基于不同基质的强化生物除磷系统中生化反应机理研究进展

阐述了3种不同基质(乙酸盐、丙酸盐和葡萄糖)在强化生物除磷系统中的生化模型反应机理.重点介绍了聚磷菌(PAOs)、聚糖菌(GAOs)和产乳酸菌在厌氧/好氧条件下对能量及还原力(NADH2)的利用方式;聚-β-羟基链烷酸盐(PHA)的合成方式及存在形式,糖原、乳酸(L型)的代谢途径等.虽然控制基质条件可以实现稳定的强化生物除磷效果,但目前的生化模型并不能完全解释所有的代谢过程,今后要在分离纯种的PAOs及相关生化代谢过程中酶的活动等方面进行深入研究.     

Competition between polyphosphate- and glycogen-accumulating organisms in enhanced-biological-phosphorus-removal systems: effect of temperature and sludge age.

Temperature and sludge age were found to be important factors in determining the outcome of competition between polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating non-polyphosphate organisms (GAOs) and the resultant stability of enhanced-biological-phosphorus removal (EBPR). At 20 degrees C and a 10-day sludge age, PAOs were dominant in an anaerobic/aerobic (A/O) sequencing-batch reactor (SBR), as a result of their higher anaerobic-acetate-uptake rate and aerobic-biomass yield than GAOs. However, at 30 degrees C and a 10-day sludge age, GAOs were able to outcompete PAOs in the A/O SBR because of their higher anaerobic-acetate-uptake rate than PAOs. At 30 degrees C and a 5-day sludge age, GAOs coexisted with PAOs in the A/O SBR, resulting in unstable EBPR performance. At 30 degrees C, reducing the sludge age from 5 to 3 days improved the EBPR efficiency drastically, and the EBPR performance was stable. The maximum specific-anaerobic-acetate-uptake rates of GAO-enriched sludge were affected by temperature with the Arrhenius temperature coefficient theta of 0.042 (degrees C(-1) between 10 and 30 degrees C. The effect of sludge age (5 and 10 days) on the maximum specific-anaerobic-acetate-uptake rates of GAO-enriched activated sludge, however, was not significant. For the PAO-enriched activated sludge, the maximum specific-anaerobic-acetate-uptake rate did not change significantly between 20 and 30 degrees C, but significantly increased from 0.38 to 0.52 mmol-C/ mmol-C/h as the sludge age decreased from 10 to 3 days at 30 degrees C.     

Functionally relevant microorganisms to enhanced biological phosphorus removal performance at full-scale wastewater treatment plants in the United States

The abundance and relevance ofAccumulibacter phosphatis (presumed to be polyphosphate-accumulating organisms [PAOs]), Competibacter phosphatis (presumed to be glycogen-accumulating organisms [GAOs]), and tetrad-forming organisms (TFOs) to phosphorus removal performance at six full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants were investigated. Coexistence of various levels of candidate PAOs and GAOs were found at these facilities. Accumulibacter were found to be 5 to 20% of the total bacterial population, and Competibacter were 0 to 20% of the total bacteria population. The TFO abundance varied from nondetectable to dominant. Anaerobic phosphorus (P) release to acetate uptake ratios (P(rel)/HAc(up)) obtained from bench tests were correlated positively with the abundance ratio of Accumulibacter/(Competibacter +TFOs) and negatively with the abundance of (Competibacter +TFOs) for all plants except one, suggesting the relevance of these candidate organisms to EBPR processes. However, effluent phosphorus concentration, amount of phosphorus removed, and process stability in an EBPR system were not directly related to high PAO abundance or mutually exclusive with a high GAO fraction. The plant that had the lowest average effluent phosphorus and highest stability rating had the lowest P(rel)/HAc(up) and the most TFOs. Evaluation of full-scale EBPR performance data indicated that low effluent phosphorus concentration and high process stability are positively correlated with the influent readily biodegradable chemical oxygen demand-to-phosphorus ratio. A system-level carbon-distribution-based conceptual model is proposed for capturing the dynamic competition between PAOs and GAOs and their effect on an EBPR process, and the results from this study seem to support the model hypothesis.     

