Krishna R. Pagilla

Use of Genetically Engineered Microorganisms (GEMs) for the Bioremediation of Contaminants

ABSTRACT This paper presents a critical review of the literature on the application of genetically engineered microorganisms (GEMs) in bioremediation. The important aspects of using GEMs in bioremediation, such as development of novel strains with desirable properties through pathway construction and the modification of enzyme specificity and affinity, are discussed in detail. Particular attention is given to the genetic engineering of bacteria using bacterial hemoglobin (VHb) for the treatment of aromatic organic compounds under hypoxic conditions. The application of VHb technology may advance treatment of contaminated sites, where oxygen availability limits the growth of aerobic bioremediating bacteria, as well as the functioning of oxygenases required for mineralization of many organic pollutants. Despite the many advantages of GEMs, there are still concerns that their introduction into polluted sites to enhance bioremediation may have adverse environmental effects, such as gene transfer. The extent of horizontal gene transfer from GEMs in the environment, compared to that of native organisms including benefits regarding bacterial bioremediation that may occur as a result of such transfer, is discussed. Recent advances in tracking methods and containment strategies for GEMs, including several biological systems that have been developed to detect the fate of GEMs in the environment, are also summarized in this review. Critical research questions pertaining to the development and implementation of GEMs for enhanced bioremediation have been identified and posed for possible future research.

Effect of oxic and anoxic conditions on nitrous oxide emissions from nitrification and denitrification processes

A lab‐scale sequencing batch reactor fed with real municipal wastewater was used to study nitrous oxide (N2O) emissions from simulated wastewater treatment processes. The experiments were performed under four different controlled conditions as follows: (1) fully aerobic, (2) anoxic–aerobic with high dissolved oxygen (DO) concentration, (3) anoxic–aerobic with low DO concentration, and 4) intermittent aeration. The results indicated that N2O production can occur from both incomplete nitrification and incomplete denitrification. N2O production from denitrification was observed in both aerobic and anoxic phases. However, N2O production from aerobic conditions occurred only when both low DO concentrations and high nitrite concentration existed simultaneously. The magnitude of N2O produced via anoxic denitrification was lower than via oxic denitrification and required the presence of nitrite. Changes in DO, ammonium, and nitrite concentrations influenced the magnitude of N2O production through denitrification. The results also suggested that N2O can be produced from incomplete denitrification and then released to the atmosphere during aeration phase due to air stripping. Therefore, biological nitrogen removal systems should be optimized to promote complete nitrification and denitrification to minimize N2O emissions. Biotechnol. Bioeng. 2011;108:2036–2045.

AEROBIC THERMOPHILIC AND ANAEROBIC MESOPHILIC TREATMENT OF SWINE WASTE

Laboratory experiments were conducted to investigate two-stage aerobic thermophilic and anaerobic mesophilic treatment of swine waste. The two-stage system included a 1-day sludge retention time (SRT) aerobic thermophilic reactor operating at 628C and with 1.0 mg dissolved oxygen/l, followed by a 5, 9, and 14-day SRT anaerobic mesophilic digester operating at 378C. A single stage anaerobic mesophilic digester operating at 6, 10, and 15-day SRT and 378C was used as the control. Feed swine waste slurry average composition included total solids (TS)=4.3%; volatile solids (VS)=67% of TS; supernatant chemical oxygen demand (COD)=14,330 mg/l; fecal coliform density=7.2 10 8 MPN/g TS; pH=7.1; and alkalinity=2700 mg CaCO3/l. Two-stage system operating at 6, 10, and 15-day system SRT reduced VS by 46, 54, and 61%, respectively, and was significantly better than the control at each SRT. Supernatant COD reduction by the two-stage system (56-67%) was significantly better than that obtained in the control (44-60%). Fecal coliform density was reduced to <10 3 MPN/g TS at all SRT by the two-stage system, whereas, the control did not reduce the fecal coliform density below 10 5 MPN/g TS at all SRT. The two-stage system anaerobic digester produced 0.56-0.64 m 3 CH4/kg VS destroyed compared to lower levels of 0.47-0.51 m 3 CH4/kg VS destroyed by the control, both operating at 6, 10, and 15-day system SRT. The methane gas production by the two- stage system of 0.26, 0.32, and 0.39 m 3 /kg VS fed at 6, 10, and 15-day system SRT, respectively, was significantly higher than that by the control system (0.17, 0.22, and 0.25 m 3 /kg VS fed at 6, 10, and 15- day SRT, respectively). The biogas produced by the two-stage system anaerobic digester contained 353-387 ppm (v/v) H2S content compared to 569-609 ppm (v/v) H2S content in the biogas from the control anaerobic digester. The time-to-filter values of the product sludge from the two-stage system (245, 197, and 158 s at 6, 10, and 15-day system SRT, respectively) were about 50% lower than those of the product sludge from the control (355, 295, and 250 s at 6, 10, and 15-day system SRT, respectively), indicating better dewaterability of the two-stage system product sludge. 7 2000 Elsevier Science Ltd. All rights reserved

