Occurrence and enrichment of 'bacterial sherpas': climb to sustainability in wastewater treatment.

The paper presents research on hemoglobin (Hb)-expressing bacteria in biological wastewater treatment systems. The outcome(s) will greatly reduce the aeration needs of wastewater treatment plants (WWTPs) and provide insight into emerging biological nitrogen removal processes using low dissolved oxygen (DO) conditions. In anthropogenic terms, the bacteria that express Hb could be considered as 'bacterial sherpas' that can function under low DO conditions. Hitherto, this functionality of bacteria has not been realized due to the initial response of the aerobic treatment stage: namely, morphology change by bacteria to filamentous forms to overcome oxygen mass transfer limitations causing bulking/foaming and nitrification inhibition. There is evidence, however, of the potential expression of Hb proteins by activated sludge (AS) bacteria. First, bacteria known to possess genes coding Hb proteins have been isolated from AS systems. Secondly, there is evidence that WWTPs are able to operate their biological processes at low DO without sludge bulking or incomplete nitrification. Our research has focused on nitrifying systems and has shown that this is due to prolonged operation at low DO conditions (0.1 mg O2/L), which allows sufficient time for bacterial acclimation. Additionally, it has been shown that enhanced Hb expression is linked to acclimation to low DO conditions.

A new approach to assess the dependency of extant half-saturation coefficients on maximum process rates and estimate intrinsic coefficients.

The Monod equation is often used to describe biological treatment processes and is the foundation for many activated sludge models. The Monod equation includes a "half-saturation coefficient" to describe the effect of substrate limitations on the process rate and it is customary to consider this parameter to be a constant for a given system. The purpose of this study was to develop a methodology, and its use to show that the half-saturation coefficient for denitrification is not constant but is in fact a function of the maximum denitrification rate. A 4-step procedure is developed to investigate the dependency of half-saturation coefficients on the maximum rate and two different models are used to describe this dependency: (a) an empirical linear model and (b) a deterministic model based on Fick's law of diffusion. Both models are proved better for describing denitrification kinetics than assuming a fixed K(NO3) at low nitrate concentrations. The empirical model is more utilitarian whereas the model based on Fick's law has a fundamental basis that enables the intrinsic K(NO3) to be estimated. In this study data was analyzed from 56 denitrification rate tests and it was found that the extant K(NO3) varied between 0.07 mgN/L and 1.47 mgN/L (5th and 95th percentile respectively) with an average of 0.47 mgN/L. In contrast to this, the intrinsic K(NO3) estimated for the diffusion model was 0.01 mgN/L which indicates that the extant K(NO3) is greatly influenced by, and mostly describes, diffusion limitations.

Characteristics and fate of organic nitrogen in municipal biological nutrient removal wastewater treatment plants.

The aim of this study was to investigate the occurrence and fate of colloidal and dissolved organic nitrogen (CON and DON) across biological nutrient removal (BNR) activated sludge bioreactors. Primary and secondary effluent total nitrogen (TN) measurements and component fractionation, CON and DON concentration profiles across BNR bioreactors, and laboratory batch experiments with the process mixed liquor were carried out at several full-scale BNR plants in northern Poland. The organic nitrogen (ON) components were divided into high CON, low CON, and DON based on sequential filtration through 1.2, 0.45 and 0.1 µm pore-size filters. The average influent DON(0.1 µm) (<0.1 µm) concentrations ranged from 1.1 g N/m(3) to 3.9 g N/m(3) and accounted for only 4-13% of total organic nitrogen. In the effluents, however, this contribution increased to 12-45% (the DON(0.1 µm) concentrations varied in a narrow range of 0.5-1.3 g N/m(3)). Conversions of ON inside the bioreactors were investigated in more detail in two largest plants, i.e. Gdansk (565,000 PE) and Gdynia (516,000 PE). Inside the two studied bioreactors, the largest reductions of the colloidal fraction were found to occur in the anaerobic and anoxic compartments, whereas an increase of DON(0.1 µm) concentrations was observed under aerobic conditions in the last compartment. Batch experiments with the process mixed liquor confirmed that DON(0.1 µm) was explicitly produced in the aerobic phase and significant amounts of ON were converted in the anoxic phase of the experiments.

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.

Nutrient removal process selection for planning and design of large wastewater treatment plant upgrade needs.

A protocol to select nutrient removal technologies that can achieve low nutrient effluents (total nitrogen (TN) < 5 mg/L and total phosphorus (TP) < 0.5 mg/L) was developed for different wastewater treatment plant (WWTP) sizes based on the research conducted during a Water Environment Research Foundation funded project. The adaptable protocol includes technology and cost assessment of feasible (pre-screened) nutrient removal technologies that are being successfully implemented at full scale. The information collected from the full scale nutrient removal plants to develop this protocol includes design, operational, performance, and cost data through a direct survey of plants, and published data. The protocol includes a "technology threshold" approach consisting of Tier I (TN < 5.0 mg/L; TP < 0.5 mg/L) and Tier II (TN < 3.0 mg/L; TP < 0.1 mg/L) effluent nutrient levels for different plant sizes. A very large WWTP (1,250,000 m(3)/day flow) in Chicago, Illinois, USA adapted this protocol for master planning and design of future nutrient removal facilities based on plant and site specific criteria.

Comparison of 2-chlorobenzoic acid biodegradation in a membrane bioreactor by B. cepacia and B. cepacia bearing the bacterial hemoglobin gene.