An observation on sludge granulation in an enhanced biological phosphorus removal process

A sequencing batch reactor (SBR) seeded with flocculated sludge and fed with synthetic wastewater was operated for an enhanced biological phosphorus removal (EBPR) process. Eight weeks after reactor startup, sludge granules were observed. The granules had a diameter of 0.5 to 3.0 mm and were brownish in color and spherical or ellipsoidal in shape. No significant change was observed in sludge granule size when operational pH was changed from 7 to 8. The 208-day continuous operation of the SBR showed that sludge granules were stably maintained with a sludge volume index (SVI) between 30 to 55 mL/g while securing a removal efficiency of 83% for carbon and 97% for phosphorus. Fluorescent in situ hybridization (FISH) confirmed the enrichment of polyphosphate accumulating organisms (PAOs) in the SBR. The observations of sludge granulation in this study encourage further studies in the development of granules-based EBPR process.     

A practical method for quantification of phosphorus- and glycogen-accumulating organism populations in activated sludge systems.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the enrichment of activated sludge with phosphorus-accumulating organisms (PAOs). The presence and proliferation of glycogen-accumulating organisms (GAOs), which compete for substrate with PAOs, may be detrimental for EBPR systems, leading to deterioration and, in extreme cases, failure of the process. Therefore, from both process evaluation and modeling perspectives, the estimation of PAO and GAO populations in activated sludge systems is a relevant issue. A simple method for the quantification of PAO and GAO population fractions in activated sludge systems is presented in this paper. To develop such a method, the activity observed in anaerobic batch tests executed with different PAO/GAO ratios, by mixing highly enriched PAO and GAO cultures, was studied. Strong correlations between PAO/GAO population ratios and biomass activity were observed (R2 > 0.97). This served as a basis for the proposal of a simple and practical method to quantify the PAO and GAO populations in activated sludge systems, based on commonly measured and reliable analytical parameters (i.e., mixed liquor suspended solids, acetate, and orthophosphate) without requiring molecular techniques. This method relies on the estimation of the total active biomass population under anaerobic conditions (PAO plus GAO populations), by measuring the maximum acetate uptake rate in the presence of excess acetate. Later, the PAO and GAO populations present in the activated sludge system can be estimated, by taking into account the PAO/GAO ratio calculated on the basis of the anaerobic phosphorus release-to-acetate consumed ratio. The proposed method was evaluated using activated sludge from municipal wastewater treatment plants. The results from the quantification performed following the proposed method were compared with direct population estimations carried out with fluorescence in situ hybridization analysis (determining Candidatus Accumulibacter Phosphatis as PAO and Candidatus Competibacter Phosphatis as GAO). The method showed to be potentially suitable to estimate the PAO and GAO populations regarding the total PAO-GAO biomass. It could be used, not only to evaluate the performance of EBPR systems, but also in the calibration of potential activated sludge mathematical models, regarding the PAO-GAO coexistence.     

Modeling external carbon addition in biological nutrient removal processes with an extension of the international water association activated sludge model

The aim of this study was to expand the International Water Association Activated Sludge Model No. 2d (ASM2d) to account for a newly defined readily biodegradable substrate that can be consumed by polyphosphate-accumulating organisms (PAOs) under anoxic and aerobic conditions, but not under anaerobic conditions. The model change was to add a new substrate component and process terms for its use by PAOs and other heterotrophic bacteria under anoxic and aerobic conditions. The Gdansk (Poland) wastewater treatment plant (WWTP), which has a modified University of Cape Town (MUCT) process for nutrient removal, provided field data and mixed liquor for batch tests for model evaluation. The original ASM2d was first calibrated under dynamic conditions with the results of batch tests with settled wastewater and mixed liquor, in which nitrate-uptake rates, phosphorus-release rates, and anoxic phosphorus uptake rates were followed. Model validation was conducted with data from a 96-hour measurement campaign in the full-scale WWTP. The results of similar batch tests with ethanol and fusel oil as the external carbon sources were used to adjust kinetic and stoichiometric coefficients in the expanded ASM2d. Both models were compared based on their predictions of the effect of adding supplemental carbon to the anoxic zone of an MUCT process. In comparison with the ASM2d, the new model better predicted the anoxic behaviors of carbonaceous oxygen demand, nitrate-nitrogen (NO3-N), and phosphorous (PO4-P) in batch experiments with ethanol and fusel oil. However, when simulating ethanol addition to the anoxic zone of a full-scale biological nutrient removal facility, both models predicted similar effluent NO3-N concentrations (6.6 to 6.9 g N/m3). For the particular application, effective enhanced biological phosphorus removal was predicted by both models with external carbon addition but, for the new model, the effluent PO4-P concentration was approximately one-half of that found from ASM2d. On a PO4-P removal percentage basis, the difference was small, that is, 94.1 vs. 97.1%, respectively, for the ASM2d and expanded ASM2d.     

