Ex situ slurry phase bioremediation of chrysene contaminated soil with the function of metabolic function: process evaluation by data enveloping analysis (DEA) and Taguchi design of experimental methodology (DOE)

Bioremediation of chrysene in soil matrix was evaluated in soil slurry phase bioreactor in conjugation with metabolic functions (aerobic, anoxic and anaerobic), microenvironment (single and mixed) conditions and nature of mixed consortia (native/resident mixed microflora and bioaugmented inoculum). Twelve experiments were operated independently in agitated-batch reactor keeping all other operating conditions constant (substrate loading rate--0.084 g chrysene/kg soil-day; soil loading rate--10 kg soil/m(3)-day (3:25 soil water ratio); operating temperature--35+/-2 degrees C). Data envelopment analysis (DEA) procedure was employed to analyze the performance of experimental variations in terms of chrysene degradation and pH. The efficacy of anoxic metabolism over the corresponding aerobic and anaerobic metabolic functions was documented. Aerobic metabolic function showed effective degradation capability under mixed microenvironment after augmentation with anaerobic inoculum. Anaerobic metabolic function showed lowest degradation potential. Application of bioaugmentation showed positive influence on the chrysene degradation rate. Design of experimental methodology (DOE) by Taguchi approach was applied to evaluate the effect of four selected factors (native soil microflora, microenvironment, metabolic function and bioaugmentation) on the chrysene degradation process. The optimized factors derived from analysis depicted the requirement of native soil microflora under anoxic metabolic function using mixed microenvironment after augmenting with anaerobic inoculum for achieving effective chrysene degradation efficacy.     

Formation of Aniline as a Transient Metabolite During the Metabolism of Tetryl by a Sulfate-Reducing Bacterial Consortium

A laboratory study was conducted to determine whether tetryl (2,4,6-trinitrophenylmethylnitramine) can be degraded by an anaerobic process. The results indicated that the metabolic conversion of tetryl to aniline is possible by a sulfate-reducing bacterial (SRB) consortium. This SRB consortium metabolized tetryl by co-metabolism with pyruvate as a growth substrate. For every mole of tetryl metabolized, 1 mole of aniline was produced, and the aniline was further metabolized. This metabolic conversion of tetryl is likely to be of value in the anaerobic treatment of tetryl-contaminated soil and ground water, such as found at many military ammunition sites. Received: 18 August 1999 / Accepted: 15 September 1999     

The fate of phosphate under anoxic conditions in biological nutrient removal activated sludge systems

The nitrogen removal potential of phosphate accumulating organisms under anoxic conditions has been evaluated using a laboratory scale sequencing batch reactor fed with synthetic wastewater and operated in a sequence of anaerobic, anoxic and aerobic periods. The phosphate uptake rate under anoxic conditions was lower than that under aerobic conditions. However, in the presence of an external substrate such as glucose and acetate, the fate of phosphate was dependent on the substrate type; phosphate release occurred in the presence of nitrate as long as acetate was present and glucose did not cause any phosphate release. The nitrate uptake rate was also much lower with glucose than acetate. The results implied that poly-hydroxyalkanoates could be oxidized by nitrate and phosphate uptake during the anoxic phase should be introduced into process modeling. © Rapid Science Ltd. 1998     

Degradation of 4,5-dichloroguaiacol by soil microorganisms

No microorganisms could be isolated from chemostats or from a soil column fed with 4,5-dichloroguaiacol as the only carbon source. If guaiacol was added to chemostats with 4,5-dichloroguaiacol, either soil microbial consortia or guaiacol-degrading bacteria could dechlorinate the 4,5-dichloroguaiacol provided it was <0.2mm. A microbial consortium from farm soil removed 4,5-dichloroguaiacol under aerobic or anoxic conditions, with or without chlorolignin. Dichlorocatechol was the only 4,5-dichloroguaiacol-derived metabolite detected. In aerobic incubations, 4,5-dichlorocatechol was further degraded whereas under anoxic conditions it accumulated.     

