsor type I system promoting an interfacial tension of 0.06mN/m between the site LNAPL and the amphoteric surfactant CAS in aqueous solution at pH12. Surfactant loses to adsorption and pore media plugging were observed in the fine soil fraction. The capillary desaturation curves (CDC) obtained with the column tests suggested mixed-wettability behavior. The soil permeability strongly influenced LNAPL recovery, as expressed by the relationship obtained between capillary numbers (NCa) and hydraulic gradients. In this case, the critical NCa, marking the onset of capillary desaturation, could only be obtained with realistic hydraulic gradients in the coarse soil fraction. At those gradients, potential LNAPL recovery was 30% at the most. Unlike previously published CDCs, the relationship between NCa (log-scale) and LNAPL recovery was not linear but dependant on residual LNAPL saturation.

2D sandbox experiments of surfactant foams for mobility control and enhanced LNAPL recovery in layered soils.

The ability of surfactant foam to enhance mobility control and LNAPL recovery in a heterogeneous porous media was investigated through sandbox experiments with p-xylene used as LNAPL. A layered heterogeneous porous media was represented in a 2D sandbox filled with two layers of coarse and medium silica sand. Based on previous tests, the surfactant solution used was Ammonyx Lo at a concentration of 0.1% (w/w). The sandbox experimental program included tracer tests done under both uncontaminated and contaminated conditions, foam injection under uncontaminated conditions and surfactant solution and foam injection under contaminated conditions. Tracer tests indicated that the permeability contrast between sand layers was increased by LNAPL contamination. Foam injection under uncontaminated conditions presented a S-shaped front that indicated a better mobility control than the piston-shaped front obtained during tracer tests. During foam injection, complete sweep of the sandbox was achieved with 1.8pore volume (PV) compared to 2.8PV during tracer injection, thus indicating better mobility control with foam. Pre-flush of the contaminated sandbox with surfactant solution initiated p-xylene mobilization but no free phase was recovered at the effluent. A negligible p-xylene residual saturation was reached following foam injection in the contaminated sandbox. However, mass balance indicated a total recovery of only 48% of the initial p-xylene, thus indicating an underestimation of the recovery by volatilization. Recovery by volatilization was corrected, which gave the following proportions of LNAPL recovery mechanisms: 19% by mobilization, 16% by dissolution and 65% by volatilization. Results show the potential efficiency of foam remediation of LNAPL source zones in heterogeneous porous media. Still, further developments are needed prior to field scale application, which could benefit from in situ foam production that would simplify on-site operations.