不同电子受体对聚磷菌吸磷性能的影响研究

采用人工配水,在厌氧/好氧交替运行的序批式活性污泥反应器(SBR)中,富集了全菌数量80%以上的聚磷菌(Candi-datus Accumulibacter Phosphates)。以此为基础,研究了O2及不同浓度NO3--N、NO2--N对聚磷菌吸磷的影响。结果表明,在一定的条件下,聚磷菌可以NO3--N和NO2--N为电子受体进行缺氧吸磷;NO3--N浓度对聚磷菌的吸磷速率影响很小;聚磷菌可以低质量浓度NO2--N(≤40mg/L)为电子受体,但不能以高质量浓度NO2--N(≥80mg/L)为电子受体,而且高浓度NO2--N对聚磷菌吸磷产生抑制甚至对细菌本身存在毒害;NO2--N为电子受体时,其抑制浓度和污泥本身以及外界条件都存在很大的关系,各个研究结论不尽相同,其影响过程有待进一步的探讨。     

Application of computational fluid dynamics to closed-loop bioreactors: II. Simulation of biological phosphorus removal using computational fluid dynamics.

Based on the International Water Association's (London) Activated Sludge Model No. 2 (ASM2), biochemistry rate expressions for general heterotrophs and phosphorus-accumulating organisms (PAOs) were introduced to a previously developed, three-dimensional computational fluid dynamics (CFD) activated sludge model that characterized the mixing pattern within the outer channel of a full-scale, closed-loop bioreactor. Using acetate as the sole carbon and energy source, CFD simulations for general heterotrophs or PAOs individually agreed well with those of ASM2 for a chemostat with the same operating conditions. Competition between and selection of heterotrophs and PAOs was verified using conventional completely mixed and tanks-in-series models. Then, competition was studied in the CFD model. These results demonstrated that PAOs and heterotrophs can theoretically coexist in a single bioreactor when the oxygen input is appropriate to allow sufficient low-dissolved-oxygen zones to develop.     

Effect of dissolved oxygen on biological nutrient removal by denitrifying phosphorus-accumulating organisms in a continuous-flow system

A laboratory-scale continuous-flow system with an anaerobic/anoxic/aerobic configuration was set up to study the effect of oxygen in the internal recycle stream; of particular interest was its performance of denitrifying phosphorus-accumulating organisms (DPAOs). It was found that, by using a degas device, the dissolved oxygen in the nitrate recycle stream was effectively decreased from 0.1 +/- 0.02 to 0.01 +/- 0.01 mg/L. This provided a favorable condition for DPAOs to grow under an anoxic condition and thus be sustained successfully in the system. When the degas device was removed from the system, the dissolved oxygen concentration in the anoxic reactor increased to 0.1 +/- 0.02 mg/L. The proliferation of the denitrifying glycogen-accumulating organisms (DGAOs) population and deterioration of DPAOs performance was observed. The increased population of DGAO/GAOs, which competed for the carbon source with DPAO/ PAOs, resulted in a poor performance of biological phosphorus removal.     

The mechanism of enhanced biological phosphorus removal washout and temperature relationships.