Bioremediation of HMX-Contaminated Soil Using Soil Slurry Reactors

Soil in some parts of the Iowa Army Ammunition Plant in Burlington, Iowa, was contaminated with cyclotetramethyleneter-anitramine, commonly known as high melting explosive (HMX). A laboratory treat-ability study was conducted to find out the ability of the native soil bacteria present in the contaminated site to degrade HMX. The results indicated that the HMX can be removed effectively from soil by native soil bacteria through a co-metabolic process. Molasses, identified as an effective co-substrate, is inexpensive, and this factor makes the treatment system cost-effective. The successful operation of aerobic-anoxic soil slurry reactors in batch mode with HMX-contaminated soil showed that the technology can be scaled up for field demonstration. The HMX concentration in the contaminated soil was decreased by 97% in 4 months of reactor operation. The advantage of the slurry reactor is its simplicity of operation. The method needs only mixing and the addition of molasses as co-substrate.     

Characterization of aerobic granules by microbial density at different COD loading rates

Aerobic granules were cultivated under temporal alternating aerobic and anoxic conditions without the presence of a carrier material in a sequencing batch reactor (SBR) with a high column height/column diameter ratio. The reactor was operated for 6h per cycle (aerobic: 4.75 h, anoxic: 1.25 h). To determine a new parameter for the definition of aerobic granules, a protocol of 4,6-diamidino-2-phenylindole hydrochloride staining and fluorescence image processing was developed. The d(tm) analysis showed that the increase in the chemical oxygen demand (COD) loading rate promoted no more growth of the aerobic granules. It was inconsistent with the results of the analysis of the sludge volume index (SVI) value but matched well with the results of the COD and nitrogen removal of the SBR and the particle size distribution by LS-PSA. The optimum COD loading rate for aerobic granulation in the SBR was 2.52 kg/m(3)d. When d(tm) was correlated with the biomass concentration and the SVI value during the period of granule formation, d(tm) could be used as a more sensitive and accurate parameter for classifying aerobic granules and optimizing the operational conditions for aerobic granulation processes.     

Effect of HRT on the biological pre-denitrification process for the simultaneous removal of toxic pollutants from cokes wastewater

A lab-scale serial anoxic-aerobic reactor for the pre-denitrification process was continuously operated to efficiently and economically treat actual cokes wastewater containing various pollutants, such as phenol, ammonia, thiocyanate and cyanide compounds. The biodegradation efficiencies of the pollutants were examined by changing hydraulic retention time (HRT) as a main operating variable. The long-term operation of the pre-denitrification process reactor showed that approximately 100% phenol, approximately 100% free cyanide, approximately 100% SCN(-), 97% ammonia, 85% COD, 84% TOC (total organic carbon) and 83% TN (total nitrogen) were removed at HRT above 11.9h. Removal efficiency of total cyanides significantly decreased with a decrease in the HRT. Free cyanide and some of total cyanides were removed in anoxic reactor, whereas thiocyanate was removed in aerobic reactor. Phenol was completely removed under successive anoxic and aerobic conditions. Although actual cokes wastewater contained high concentrations of various toxic pollutants, the pre-denitrification process showed stable and successful performances in both nitrification and denitrification reactions.     

Alleviating soil sickness caused by aerobic monocropping: responses of aerobic rice to soil oven-heating