Degradation of 2-chlorobenzoic acid (2-CBA), a model recalcitrant chlorinated organic compound, by pure cultures of Burkholderia cepacia strain DNT with (transformed B. cepacia) and without (untransformed B. cepacia) the bacterial hemoglobin (Vitreoscilla hemoglobin, VHb) gene, vgb, was investigated in parallel membrane bioreactors (MBRs). This was done aseptically to prevent contamination during the operation of the MBRs. The objective was to determine whether the degradation of 2-CBA by cometabolism, using acetate as a primary carbon source, under hypoxic conditions might be enhanced for vgb-bearing cells in MBRs. The 2-CBA removal efficiency of transformed B. cepacia (97-99%) was slightly higher than that of untransformed B. cepacia (95-99%) at all stages. The average amount of chloride released from 2-CBA by transformed cells was also higher than for untransformed cells, 92-96% compared to 64-84% of the maximum theoretical amount, the exact value depending on the operating conditions. These results indicate that 2-CBA degradation/transformation is not accompanied by the stoichiometric release of chloride for the untransformed strain. The difference between percentages of 2-CBA removal and chloride release by untransformed cells was attributed to persistence, under hypoxic conditions, of the 2-CBA chlorine atom in 2-CBA metabolites. Growth of transformed cells was also significantly enhanced under hypoxic conditions compared to untransformed cells. For varying media compositions, the transformed cells reached higher cell densities (3.2-5.4 g/L) relative to untransformed cells (2.8-4.7 g/L) at food to microorganism ratios ranging from 0.44-0.59 to 0.38-0.49 g COD/g biomass-d The observed yields thus ranged from 0.16-0.20 and 0.15-0.18 g TSS/g COD for untransformed and transformed cells, respectively. The value of the yield depended on medium composition. The MBR system using vgb-containing B. cepacia maintained a high biomass concentration without oxygen limitations and provided cell-free effluent. Hence, it may be useful for treating high volumes of water contaminated with low levels of recalcitrant organics.

Low effluent nutrient technologies for wastewater treatment.

The USEPA (2001) water quality nutrient criteria will have a significant impact on water pollution control industry due to stringent N and P requirements. This paper presents an update of findings on successful total N (TN) and total P (TP) technologies being implemented at existing wastewater treatment plants (WWTP) to achieve low TN and TP effluents and some key challenges in achieving lower levels. Plants consistently achieving <5 mg TN/L and < 0.5 mg TP/L were identified from a worldwide literature search and plant data collection. Technology gaps and research needs to improve successful technologies to achieve very low TN and TP effluents are summarised in this paper. The dissolved and colloidal organic N have been identified as major challenges in achieving very low levels of TN. Technical and economic challenges to achieve very low TP effluents include alkalinity deficiency, high chemical usage, high sludge production and lack of sufficient influent BOD for biological P uptake.

Gordonia (nocardia) amarae foaming due to biosurfactant production.

Gordonia amarae, a filamentous actinomycete, commonly found in foaming activated sludge wastewater treatment plants was investigated for its biosurfactant production capability. Soluble acetate and paringly soluble hexadecane were used as carbon sources for G. amarae growth and biosurfactant production in laboratory scale batch reactors. The lowest surface tension (critical micelle concentration, CMC) of the cell-free culture broth was 55 dynes/cm when 1,900 mg/L acetate was used as the sole carbon source. The lowest surface tension was less than 40 dynes/cm when either 1% (v/v) hexadecane or a mixture of 1% (v/v) hexadecane and 0.5% (w/v) acetate was used as the carbon source. The maximum biomass concentration (the stationary phase) was achieved after 4 days when acetate was used along with hexadecane, whereas it took about 8 days to achieve the stationary phase with hexadecane alone. The maximum biosurfactant production was 3 x CMC with hexadecane as the sole carbon source, and it was 5 x CMC with the mixture of hexadecane and acetate. Longer term growth studies (approximately 35 days of culture growth) indicated that G. amarae produces biosurfactant in order to solubilize hexadecane, and that adding acetate improves its biosurfactant production by providing readily degradable substrate for initial biomass growth. This research confirms that the foaming problems in activated sludge containing G. amarae in the activated sludge are due to the biosurfactant production by G. amarae when hydrophobic substrates such as hexadecane are present.

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.

Stabilization and solidification of metal-laden wastes by compaction and magnesium phosphate-based binder.

Bench-scale and full-scale investigations of waste stabilization and volume reduction were conducted using spiked soil and ash wastes containing heavy metals such as Cd, Cr, Pb, Ni, and Hg. The waste streams were stabilized and solidified using chemically bonded phosphate ceramic (CBPC) binder, and then compacted by either uniaxial or harmonic press for volume reduction. The physical properties of the final waste forms were determined by measuring volume reduction, density, porosity, and compressive strength. The leachability of heavy metals in the final waste forms was determined by a toxicity characteristic leaching procedure (TCLP) test and a 90-day immersion test (ANS 16.1). The structural composition and nature of waste forms were determined by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively. CBPC binder and compaction can achieve 80-wt% waste loading and 39-47% reduction in waste volume. Compressive strength of final waste forms ranged from 1500 to 2000 psi. TCLP testing of waste forms showed that all heavy metals except Hg passed the TCLP limits using the phosphate-based binder. When Na2S was added to the binder, the waste forms also passed TCLP limits for Hg. Long-term leachability resistance of the final waste forms was achieved for all metals in both soil and ash wastes, and the leachability index was approximately 14. XRD patterns of waste forms indicated vermiculite in the ash waste was chemically incorporated into the CBPC matrix. SEM showed that waste forms are layered when compacted by uniaxial press and are homogeneous when compacted by harmonic press.