Eric N. Kaufman

Poly(ethylene glycol)‐modified ligninase enhances pentachlorophenol biodegradation in water–solvent mixtures

Polychlorinated hydrocarbons are prevalent environmental contaminants whose rates of biodegradation are limited by their minimal solubilities in aqueous solutions where the biological reactions take place. In this study, ligninase (LiP) from Phanerochaete chrysosporium was modified by poly(ethylene glycol) to enhance its activity and stability for the biodegradation of pentachlorophenol (PCP) in the presence of acetonitrile (MeCN), a water-miscible solvent. The modified enzyme retained 100% of its activity in aqueous solutions and showed enhanced tolerance against the organic solvent. The activity of the modified enzyme was found to be over twice that of the native enzyme in the presence of 10% (v/v) MeCN. The solubility of PCP was enhanced significantly by the addition of MeCN to aqueous solutions, such that it was over 10-fold more soluble in the presence of 15% (v/v) MeCN than in pure aqueous buffer solution (from 0.06 to 0.65 mM). Capitalizing on the enhanced substrate solubility and the increased activity of the modified enzyme, the catalytic efficiency of the modified LiP in solutions containing 15% MeCN was over 11-fold higher than that of the native enzyme in buffer solutions (pH 4.2) in unoptimized reactor systems (from 44 to 480 mol PCP/mol LiP.h). Continued research both in the use of organic solvents to increase the availability of recalcitrant contaminants and in the modification of enzymes to enhance their activity and stability in such solvents promises to dramatically affect our ability to remediate contaminated sites. Published by John Wiley & Sons.

Comparison of batchstirred and electrospray reactors for biodesulfurization of dibenzothiophene in crude oil and hydrocarbon feedstocks

Biological removal of organic sulfur from petroleum feedstocks offers an attractive alternative to conventional thermochemical treatment, because of the mild operating conditions afforded by the biocatalyst. In order for biodesulfurization to realize commercial success, reactors must be designed that allow for sufficient liquid-liquid and gas-liquid mass transfer, while simultaneously reducing operating costs. Electro-spray bioreactors were investigated for use as desulfurization reactors because of their reported operational cost savings relative to mechanically agitated reactors. Unlike batch-stirred reactors, which mix the biocatalystcontaining aqueous phase with the organic feedstock by imparting momentum to the entire bulk solution, electro-spray reactors have the potential for tremendous cost savings, creating an emulsion <5 (μm in diameter, at a cost of only 3 W/L. Power law relationships indicate that mechanically stirred reactors would require 100-1000-fold more energy to create such a fine emulsion, but these relationships generally do not account for the effect of endogenously produced surfactant in the system. Here, the rates dibenzothiophene (DBT) oxidation to 2-hydroxybiphenyl (2-HBP) in hexadecane, byRhodococcus sp IGTS8 are compared in the two reactor systems. Desulfurization rates ranged from 1.0 to 5.0 mg 2-HBP/(dry g cells · h), independent of the reactor employed. The batch-stirred reactor was capable of forming a very fine emulsion in the presence of the biocatalyst IGTS8, similar to that formed in the emulsion phase contactor (EPTM), presumably because the biocatalyst produces its own surfactant. Although EPC did not prove to be advantageous for the IGTS8 desulfurization system, it may prove advantageous for systems that do not produce surface-active bioagents, in addition to being mass-transport limited.

Screening of resins for use in a biparticle fluidized-bed bioreactor for the continuous fermentation and separation of lactic acid

A continuous particle fluidized-bed reactor is being developed for the simultaneous fermentation and purification of lactic acid. Unlike conventional fermentation schemes, the biparticle fluidized bed does not require the addition of salts for reactor pH control and product separation as the inhibitory product is removed directly from the reactor. This minimizes process waste and enhances reactor efficiency. This work has screened a series of adsorbents for possible utilization in the biparticle fluidized-bed fermentation of lactic acid by immobilizedLactobacillus delbreuckii. Capacity, specificity, regenerability, and kinetics were investigated. Although a resin exhibiting all of the desired properties has yet to be found, Reillex 425 appears satisfactory and will be utilized in initial process demonstration as resin screening continues.