“Aerobic rice” system is the cultivation of nutrient-responsive cultivars in nonflooded and nonsaturated soil under supplemental irrigation. It is intended for lowland areas with water shortage and for favorable upland areas with access to supplementary irrigation. Yield decline caused by soil sickness has been reported with continuous monocropping of aerobic rice grown under nonflooded conditions. The objective of this study was to determine the growth response of rice plant to oven heating of soil with a monocropping history of aerobic rice. A series of pot experiments was conducted with soils from fields where rice has been grown continuously under aerobic or anaerobic (flooded) conditions. Soil was oven heated at different temperatures and for various durations. Plants of Apo, an upland variety that does relatively well under the aerobic conditions of lowland, were grown aerobically without fertilizer inputs in all six experiments. Plants were sampled during vegetative stage to determine stem number, plant height, leaf area, and total biomass. Heating of soil increased plant growth greatly in soils with an aerobic history but a relatively small increase was observed in soils with a flooded history as these plants nearly reached optimum growth. A growth increase with continuous aerobic soil was already observed with heating at 90°C for 12 h and at 120°C for as short as 3 h. Maximum plant growth response was observed with heating at 120°C for 12 h. Leaf area was most sensitive to soil heating, followed by total biomass and stem number. We conclude that soil heating provides a simple and quick test to determine whether a soil has any sign of sickness that is caused by continuous cropping of aerobic rice.     

Biological treatment of low-salinity shrimp aquaculture wastewater using sequencing batch reactor

In order to improve the water quality in shrimp aquaculture operated under low-salinity conditions, a sequencing batch reactor (SBR) was tested for treatment of the wastewater. This water from the backwash of a single-bead filter from the Waddell Mariculture Center, South Carolina, contained high concentrations of carbon and nitrogen and was successfully treated using the SBR. By operating the reactor sequentially in aerobic, anoxic and aerobic modes, nitrification and denitrification were achieved, as well as removal of carbon. Specifically, the initial chemical oxygen demand (COD) concentration of 1201 mg l⁻¹ was reduced to 32 mg l⁻¹ within 8 days of reactor operation. Ammonia in the sludge was nitrified within 3 days. The denitrification of nitrate was achieved by the anoxic process and total removal of nitrate was observed.     

Treatment of raw and ozonated oil sands process-affected water under decoupled denitrifying anoxic and nitrifying aerobic conditions: a comparative study

Batch experiments were performed to evaluate biodegradation of raw and ozonated oil sands process-affected water (OSPW) under denitrifying anoxic and nitrifying aerobic conditions for 33 days. The results showed both the anoxic and aerobic conditions are effective in degrading OSPW classical and oxidized naphthenic acids (NAs) with the aerobic conditions demonstrating higher removal efficiency. The reactors under nitrifying aerobic condition reduced the total classical NAs of raw OSPW by 69.1 %, with better efficiency for species of higher hydrophobicity. Compared with conventional aerobic reactor, nitrifying aerobic condition substantially shortened the NA degradation half-life to 16 days. The mild-dose ozonation remarkably accelerated the subsequent aerobic biodegradation of classical NAs within the first 14 days, especially for those with long carbon chains. Moreover, the ozone pretreatment enhanced the biological removal of OSPW classical NAs by leaving a considerably lower final residual concentration of 10.4 mg/L under anoxic conditions, and 5.7 mg/L under aerobic conditions. The combination of ozonation and nitrifying aerobic biodegradation removed total classical NAs by 76.5 % and total oxy-NAs (O3–O6) by 23.6 %. 454 Pyrosequencing revealed that microbial species capable of degrading recalcitrant hydrocarbons were dominant in all reactors. The most abundant genus in the raw and ozonated anoxic reactors was Thauera (~56 % in the raw OSPW anoxic reactor, and ~65 % in the ozonated OSPW anoxic reactor); whereas Rhodanobacter (~40 %) and Pseudomonas (~40 %) dominated the raw and ozonated aerobic reactors, respectively. Therefore, the combination of mild-dose ozone pretreatment and subsequent biological process could be a competent choice for OSPW treatment.     