Organic nitrogen transformations in a 4-stage Bardenpho nitrogen removal plant and bioavailability/biodegradability of effluent DON.

Nitrogen species, specifically, the fate and occurrence of organic nitrogen (ON) within a 4-stage Bardenpho process bioreactor producing low total nitrogen (TN) effluents were investigated in this study. The results showed release of ON in primary anoxic zone and no ON release in the first aerobic zone of the process. The research included investigation of biodegradability/bioavailability of wastewater-derived effluent dissolved ON (DON). The final-effluent DON utilization was evaluated by two different bioassay protocols in the presence and absence of nitrate. About 28-57% of the effluent DON was bioavailable/biodegradable. Bioavailable (to algae and bacteria) DON (ABDON) and biodegradable (to bacteria) DON (BDON) results did not show significant differences in terms of quantity, but DON utilization rates by ABDON (0.13 day(-1)) protocol were higher than that of the BDON (0.04 day(-1)) protocol in the nitrate-removal samples. As a result, ABDON requires a shorter time to exert the bioavailable fraction due to symbiotic relationship between algae and bacteria. In the nitrate-containing samples, it appears that nitrate competes with labile DON as a nitrogen source to microorganisms in both ABDON and BDON protocols. The first order decay rate of DON in the presence of nitrate was 0.11 day(-1) and 0.02 day(-1) for ABDON and BDON, respectively.

Nitrogen speciation in wastewater treatment plant influents and effluents—the US and Polish case studies

The fate of N species, particularly dissolved organic nitrogen (DON), through process trains of a wastewater treatment plant (WWTP) was investigated. In this study, three fully nitrifying plants in Illinois, USA and biological nutrient removal (BNR) plants in northern Poland were sampled for N characterization in the primary and secondary effluents as a function of the particle size distribution. The correlations between dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) concentrations were examined. The key findings are that DON becomes significant portion (about 20%) of the effluent N, reaching up to 50% of effluent total N in one of the Polish plants. The DON constituted 56-95% of total ON (TON) in the secondary effluents, whereas in the Polish plants the DON contribution was substantially lower (19-62%) and in one case (Gdansk WWTP) colloidal ON was the dominating fraction (62% of TON). The DOC to DON ratio in the US plants is significantly lower than that in the receiving waters indicating potential for deterioration of receiving water quality. In Polish plants, the influent and effluent C:N ratios are similar, but not in the US plants.

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen. Journal of Industrial Microbiology & Biotechnology (2001) 27, 27–33.

Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla

The hemoglobin from the bacterium Vitreoscilla (VHb) is the first microbial hemoglobin that was conclusively identified as such (in 1986). It has been extensively studied with respect to its ligand binding properties and mechanisms, structure, biochemical functions, and the mechanisms by which its expression is controlled. In addition, cloning of its gene (vgb) into a variety of heterologous hosts has proved that its expression results substantial increases in production of a variety of useful products and ability to degrade potentially harmful compounds. Recent studies (since 2005) have added significant knowledge to all of these areas and shown the broad range of biotechnological applications in which VHb can have a positive effect.