In this study, the combined effects of temperature and solids retention time (SRT) on enhanced biological phosphorus removal (EBPR) performance and the mechanism of EBPR washout were investigated. Two pilot-scale University of Cape Town (South Africa) systems fed with synthetic wastewater were operated at 5 and 10 degrees C. The results showed that the phosphorus removal performance was optimum at total SRT ranges of 16 to 24 days and 12 to 17 days for 5 and 10 degrees C, respectively, and steady-state phosphorus removal was greater at the lower temperature. Higher SRT values of up to 32 days at 5 degrees C and 25 days at 10 degrees C slightly reduced EBPR performance as a result of increased extent of endogenous respiration, which consumed internally stored glycogen, leaving less reducing power for poly-hydroxy alkanoate (PHA) formation in anaerobic stages. The phosphorus-accumulating organism (PAO) washout SRTs of the systems were determined as 3.5 days at 5 degrees C and 1.8 days at 10 degrees C, considerably less than the washout SRTs of nitrifiers. Polyphosphorus, the main energy reserve of the EBPR bacterial consortium, was not completely depleted, even at washout points. The inability of EBPR biomass to use glycogen to generate reducing power for PHA formation was the major reason for washout. The results not only suggest that glycogen mechanism is the most rate-limiting step in EBPR systems, but also that it is an integral part of EBPR biochemistry, as proposed originally by Mino et al. (1987), and later others (Pereira et al., 1996, Erdal et al., 2002; Erdal, Z. K., 2002). The aerobic washout SRT values (2.1 and 1.2 days for 5 and 10 degrees C, respectively) of this study did not fit the linear line for PAO washout developed by Mamais and Jenkins (1992). Perhaps this was because the feeds used during this study were chemical-oxygen-demand-limited (acetate-based synthetic feed), whereas the feeds used for their study were phosphorus-limited (external acetate added to domestic wastewater), resulting in different ratios of PAOs and nonPAOs in the biomass.     

Specificity and potential applications of the biochemical acidogenic potential method for the anaerobic characterization of wastewater.

The biochemical acidogenic potential (BAP) test is an anaerobic characterization method for wastewater. Fermentable organic fractions are obtained through modeling BAP test results. This method was compared to more common fractionation methods such as settling, coagulation, and respirometry, but no direct relationship was found. Biochemical acidogenic potential testing was thus considered to bring new and complementary information. The settleable matter accounted for approximately 50% of the fermentable matter, with a rate comparable to that of aerobic hydrolysis, suggesting a potential assimilable carbon source that could be liberated in sewers or in anaerobic processes. It was also observed that respirometry could underestimate the amount of fermentable substrates while overestimating that of hydrolyzable matter and of heterotrophic biomass involved in anaerobic processes. The BAP fractions are related to the wastewater capacity to produce volatile fatty acids, which are the main substrates of the micro-organisms responsible for enhanced biological phosphorus removal (EBPR). The potential contribution of the BAP fractionation to assist the design, operation, and modeling of the activated-sludge EBPR processes was discussed.     

Effect of initial pH control on enhanced biological phosphorus removal from wastewater containing acetic and propionic acids

In the literature most of the studies on the effect of pH on enhanced biological phosphorous removal were conducted with the acetate wastewater, and the pH was controlled during the entire anaerobic and aerobic stages. This paper investigated the influence of anaerobic initial pH control, which will be more practical than the entire process pH control strategy, on enhanced biological phosphorus removal from wastewater containing acetic and propionic acids. Typical pH profile showed that both the initial alkaline and acidic pH tended to neutralize due to the consumption of short-chain fatty acid (SCFA) and intracellular pH regulation by polyphosphate accumulating organisms (PAOs). It was observed that the glycogen degradation and polyhydroxyalkanoates (PHA) accumulation decreased with increasing initial pH, which disagreed with previous reports. In the literature the metabolisms of both glycogen and PHA by PAOs in the acetate wastewater were independent of pH. An anaerobic mechanism model was proposed to explain the intra- and extra-cellular pH buffer nature of PAOs, and to address the reasons for increased polyphosphate degradation and decreased PHA synthesis and glycogen degradation at higher pH. The optimal initial pH for higher soluble ortho-phosphorus (SOP) removal efficiency should be controlled between 6.4 and 7.2. This pH control strategy will be easier to use in practice of wastewater treatment plant.     

Biological phosphate uptake and release: effect of pH and magnesium ions.

Enhanced biological phosphorus removal (EBPR) is based on poly-phosphate accumulating organisms' (PAOs) unique features of "luxury" phosphate uptake during aerobic conditions and phosphate release in anaerobic conditions. It is believed that poly-phosphate accumulation is accompanied by the uptake and accumulation of potassium ions (K+) and magnesium ions (Mg2+). The release of phosphate under anaerobic conditions is also accompanied by the release of both cations. The objective of this research was to evaluate the effect of pH and Mg2+ on the biological phosphate uptake and release behavior of activated sludge mixed liquor during aeration and sedimentation. Research results indicate that Mg2+, supplied either by magnesium chloride (MgCl2) or magnesium hydroxide [Mg(OH)2], stimulated phosphate uptake during the aeration period, while pH increase, caused by the application of Mg(OH)2, enhanced phosphate release during the sedimentation period. It is also noted in our experiments with MgCl2 that Mg2+ slightly inhibited anaerobic phosphate release.     