Biodesulfurization of Flue Gases and Other Sulfate/Sulfite Waste Streams Using Immobilized Mixed Sulfate-Reducing Bacteria†

Sulfur dioxide (SO2) is one of the major pollutants in the atmosphere that cause acid rain. Microbial processes for reducing SO2 to hydrogen sulfide (H2S) have previously been demonstrated by utilizing mixed cultures of sulfate‐reducing bacteria (SRB) with municipal sewage digest as the carbon and energy source. To maximize the productivity of the bioreactor for SO2 reduction in this study, various immobilized cell bioreactors were investigated: a stirred tank with SRB flocs and columnar reactors with cells immobilized in either κ‐carrageenan gel matrix or polymeric porous BIO‐SEP beads. The maximum volumetric productivity for SO2 reduction in the continuous stirred‐tank reactor (CSTR) with SRB flocs was 2.1 mmol of SO2/(h·L) . The κ‐carrageenan gel matrix used for cell immobilization was not durable at feed sulfite concentrations greater than 2000 mg/L (1.7 mmol/(h·L) ) . A columnar reactor with mixed SRB cells that had been allowed to grow into highly stable BIO‐SEP polymeric beads exhibited the highest sulfite conversion rates, in the range 16.5 mmol/(h·L) (with 100% conversion) to 20 mmol/(h·L) (with 95% conversion) . The average specific activity for sulfite reduction in the column, in terms of dry weight of SRB biomass, was 9.5 mmol of sulfite/(h·g) . In addition to flue gas desulfurization, potential applications of this microbial process include the treatment of sulfate/sulfite‐laden wastewater from the pulp and paper, petroleum, mining, and chemical industries.

Development of an electro-spray bioreactor for crude oil processing☆

Biological removal of organic sulfur from petroleum feedstocks offers an attractive alternative to conventional thermochemical treatment due to the mild operating conditions afforded by the biocatalyst. In order for biodesulfurization to realize commercial success, it will be necessary to design reactors that allow for sufficient liquid/liquid and gas/liquid mass transfer while simultaneously reducing operating costs. In this study, the use of electric field contactors for the biodesulfurization of the model compound dibenzothiophene (DBT) as well as actual crude oil was investigated. The emulsion phase contactor (EPC) creates an emulsion of aqueous biocatalyst in the organic phase by concentrating forces at the liquid/liquid interface rather than by imparting energy to the bulk solution as is done in impeller-based reactors. Characterization of emulsion quality and determination of rates of DBT oxidation to 2-hydroxybiphenyl (2-HBP) were performed for both batch stirred reactors (BSR) and the EPC. The EPC was capable of producing aqueous droplets of about 5 μm in diameter using 3 W/1 whereas the impeller-based reactor formed droplets between 100 and 200 μm with comparable power consumption. The presence of electric fields was not found to adversely affect biocatalytic activity. Despite the greater surface area for reaction afforded by the EPC, rates of DBT oxidation in both reactors were similar, demonstrating that the biocatalyst used (Rhodococcus sp. IGTS8) was not active enough to be mass transport limited. The EPC is expected to have tremendous impact on reactor operating costs and biocatalyst utilization once advances in biocatalyst development provide systems that are mass transport limited.

Sulfur specificity in the bench-scale biological desulfurization of crude oil by Rhodococcus IGTS8†

Biological removal of organic sulfur from petroleum feedstocks may offer an attractive alternative to conventional thermochemical treatment due to the mild operating conditions and greater reaction specificity afforded by the nature of biocatalysis. Previous investigations have either reported the desulfurization of model sulfur compounds in organic solvents or gross desulfurization of crude oil without data on which sulfur species were being removed. This study reports initial sulfur speciation data for thiophenic sulfur compounds present in crude oil which may be used as a guide both as to which species are treated by the biocatalyst investigated as well as to where biocatalyst development is needed to improve the extent of biological desulfurization when applied to whole crudes. Biodesulfurization of two different crude oils in the 22-31° API specific gravity range with total sulfur contents between 1 and 2% is demonstrated in 1-dm 3 batch stirred reactors using wild type Rhodococcus sp IGTS8. While analysis of the crudes before and after biodesulfurization did not reveal a decrease in total sulfur, GC-MS did reveal significant (43-99%) desulfurization of dibenzothiophenes (DBT) and substituted DBTs. Fractionation of the whole crude, followed by analysis using gas chromatography-sulfur chemiluminescence detection (GC-SCD) of the aromatic fraction of the Van Texas crude oil, demonstrated a reduction of sulfur in this fraction from 3.8% to 3.2%. This research indicates that IGTS8 may be capable of biodesulfurization of refined products such as gasoline and diesel whose predominant sulfur species are dibenzothiophenes. Further biocatalyst development would be needed for effective treatment of the spectrum of sulfur-bearing compounds present in whole crudes.

Use of a biparticle fluidized-bed bioreactor for the continuous and simultaneous fermentation and purification of lactic acid

A continuous biparticle fluidized-bed reactor is developed for the simultaneous fermentation and purification of lactic acid. In this processing scheme, bacteria are immobilized in gelatin beads and are fluidized in a columnar reactor. Solid particles with sorbent capacity for the product are introduced at the top of the reactor, and fall counter currently to the biocatalyst, effectingin situ removal of the inhibitory product, while also controlling reactor pH at optimal levels. Initial long-term fermentation trials using immobilizedLactobacillus delbreuckii have demonstrated a 12-fold increase in volumetric productivity during absorbent addition as opposed to control fermentations in the same reactor. Unoptimized regeneration of the loaded sorbent has effected at least an eightfold concentration of lactic acid and a 68-fold enhancement in separation from glucose compared to original levels in the fermentation broth. The benefits of this reactor system as opposed to conventional batch fermentation are discussed in terms of productivity and process economics.

