Effects of Triton X-100 and Quillaya Saponin on the ex situ bioremediation of a chronically polychlorobiphenyl-contaminated soil

The possibility of enhancing the ex situ bioremediation of a chronically polychlorinated biphenyl (PCB)-contaminated soil by using Triton X-100 or Quillaya Saponin, a synthetic and a biogenic surfactant, respectively, was studied. The soil, which contained about 350 mg/kg of PCBs and indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids, was amended with inorganic nutrients and biphenyl, saturated with water and treated in aerobic batch slurry- and fixed-phase reactors. Triton X-100 and Quillaya Saponin were added to the reactors at a final concentration of 10 g/l at the 42nd day of treatment, and at the 43rd and 100th day, respectively. Triton X-100 was not metabolised by the soil microflora and it exerted inhibitory effects on the indigenous bacteria. Quillaya Saponin, on the contrary, was readily metabolised by the soil microflora. Under slurry-phase conditions, Triton X-100 negatively influenced the soil bioremediation process by affecting the availability of the chlorobenzoic acid degrading indigenous bacteria, whereas Quillaya Saponin slightly enhanced the biological degradation and dechlorination of the soil PCBs. In the fixed-phase reactors, where both the surfactant availability and the mixing of the soil were lower, Triton X-100 did not exert inhibitory effects on the soil biomass and enhanced significantly the soil PCB depletion, whereas Quillaya Saponin did not influence the bioremediation process. Received: 28 April 1998 / Received last revision: 15 July 1998 / Accepted: 29 July 1998     

Soya lecithin effects on the aerobic biodegradation of polychlorinated biphenyls in an artificially contaminated soil

The effects of the phytogenic surfactant soya lecithin (SL) on the aerobic biodegradation of polychlorinated biphenyls (PCBs) spiked into a synthetic soil were studied. Soil was spiked with both biphenyl (4 g/kg) and Fenclor 42 (1,000 mg/kg) and treated in aerobic batch slurry-phase microcosms (17.5% w/v). Microcosms were prepared either with or without the exogenous aerobic PCB-dechlorinating bacterial co-culture ECO3 (inoculum:10(8) CFU/mL). In some inoculated microcosms, SL was added at 15 or 30 g/kg. Indigenous bacteria having the capability of metabolizing biphenyl and 2-chlorobenzoic acid were found to develop in the microcosms during the experiment, and were responsible for the significant PCB biodegradation and dechlorination observed in the uninoculated controls. The addition of ECO3 bacteria resulted in only a slight PCB biodegradation increase. In the presence of SL, a higher availability of biphenyl- and chlorobenzoic acid-degrading bacteria and higher PCB biodegradation and dechlorination yields were observed; the effects increased proportionally with the concentration of the applied SL. A significant decrease of soil ecotoxicity was also revealed in SL-supplemented microcosms. At both concentrations, SL was found to be a good carbon source for both the indigenous and ECO3 bacteria, as well as a product capable of enhancing the PCB bioavailability in the microcosms.     

Effects of humic substances and soya lecithin on the aerobic bioremediation of a soil historically contaminated by polycyclic aromatic hydrocarbons (PAHs)

The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) strongly reduces their bioavailability in aged contaminated soils, thus limiting their bioremediation. The biodegradation of PAHs in soils can be enhanced by employing surface-active agents. However, chemical surfactants are often recalcitrant and exert toxic effects in the amended soils. The effects of two biogenic materials as pollutant-mobilizing agents on the aerobic bioremediation of an aged-contaminated soil were investigated here. A soil historically contaminated by about 13 g kg(-1) of a large variety of PAHs, was amended with soya lecithin (SL) or humic substances (HS) at 1.5% w/w and incubated in aerobic solid-phase and slurry-phase reactors for 150 days. A slow and only partial biodegradation of low-molecular weight PAHs, along with a moderate depletion of the initial soil ecotoxicity, was observed in the control reactors. The overall removal of PAHs in the presence of SL or HS was faster and more extensive and accompanied by a larger soil detoxification, especially under slurry-phase conditions. The SL and HS could be metabolized by soil aerobic microorganisms and enhanced the occurrence of both soil PAHs and indigenous aerobic PAH-degrading bacteria in the reactor water phase. These results indicate that SL and HS are biodegradable and efficiently enhance PAH bioavailability in soil. These natural surfactants significantly intensified the aerobic bioremediation of a historically PAH-contaminated soil under treatment conditions similar to those commonly employed in large-scale soil bioremediation.     