剩余污泥水解酸化液磷去除的影响因素研究

城市污水厂剩余污泥水解酸化后可产生高浓度挥发性有机酸(VFAs),其中的乙酸和丙酸是增强生物除磷(EBPR)工艺的有利基质.但水解酸化液中含有大量的磷,如不进行处理就作为碳源回用到污水处理工艺中,势必增加除磷负荷.利用鸟粪石沉淀法可以去除污水中的磷.对城市污水厂剩余污泥水解酸化液形成鸟粪石的影响因素进行了试验研究.结果表明,在最佳工艺条件下,正磷和总磷的去除率分别可达92.5%和83.8%.     

Molecular characterization of denitrifying bacteria isolated from the anoxic reactor of a modified DEPHANOX plant performing enhanced biological phosphorus removal

Enhanced Biological Phosphorus Removal (EBPR) under anoxic conditions was achieved using a Biological Nutrient Removal (BNR) system based on a modification of the DEPHANOX configuration. Double-probe Fluorescence in Situ Hybridization (FISH) revealed that Polyphosphate Accumulating Organisms (PAOs) comprised 12.3 +/- 3.2% of the total bacterial population in the modified DEPHANOX plant. The growing bacterial population on blood agar and Casitone Glycerol Yeast Autolysate agar (CGYA) medium was 16.7 +/- 0.9 x 10(5) and 3.0 +/- 0.6 x 10(5) colony forming units (cfu) mL(-1) activated sludge, respectively. A total of 121 bacterial isolates were characterized according to their denitrification ability, with 26 bacterial strains being capable of reducing nitrate to gas. All denitrifying isolates were placed within the alpha-, beta-, and gamma-subdivisions of Proteobacteria and the family Flavobacteriaceae. Furthermore, a novel denitrifying bacterium within the genus Pseudomonas was identified. This is the first report on the isolation and molecular characterization of denitrifying bacteria from EBPR sludge using a DEPHANOX-type plant.     

Effect of initial pH control on biological phosphorus removal induced by the aerobic/extended-idle regime

Recently, it has been reported that biological phosphorus removal (BPR) can be induced by an aerobic/extended-idle (AEI) regime. This study further investigated the effect of initial pH ranging from 6.6 to 8.2 on BPR in the AEI process, and compared the BPR performance between the AEI and the anaerobic/oxic (A/O) regimes under their optimal initial pH value. Experimental results firstly showed that phosphorus removal linearly increased with initial pH increasing from 6.6 to 7.8, but slightly decreased when initial pH increased from 7.8 to 8.2. The optimal initial pH should be controlled at 7.8, and the phosphorus removal at initial pH 7.8 was approximately 1.7-time than that at initial pH 6.6. The mechanism studies showed that the biomass cultured at initial pH 7.8 contained more polyphosphate accumulating organisms (PAOs), lower glycogen accumulating organisms (GAOs), and had higher activities of exopolyphosphatase and polyphosphate kinase than that cultured at initial pH 6.6. Cyclic studies revealed that initial pH control affected the transformations of intracellular polyhydroxyalkanoates and glycogen, which might thereby influence microbial competition between PAOs and GAOs. Then, BPR performance between the AEI and the A/O regimes by adjusting initial pH at 7.8 was also compared. The results showed the AEI regime could drive a better BPR than the generally accepted A/O regime (98% vs 88%). Finally, controlling initial pH at 7.8 to promote BPR in the AEI process was confirmed for a municipal wastewater.     