Biodegradation of 2-Chlorobenzoate by Recombinant Burkholderia Cepacia Expressing Vitreoscilla Hemoglobin Under Variable Levels of Oxygen Availability

The influence of bacterial hemoglobin, VHb, on dechlorinationand degradation of 2-chlorobenzoate (2-CBA) by recombinantBurkholderia sp. under variable oxygen availability with an initial dissolved oxygenconcentration of 0.27 mM-0.72 mM was investigated in batch and continuous culture. Abilityto express VHb was provided to recombinant Burkholderia by transformationwith the VHb gene, vgb, on plasmid pSC160. 100% of 0.5 mM CBA was degraded incultures with 85% and 70% of total volume as headspace air in closed reactorsby both wild type and recombinant Burkholderia. The recombinant cultures were able todechlorinate and degrade 100% of the 2-CBA in less than 48 hours at 30 °Ccompared to more than 120 hours for wild type cultures. The rate and extent of CBAdegradation by recombinant cultures with 40% of total volume as headspace air was higher than thoseachieved by wild type cells at the end of the 168 hours of incubation period, 98and 73%, respectively. The chloride released: CBA degraded molar ratio for cultures with 40%of total volume headspace air was nearly stoichiometric (molar ratio = 1.0) for recombinantstrains, whereas it was non-stoichiometric (molar ratio = 0.24)for wild type cells. The results suggest a suicidal meta-pathway for wild type cells and a complete dechlorinationand degradation pathway for recombinant cells under hypoxic conditions.The degradation and dechlorination ability of both types of cells was alsoinvestigated in continuous reactor studies by varying the dilution rate under hypoxicconditions. Regarding potential of the recombinant strain for 2-CBA degradation in eitheropen ecosystems or closed bioreactor bioremediation systems, the stability of the plasmidcontaining vgb in the recombinant cells was also studied; the plasmid was100% stable at 0.025 h-1 dilution rate (∼1.7 d hydraulic retention time),even after one month.

Model development and simulation for predicting risk of foaming in anaerobic digestion systems.

Although there is not a complete agreement on the causes of foaming in anaerobic digestion, experts and operators do have valuable empirical knowledge of key factors. Based on this knowledge, a model for calculating the risk of foaming in anaerobic digestion systems due to microbiological causes has been developed. Organic loading rate, variation in organic loading rate, and the presence of filamentous microorganisms in the activated sludge system, used as a feed source for the digester, have been selected as the inputs of a knowledge-based model designed to provide as output the risk of foaming in an anaerobic digester. The performance of the model is demonstrated by means of a case study using the IWA Benchmark Simulation Model No. 2 as a framework, where risk of foaming is used as a new evaluation criterion. The simulated results of an open-loop configuration and two closed-loop control strategies illustrate the usefulness of this knowledge-based approach as a means of estimating the risk of foaming in anaerobic digestion.

Effluent dissolved organic nitrogen and dissolved phosphorus removal by enhanced coagulation and microfiltration.

Plants aiming to achieve very low effluent nutrient levels (<3 mg N/L for N, and <0.1 mg P/L for P) need to consider removal of effluent fractions hitherto not taken into account. Two of these fractions are dissolved organic nitrogen (DON) and dissolved non-reactive phosphorus (DNRP) (mainly composed of organic phosphorus). In this research, enhanced coagulation using alum (at doses commonly employed in tertiary phosphorus removal) followed by microfiltration (using 0.22 μm pore size filters) was investigated for simultaneous effluent DON and dissolved phosphorus (DP) fractions removal. At an approximate dose of 3.2 mg Al(III)/L, corresponding to 1.5 Al(III)/initial DON-N and 3.8 Al(III)/initial DP-P molar ratios, maximum simultaneous removal of DON and DP was achieved (69% for DON and 72% for DP). At this dose, residual DON and DP concentrations were found to be 0.3 mg N/L and 0.25 mg P/L, respectively. Analysis of the trends of removal revealed that the DNRP removal pattern was similar to that commonly reported for dissolved reactive phosphorus. Since this study involved intensive analytical work, a secondary objective was to develop a simple and accurate measurement protocol for determining dissolved N and P species at very low levels in wastewater effluents. The protocol developed in this study, involving simultaneous digestion for DON and DNRP species, was found to be very reliable and accurate based on the results.