Effects of humic substances on the bioavailability and aerobic biodegradation of polychlorinated biphenyls in a model soil.

The very high hydrophobicity of polychlorinated biphenyls (PCBs) strongly reduces their bioavailability in aged contaminated soils, thus limiting their bioremediation. The biodegradability of PCBs in heavily contaminated soils can be significantly enhanced by soil treatment with surface-active agents. In this work, the effects of naturally occurring surfactants such as humic substances (HS) on the aerobic biodegradation of PCBs in a model soil were studied. The soil was amended with biphenyl (4 g/kg), Fenclor 42 (1,000 mg/kg), the aerobic PCB-biodegrading bacterial co-culture ECO3 (inoculum: 10(8)CFU/mL), and treated in aerobic batch slurry-phase conditions (17.5% w/v) with and without the addition of HS at the rates of 1.5 and 3.0% (w/w). Low PCBs biodegradation and dechlorination yields were observed in the HS-free microcosms, probably as a result of the rapid disappearance of inoculated bacteria. The presence of HS influenced significantly the activity of the specialized biomass and the biodegradation of PCBs in the microcosms. The microcosms that received HS at the 1.5% rate showed a higher persistence of the specialized bacteria and yields of PCB biodegradation and dechlorination about 150 and 100%, respectively, larger than those found for the HS-free microcosms. Lower stimulating effects were observed in the microcosms added with the HS at 3.0% rate. These effects were attributed to an increased solubilization of PCBs in the hydrophobic domains of the humic supramolecular associations and to a different accessibility of PCBs by the specialized bacteria at the different rates of HS addition. Although the slurry-phase treatment generally showed a decrease of the original soil ecotoxicity, the addition of the originally non-toxic HS decreased soil ecotoxicity for the Collembola animal biomarker and increased that towards the Lepidium sativum vegetal biomarker.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Production of swainsonine from Metarhizium anisopliaein stirred-tank and air-lift reactors

Growth of Metarhizium anisopliaein modified starch-casein medium produced 61, 50, and 58 mg swainsonine/l when cultured in shaken flasks, stirred-tank and air-lift reactors respectively. Over approximately 45 h, the maximum swain-sonine specific productivity was 0.47 mg/g.h in a stirred tank reactor and 0.32 mg/g.h in an air-lift reactor. After 120 h, increasing broth viscosity was encountered in the latter fermenter.     

Intensification of the aerobic bioremediation of an actual site soil historically contaminated by polychlorinated biphenyls (PCBs) through bioaugmentation with a non acclimated, complex source of microorganisms

Background  

The biotreatability of actual-site polychlorinated biphenyl (PCB)-contaminated soils is often limited by their poor content of autochthonous pollutant-degrading microorganisms. In such cases, inoculation might be the solution for a successful bioremediation. Some pure and mixed cultures of characterized PCB degrading bacteria have been tested to this purpose. However, several failures have been recorded mostly due to the inability of inoculated microbes to compete with autochthonous microflora and to face the toxicity and the scarcity of nutrients occurring in the contaminated biotope. Complex microbial systems, such as compost or sludge, normally consisting of a large variety of robust microorganisms and essential nutrients, would have better chances to succeed in colonizing degraded contaminated soils. However, such sources of microorganisms have been poorly applied in soil bioremediation and in particular in the biotreatment of soil with PCBs. Thus, in this study the effects of Enzyveba, i.e. a consortium of non-adapted microorganisms developed from composted material, on the slurry- and solid-phase aerobic bioremediation of an actual-site, aged PCB-contaminated soil were studied.     