Simultaneous removal of carbon and nitrate in an airlift bioreactor

This paper presents the integrated removal of carbon (measured as chemical oxygen demand i.e. COD) and NO(x)-N by sequentially adapted sludge, studied in an airlift reactor (ALR). Simultaneous removal of COD and nitrate occurs by denitrification (anoxic) and oxidation (aerobic). Aerobic (riser) and anoxic (remaining part) conditions prevail in different parts of the reactor. Studies were carried out in a 42 L ALR operated at low aeration rate to maintain anoxic and aerobic conditions as required for denitrification and COD removal, respectively. The sludge was adapted sequentially to increasing levels of NO(x)-N and COD over a period of 45 days. Nitrate removal efficiency of the sludge increased due to adaptation and degraded 900 ppm NO(3)-N completely in 2h (initially the sludge could not degrade 100 ppm NO(3)-N). The performance of the adapted sludge was tested for the degradation of synthetic waste with COD/N loadings in the range of 4-10. The reduction of COD was significantly faster in the presence of NO(x)-N and was attributed to the availability of oxygen from NO(x)-N and distinct conditions in the reactor. This hypothesis was justified by the material balance of COD.     

Redox fluctuation structures microbial communities in a wet tropical soil

Frequent high-amplitude redox fluctuation may be a strong selective force on the phylogenetic and physiological composition of soil bacterial communities and may promote metabolic plasticity or redox tolerance mechanisms. To determine effects of fluctuating oxygen regimens, we incubated tropical soils under four treatments: aerobic, anaerobic, 12-h oxic/anoxic fluctuation, and 4-day oxic/anoxic fluctuation. Changes in soil bacterial community structure and diversity were monitored with terminal restriction fragment length polymorphism (T-RFLP) fingerprints. These profiles were correlated with gross N cycling rates, and a Web-based phylogenetic assignment tool was used to infer putative community composition from multiple fragment patterns. T-RFLP ordinations indicated that bacterial communities from 4-day oxic/anoxic incubations were most similar to field communities, whereas those incubated under consistently aerobic or anaerobic regimens developed distinctly different molecular profiles. Terminal fragments found in field soils persisted either in 4-day fluctuation/aerobic conditions or in anaerobic/12-h treatments but rarely in both. Only 3 of 179 total fragments were ubiquitous in all soils. Soil bacterial communities inferred from in silico phylogenetic assignment appeared to be dominated by Actinobacteria (especially Micrococcus and Streptomycetes), "Bacilli," "Clostridia," and Burkholderia and lost significant diversity under consistently or frequently anoxic incubations. Community patterns correlated well with redox-sensitive processes such as nitrification, dissimilatory nitrate reduction to ammonium (DNRA), and denitrification but did not predict patterns of more general functions such as N mineralization and consumption. The results suggest that this soil's indigenous bacteria are highly adapted to fluctuating redox regimens and generally possess physiological tolerance mechanisms which allow them to withstand unfavorable redox periods.     

Ex situ bioremediation of pyrene contaminated soil in bio-slurry phase reactor operated in periodic discontinuous batch mode: Influence of bioaugmentation

Ex situ treatment of simulated pyrene-contaminated soil was studied in bio-slurry phase reactors operated in periodic discontinuous batch mode under anoxic–aerobic–anoxic–anoxic microenvironment. Experiments were performed in six different bio-slurry phase reactors (retention time of 120 h; soil loading rate of 20 kg soil/m³-day; operating temperature at 28±2 °C) by varying substrate concentration (substrate loading rate (SLR), 0.12, 0.24 and 0.36 g pyrene/kg soil-day) and bioaugmentation application (domestic sewage inoculum; CFU—2×10⁶). The performance of slurry phase reactors was found to be dependent on the applied SLR and application of bioaugmentation (domestic sewage as augmented inoculum). Control reactor (killed control) showed only 6% of pyrene degradation while the non-augmented reactor showed an efficiency of 34% (substrate degradation rate (SDR)—0.0165 g pyrene/kg soil-day). In the case of augmented reactors, the system operated with low SLR showed a pyrene degradation efficiency of almost 90% (SDR—0.04 g pyrene/kg soil-day) and the reactor with high SLR showed 50% (SDR—0.025 g pyrene/kg soil-day) of pyrene degradation indicating the dependence of performance on the substrate concentration. Colony forming units (CFUs) variation was in good agreement with the performance of the reactors with respect to pyrene degradation. On the whole, pyrene degradation rate was greater in the augmented reactors compared to non-augmented reactors.     