活性污泥体系中聚糖菌的富集与鉴定

活性污泥体系中,聚糖菌(GAOs)在厌氧环境下与聚磷菌(PAOs)形成对底物的竞争关系,对聚糖菌的研究对于优化生物除磷工艺有重要意义。以葡萄糖为惟一碳源,在磷限制条件下,利用特殊运行方式对活性污泥进行驯化培养出了稳定的聚糖菌颗粒污泥,厌氧阶段磷释放量与有机物吸收量浓度(mg/L)比从7.4%下降为0.25%。从培养好的活性污泥反应器中分离获得2株聚糖菌,经菌落PCR和16S rRNA序列分析确定了所得聚糖菌菌株G1和菌株G2分别是枯草芽孢杆菌(Bacillus subtilis)和解鸟氨酸克雷伯氏菌(Klebsiella ornithinolytica)。     

Recovery of polyhydroxyalkanoate from activated sludge in an enhanced biological phosphorus removal bench-scale reactor.

A sequencing batch reactor was used to study the possibility of harvesting polyhydroxyalkanoate (PHA) from enhanced biological phosphorus removal (EBPR) processes without compromising treatment quality. Because, in EBPR, the highest PHA concentrations are observed after exposure of the sludge to anaerobic conditions, PHA accumulation was evaluated with collection of waste activated sludge (WAS) at the end of the anaerobic stage, in addition to the traditional removal after the aerobic stage. The system achieved good phosphorus removal, regardless of the point of WAS collection. When sludge was harvested at the end of the anaerobic stage, the PHA content of the sludge ranged from 7 to 16 mg PHA/100 mg mixed liquor volatile suspended solids. Although this level of PHA production is below levels obtained with pure cultures, the demonstrated ability to harvest PHA, while simultaneously satisfying phosphorus removal in an EBPR process, is a key initial step towards of the use of wastewater treatment plants for PHA production.     

Comparison of nitrification rates in conventional and membrane-assisted biological nutrient removal processes

A membrane-assisted and a conventional activated sludge system, both operated in an enhanced biological phosphorus removal (EBPR) mode and under identical operating conditions, were studied to investigate the effect of the membrane solids-liquid separation on nitrification activity. Both the membrane EBPR (MEBPR) and conventional EBPR (CEBPR) processes achieved stable and complete removal of ammonium-nitrogen from the influent wastewater. However, when the intrinsic nitrification activity was assessed in offline batch tests, the CEBPR mixed liquor exhibited 15 to 75% greater nitrification potential than the MEBPR counterpart. These results were further validated by monitoring nitrification rates of conventional mixed liquor as it evolved toward a membrane mixed liquor. It was also demonstrated that the larger aerobic mass fraction of the MEBPR system could not be the only factor influencing the reduced intrinsic nitrification rate. The present study strongly suggests that the presence of a membrane solids-liquid separation per se may be sufficient to alter the nitrification kinetics of an EBPR mixed liquor and that this possibility should be considered in arriving at an appropriate process design.     

Optimization of enhanced biological phosphorus removal after periods of low loading.

Enhanced biological phosphorus removal is a well-established technology for the treatment of municipal wastewater. However, increased effluent phosphorus concentrations have been reported after periods (days) of low organic loading. The purpose of this study was to evaluate different operating strategies to prevent discharge of effluent after such low-loading periods. Mechanisms leading to these operational problems have been related to the reduction of polyphosphate-accumulating organisms (PAOs) and their storage compounds (polyhydroxy alkanoates [PHA]). Increased effluent phosphorus concentrations can be the result of an imbalance between influent loading and PAOs in the system and an imbalance between phosphorus release and uptake rates. The following operating conditions were tested in their ability to prevent a reduction of PHA and of overall biomass during low organic loading conditions: (a) unchanged operation, (b) reduced aeration time, (c) reduced sludge wastage, and (d) combination of reduced aeration time and reduced sludge wastage. Experiments were performed in a laboratory-scale anaerobic-aerobic sequencing batch reactor, using acetate as the carbon source. Without operational adjustments, phosphorus-release rates decreased during low-loading periods but recovered rapidly. Phosphorus-uptake rates also decreased, and the recovery typically required several days to increase to normal levels. The combination of reduced aeration time and reduced sludge wastage allowed the maintenance of constant levels of both PHA and overall biomass. A mathematical model was used to explain the influence of the tested operating conditions on PAO and PHA concentrations. While experimental results were in general agreement with model predictions, the kinetic expression for phosphorus uptake deviated significantly for the first 24 hours after low-loading conditions. Mechanisms leading to these deviations need to be further investigated.