Optimization of agitation for production of swainsonine from Metarhizium anisopliae in stirred tank and airlift reactors

The optimal agitation rate for production of swainsonine from Metarhizium anisopliae grown in batch stirred tank reactors (2 to 20 l) was 400 rpm with a mixed hyphal and pelleted morphology where the specific swainsonine production rate was 9×10^–2 mg g^–1 cell dry wt h from 87 to 142 h. Culture of the fungus in a 6-l airlift reactor produced loose pellets and the production of swainsonine started at least 24 h earlier than in the stirred tank reactor. The final yield (5.9 mg swainsonine g^–1 cell dry wt) after 168 h in the airlift reactor was 18% less than those obtained in the stirred tank reactor with an agitation rate of 400 rpm.     

Influence of bioreactor design on the rate and amount of curdlan-type exopolysaccharide production byAlcaligenes faecalis

Summary The amount of polymer recovered during lab-scale batch production of curdlan-type polysaccharide byAlcaligenes faecalis (ATCC 31749) was increased by 46% through the manipulation of the vessel configuration. When standard turbine impellers were used to provide mixing and agitation the specific rate of production, Qp, decreased significantly after 40–50 hours elapsed fermentation time, EFT. The Qp remained at a high level throughout the entire time course of production (90 hours) when (i) a propeller was substituted for a flat-blade turbine impeller in a conventional baffled stirred tank reactor, or (ii) agitation and mixing were accomplished in a non-rotary vibro-fermenter.     

Use of exogenous specialised bacteria in the biological detoxification of a dump site‐polychlorobiphenyl‐contaminated soil in slurry phase conditions

The possibility of biologically detoxifying a contaminated soil from an Italian dump site containing about 1500 mg/kg (in dry soil) of polychlorinated biphenyls was studied in the laboratory in this work. The soil, which contained indigenous aerobic bacteria capable of growing on biphenyl or on monochlorobenzoic acids at concentration of about 300 CFU per g of air‐dried soil, was amended with inorganic nutrients, saturated with water and treated in aerobic 3‐L batch slurry reactors (soil suspension at 20% w/v). Either Pseudomonas sp. CPE1 strain, capable of cometabolising low‐chlorinated biphenyls into chlorobenzoic acids, or a bacterial co‐culture capable of aerobically dechlorinating polychlorobiphenyls constituted by this bacterium and the two chlorobenzoic acid degrading bacteria Pseudomonas sp. CPE2 strain and Alcaligenes sp. CPE3 strain, were used as inocula (final concentration of about 10⁸ CFU/mL for each bacterium), in the absence and in the presence of biphenyl (4 g/kg of air dried soil). Significant soil polychlorobiphenyl depletions were observed in all the reactors after 119 days of treatment. The soil inoculation with the sole CPE1 was found to slightly enhance the polychlorobiphenyl depletions (about 20%) and the soil detoxification; the effect was higher in the presence of biphenyl. The use of the polychlorobiphenyl mineralising bacterial co‐culture as inoculum resulted in a strong enhancement of the depletions of both the soil polychlorobiphenyls (from 50 to 65%) and of the original soil ecotoxicity. The bacterial biomass inoculated was found to implant into the soil; the higher specialised biomass availability thus reached in the inoculated soil was probably responsible of a more extensive biodegradation of polychlorobiphenyls and therefore of the higher detoxification yields observed in the inoculated reactors. The soil ecotoxicity, measured through two different soil contact assays, i.e., the Lepidium sativum germination test and the Collembola mortality test, was often found to decrease proportionally with the soil polychlorobiphenyl concentration. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 240–249, 1999.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor

 A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of Taxus cuspidata (Japanese yew) on a pilot-scale. Rapidly growing cell lines were selected from callus cultures derived from immature embryos of yew. The cells were inoculated in 20-l capacity bioreactors of different types to test the growth performance. The models of small-scale bioreactors incorporated in this study included a balloon-type bubble bioreactor (BTBB), a bubble-column bioreactor (BCB), a BCB with a split-plate internal loop, a BCB with a concentric draught-tube internal loop, a BCB with a fluidized bed bioreactor, and two different models of stirred tank reactors. Among the reactors, BTBB appeared to be the most efficient in promoting cell growth. The doubling time of cell growth in BTBB was 12 days with a 30% inoculation cell density. The optimum time for medium replacement or feeding was 12–15 days after inoculation as determined by monitoring both the levels of sugars and medium conductivity. When yew tree cells were grown in different sizes (100–500-l) of BTBBs, more than 70% cell viability was recorded at the time of harvest. The growth pattern of the cells in the pilot-scale BTBB appeared to be the same as that of cells in the 20-l bioreactors. Approximately 3 mg/l of taxol and 74 mg/l total taxanes were obtained after 27 days of culture. Received: 6 April 1999 / Revision received: 23 August 1999 / Accepted: 31 August 1999     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.     