Neural network modeling for nutrient dynamics in a recycling piggery slurry treatment system

A recycling reactor system operated under sequential anoxic and oxic conditions was evaluated, in which the nutrients of piggery slurry were anaerobically and aerobically treated and then a portion of the effluent was recycled to the pigsty. The most dominant aerobic heterotrophs from the reactor were Alcaligenes faecalis (TSA-3), Brevundimonas diminuta (TSA-1) and Abiotrophia defectiva (TSA-2) in decreasing order, whereas lactic acid bacteria, LAB (MRS-1, etc.) were most dominantly observed in the anoxic tank. Here we have tried to model the nutrient removal process for each tank in the system based on population densities of heterotrophic and LAB. Principal component analysis (PCA) was first applied to delineate a relationship between input (microbial densities and treatment parameters such as population densities of heterotrophic and LAB, suspended solids (SS), COD, NH₄ ⁺–N, ortho-phosphorus, and total phosphorus) and output. Multi-layer neural networks using an error back-propagation learning algorithm were then employed to model the nutrient removal process for each tank. PCA filtration of microbial densities as input data was able to enhance generalization performance of the neural network, and this has led to a better prediction of the measured data. Neural networks independently trained for each treatment tank and the combined analysis of the subsequent tank data allowed a successful prediction of the treatment system for at least 2 days.     

Functionally redundant cellobiose-degrading soil bacteria respond differentially to oxygen

The availability of oxygen (O(2)) in aerated (i.e., water-unsaturated) soils affects the metabolic activities of aerobic and anaerobic soil prokaryotes that degrade plant-derived saccharides. Fluctuating availabilities of O(2) were imposed on agricultural soil slurries supplemented with cellobiose. Slurries were subjected to oxic conditions (48 h), followed by an anoxic period (120 h) and a final oxic period (24 h). Redox potential was stable at 500 mV during oxic periods but decreased rapidly (within 10 h) under anoxic conditions to -330 mV. The consumption of cellobiose occurred without apparent delay at all redox potentials. The metabolic activities of seven previously identified saccharolytic family-level taxa of the investigated soil were measured with newly designed quantitative PCR assays targeting the 16S rRNA. Four taxa responded to the experimental conditions. The amounts of rRNAs of Micrococcaceae and Cellulomonadaceae (Actinobacteria) increased under oxic conditions. In contrast, the RNA contents of Clostridiaceae (cluster I, Firmicutes) and two uncultured family-level-taxa, i.e., "Cellu" and "Sphingo" (both Bacteroidetes) increased under anoxic conditions. That the degradation of cellobiose was independent of the availability of O(2) and that redox potentials decreased in response to anaerobic activities indicated that the degradation of cellobiose was linked to functionally redundant cellobiose-degrading taxa capable of altering redox conditions.     

Oxidative consumption of nitric oxide by heterotrophic bacteria in soil

Abstract: Uptake rate constants for nitric oxide were measured in a neutral calcic cambisol (KBE) and an acidic luvisol (PBE). The NO uptake was higher under oxic than under anoxic incubation conditions by a factor of about three. Gassing the soils with air containing 10 ppmv NO resulted in the accumulation of nitrate which accounted for 57–94% of the NO consumed. Aerobic heterotrophic bacteria were isolated on glucose-yeast extract medium from soil dilutions corresponding to a most probable number of 10⁸–10⁹ bacteria per gram dry weight soil. One of the isolates (strain PS88, a Pseudomonas sp.) exhibited NO consumption activity that was much higher under oxic than anoxic incubation conditions. When sterile KBE amended with strain PS88 was gassed with air containing 10 ppmv NO, 88% of the consumed NO was recovered as nitrate and nitrite. A screening of various bacteria obtained from culture collections showed a widespread ability for consumption of low NO concentrations. Our results indicate that NO consumption in soil is not only possible by reductive denitrification, but also by oxidation due to aerobic heterotrophic bacteria such as strain PS88.     

Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil

The use of an indigenous microbial consortium, pollutant-acclimated and attached to soil particles (activated soil), was studied as a bioaugmentation method for the aerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil. A 125-l completely mixed soil slurry (10% soil) bioreactor was used to produce the activated soil biomass. Results showed that the bioreactor was very effective in producing a PCP-acclimated biomass. Within 30 days, PCP-degrading bacteria increased from 10⁵ cfu/g to 10⁸ cfu/g soil. Mineralization of the PCP added to the reactor was demonstrated by chloride accumulation in solution. The soil-attached consortium produced in the reactor was inhibited by PCP concentrations exceeding 250 mg/l. This high level of tolerance was attributed to the beneficial effect of the soil particles. Once produced, the activated soil biomass remained active for 5 weeks at 20 °C and for up to 3 months when kept at 4 °C. The activated attached soil biomass produced in the completely mixed soil slurry bioreactor, as well as a PCP-acclimated flocculent biomass obtained from an air-lift immobilized-soil bioreactor, were used to stimulate the bioremediation of a PCP-impacted sandy soil, which had no indigenous PCP-degrading microorganisms. Bioaugmentation of this soil by the acclimated biomass resulted in a 99% reduction (from 400 mg/kg to 5 mg/kg in 130 days) in PCP concentration. The PCP degradation rates obtained with the activated soil biomass, produced either as a biomass attached to soil particles or as a flocculent biomass, were similar. Received: 31 March 1997 / Received revision: 22 July 1997 / Accepted: 25 August 1997     

Aerobic versus anaerobic metabolism of halogenated anilines by aParacoccus sp.

AParacoccus sp. which transforms aniline and different halogen-substituted derivatives under aerobic and anaerobic conditions was isolated from the soil. In experiments with¹⁴C-ring-labeled 4-chloroaniline, approximately 60% of the radioactive material disappeared from the growth medium after incubation under anaerobiosis within 48 hr, but under aerobic conditions no decrease of radioactivity in the growth medium was observed, although 4-chloroaniline was completely metabolized. Acetylation appears to constitute, especially under aerobic conditions, a major transformation mechanism by the bacterium, since almost 50% of the acetylated compound could be detected and identified if aniline, 2-, 3-, and 4-chloroaniline served as substrate. The formation of different metabolites under aerobic and anaerobic conditions clearly indicates the existence of two separate pathways in the metabolism of aniline compounds depending on the oxygen status of the environment.     

Selection and dominance mechanisms of denitrifying phosphate-accumulating organisms in biological phosphate removal process

A sequencing batch reactor under different electron acceptor conditions was operated serially to investigate the selection and dominance mechanisms of denitrifying phosphate-accumulating organisms (DNPAOs) in a biological nutrient removal process. The presence of a small amount of NO^– ₃ at the start of the anaerobic phase stimulated the selection of DNPAOs in an anaerobic/aerobic system, and switching O₂ to NO^– ₃ as an electron acceptor enhanced the activity of anoxic phosphate uptake.     

Atypical polyphosphate accumulation by the denitrifying bacterium Paracoccus denitrificans

Polyphosphate accumulation by Paracoccus denitrificans was examined under aerobic, anoxic, and anaerobic conditions. Polyphosphate synthesis by this denitrifier took place with either oxygen or nitrate as the electron acceptor and in the presence of an external carbon source. Cells were capable of poly-beta-hydroxybutyrate (PHB) synthesis, but no polyphosphate was produced when PHB-rich cells were incubated under anoxic conditions in the absence of an external carbon source. By comparison of these findings to those with polyphosphate-accumulating organisms thought to be responsible for phosphate removal in activated sludge systems, it is concluded that P. denitrificans is capable of combined phosphate and nitrate removal without the need for alternating anaerobic/aerobic or anaerobic/anoxic switches. Studies on additional denitrifying isolates from a denitrifying fluidized bed reactor suggested that polyphosphate accumulation is widespread among denitrifiers.