Recycling of FGD gypsum to calcium carbonate and elemental sulfur using mixed sulfate‐reducing bacteria with sewage digest as a carbon source

A combined chemical and biological process for the recycling of flue gas desulfurization (FGD) gypsum into calcium carbonate and elemental sulfur is demonstrated. In this process, a mixed culture of sulfate-reducing bacteria (SRB) utilizes sewage digest as its carbon source to reduce FGD gypsum to hydrogen sulfide. The sulfide is then oxidized to elemental sulfur via reaction with ferric sulfate, and accumulating calcium ions are precipitated to calcium carbonate using carbon dioxide. Employing anaerobically digested-municipal sewage sludge (AD-MSS) medium as a carbon source, SRB in serum bottles demonstrated an FGD gypsum reduction rate of 8 mg dm -3 h -1 (10 9 cells) -1 . A chemostat with continuous addition of both AD-MSS medium and gypsum exhibited sulfate reduction rates as high as 1.3 kg FGD gypsum m -3 day -1 . The increased biocatalyst density afforded by cell immobilization in a columnar reactor allowed a productivity of 152 mg SO 4 dm -3 h -1 or 6.6kg FGD gypsum m -3 day -1 . Both reactors demonstrated 100% conversion of sulfate, with 75-100% recovery of elemental sulfur and as high as 70% COD utilization. Calcium carbonate was recovered from the reactor effluent upon precipitation using carbon dioxide. The formation of two marketable products-elemental sulfur and calcium carbonate-from FGD gypsum sludge, combined with the use of a low-cost carbon source and further improvements in reactor design, promises to offer an attractive alternative to the landfilling of FGD gypsum.

Continuous and simultaneous fermentation and recovery of lactic acid in a biparticle fluidized-bed bioreactor

A continuous biparticle fluidized-bed reactor (BFBR) is developed for the simultaneous fermentation and recovery of lactic acid. In this processing scheme, bacteria are immobilized in gelatin beads and are fluidized in a columnar reactor. Solid particles (weak-base resin IRA-35) with sorbent capacity for the product are introduced at the top of the reactor and fall countercurrently to the biocatalyst, effectingin situ removal of the inhibitory lactic acid while also controlling reactor pH at optimal levels. One-week-long fermentation trials using immobilizedLactobacillus delbreuckii with sorbent addition demonstrated a volumetric productivity (6.9 g/L·h) at least 16-fold higher than that of a free-cell batch fermentation with base pH control and identical biomass concentration and medium composition. Regeneration of the loaded sorbent from the BFBR has effected a 35-fold concentration of lactic acid compared with original levels in the fermentation broth (70 vs 2 g/L). Lactic acid concentrations as high as 610 g/L have been observed when the loading solution contained 50 g/L lactic acid. Rich medium formulations did not seem to increase BFBR performance. The benefits of this reactor system, as opposed to conventional batch fermentation, are discussed in terms of productivity and process economics.

Enzymatic catalysis in organic solvents: Polyethylene glycol modified hydrogenase retains sulfhydrogenase activity in toluene

Naturally occurring enzymes may be modified by covalently attaching hydrophobic groups that render the enzyme soluble and active in organic solvents, and have the potential to greatly expand applications of enzymatic catalysis. The reduction of elemental sulfur to hydrogen sulfide by a hydrogenase isolated from Pyrococcus furiosus has been investigated as a model system for organic biocatalysis. While the native hydrogenase catalyzed the reduction of sulfur to H(2)S in aqueous solution, no activity was observed when the aqueous solvent was replaced with anhydrous toluene. Hydrogenase modified with PEG p-nitrophenyl carbonate demonstrated its native biocatalytic ability in toluene when the reducing dye, benzyl viologen, was also present. Neither benzyl viologen nor PEG p-nitrophenyl carbonate alone demonstrated reducing capability. PEG modified cellulase and benzyl viologen were also incapable of reducing sulfur to H(2)S, indicating that the enzyme itself, and not the modification procedure, is responsible for the conversion in the nonpolar organic solvent. Sulfide production in toluene was tenfold higher than that produced in an aqueous system with equal enzyme activity, demonstrating the advantages of organic biocatalysis. Applications of bio-processing in nonaqueous media are expected to provide significant advances in the areas of fossil fuels, renewable feedstocks, organic synthesis, and environmental control technology. (c) 1996 John Wiley & Sons, Inc.