Production of tropane alkaloids by transformed root cultures of Atropa belladonna in stirred bioreactors with a stainless steel net

A scale up of transformed root cultures of Atropa belladonna from a 300-ml flask to a 30-l tank was accomplished without any reduction in alkaloid productivity. Cutting treatment of seed cultures showed no distinct effect on root growth, morphology, and alkaloid content in conical flasks during 1 month of culture. Randomly cut roots thus grown were further cultivated in 3-l and 30-l modified stirred bioreactors for a scale-up culture. After 1 month of culture, 1490 mg of tropane alkaloids was produced by a 30-l culture of A. belladonna transformed roots. These roots contained the same level of atropine (5.4 mg/ g dw) as the roots of this plant grown in the field for 12 months and still contained a considerable amount of other alkaloids including 1.6 mg/g dw of 6-β-hydroxyhyoscyamine, 0.9 mg/g dw of scopolamine, and 2.0 mg/g dw of littorine. Received: 12 June 1998 / Revision received: 31 August 1998 / Accepted: 27 October 1998     

Methyl-beta-cyclodextrin-enhanced solubilization and aerobic biodegradation of polychlorinated biphenyls in two aged-contaminated soils

The bioremediation of aged polychlorinated biphenyl (PCB)-contaminated soils is adversely affected by the low bioavailability of the pollutants. Randomly methylated-beta-cyclodextrins (RAMEB) were tested as a potential PCB-bioavailability-enhancing agent in the aerobic treatment of two aged-contaminated soils. The soils, contaminated by about 890 and 8500 mg/kg of Aroclor 1260 PCBs, were amended with biphenyl (4 g/kg), inorganic nutrients (to adjust their C:N ratio to 20:1), and variable amounts of RAMEB (0%, 0.5%, or 1.0% [w/w]) and treated in both aerobic 3-L solid-phase reactors and 1.5-L packed-bed loop reactors for 6 months. Notably, significant enhancement of the PCB biodegradation and dechlorination, along with a detectable depletion of the initial soil ecotoxicity, were generally observed in the RAMEB-treated reactors of both soils. RAMEB effects were different in the two soils, depending upon the treatment conditions employed, and generally increased proportionally with the concentration at which RAMEB was applied. RAMEB, which was slowly metabolized by the soil's aerobic microorganisms, was found to markedly enhance the occurrence of the indigenous aerobic, cultivable biphenyl-growing bacteria harboring genes homologous to those of two highly specialized PCB degraders (i.e., bphABC genes of Pseudomonas pseudoalcaligenes KF707 and bphA1A2A3A4BC1 genes of Rhodococcus globerulus P6) and chlorobenzoic acid-degrading bacteria as well as the occurrence of PCBs in the water phase of the soil reactors. These findings indicate that RAMEB enhanced the aerobic bioremediation of the two soils by increasing the bioavailability of PCBs and the occurrence of specialized bacteria in the soil reactors.     

Enhancement of PCB degradation by Burkholderia xenovorans LB400 in biphasic systems by manipulating culture conditions

Two-phase partitioning bioreactors (TPPBs) can be used to biodegrade environmental contaminants after their extraction from soil. TPPBs are typically stirred tank bioreactors containing an aqueous phase hosting the degrading microorganism and an immiscible, non-toxic and non-bioavailable organic phase functioning as a reservoir for hydrophobic compounds. Biodegradation of these compounds in the aqueous phase results in thermodynamic disequilibrium and partitioning of additional compounds from the organic phase into the aqueous phase. This self-regulated process can allow the delivery of large amounts of hydrophobic substances to degrading microorganisms. This paper explores the reactor conditions under which the polychlorinated biphenyl (PCB) degrader Burkholderia xenovorans LB400 can degrade significant amounts of the PCB mixture Aroclor(R) 1242. Aroclor(R) degradation was found to stall after approximately 40 h if no carbon source other than PCBs was available in the reactor. Sodium pyruvate was found to be a suitable carbon source to maintain microbial activity against PCBs and to function as a substrate for additional cell growth. Both biphenyl (while required during the inoculum preparation) and glucose had a negative effect during the Aroclor(R) degradation phase. Initial Aroclor(R) 1242 degradation rates in the presence of pyruvate were high (6.2 mg L(-1) h(-1)) and 85% of an equivalent concentration of 100 mg Aroclor(R) 1242 per L aqueous phase could be degraded in 48 h, which suggest that solvent extraction of PCBs from soil followed by their biodegradation in TPPBs might be a feasible remediation option.     

Impact of Anaerobic and Aerobic Processes on PolyChloroBiphenyl Removal in Contaminated Sewage Sludge

Aerobic and anaerobic biodegradation of six priority PCBs was investigated in continuous stirred tank reactors fed with naturally contaminated sewage sludge. Anaerobic and aerobic abiotic losses were higher for the lightly chlorinated PCBs but remained for all PCBs below 20%. Under strict methanogenic conditions, PCB removals were about 40% whatever PCB molecular weight or their degree of chlorination. However, considering abiotic losses, the heaviest PCBs were more efficiently anaerobically biodegraded probably because of higher dechlorination rates. The aerating sludge process enhanced removal of the lightest chlorinated PCBs from 40% up to 100%, while removal rates of the heaviest PCBs remained around 40%. Although the mesophilic aerobic process exhibits better removal efficiencies because of operating conditions, the results suggest that PCB biodegradation was strongly limited by their bioavailability in naturally contaminated sludge, under both redox conditions. Indeed, since PCB removal was closely linked to the solid reduction rates, PCB bioavailability was likely the limiting factor for biodegradation. As a consequence, the raw PCB concentrations (in mg kg^–1_{dry weight}) which are concerned by legislative procedures did not decrease sufficiently in both processes to reach a limit value fulfilling the current French/European regulation about PCB contents in sewage sludge before spreading on agricultural land.     

Comparison of the performances of stirred tank and airlift tower loop reactors.

Following a consideration of the prerequisites for reactor comparison and the fundamental differences between stirred tank and airlift tower loop reactors, their performances are compared for the production of secondary metabolites: penicillin V by Penicillium chrysogenum, cephalosporin C by Cephalosporium acremonium, and tetracycline by Streptomyces aureofaciens. In stirred tank reactors, cell mass concentrations, volumetric productivities, and specific power inputs are higher than in airlift tower loop reactors. In the latter, efficiencies of oxygen transfer are higher, and specific productivities with regard to power input, substrate and oxygen consumptions, and yield coefficients of product formation with regard to substrate and oxygen consumptions are considerably higher than in stirred tank reactors. The prerequisites for improved performance are discussed.     

Effect of mode of operation of bioreactors on the biosynthesis of penicillin amidase in Escherichia coli

Different operational mode of bioreactors influence the biosynthesis of the enzyme and related products as well as the growth of industrial microorganisms. This communication deals with the effect of mode of operation of various bioreactors with different geometric configurations, viz., batch (includes commercially available batch stirred tank, and custom-designed cylindrical and tapered reactors), batch-fed, continuous flow stirred tank reactors on the biosynthesis of penicillin amidase in Escherichia coli. Experimental findings show that the biosynthesis of penicillin amidase in E. coli show a little variation among batch reactor modes and significant variation on the continuous mode of operation. Further analysis show that the different reactor modes also influence periplasmic localization of the enzyme in the cell.     

Production of optically pure L-alanine in batch and continuous stirred tank reactors using immobilized Pseudomonas sp.

A process to obtain optically pure l-alanine has been developed using batch and continuous stirred tank reactors with a new l-aminoacylase-producing bacterium Pseudomonas sp. BA2 immobilized in calcium alginate beads coated with glutaraldehyde. The maximum production of l-alanine in a continuous stirred tank reactor was 11.26 g after 2 days of operation which is higher than that previously reported.