Application of Raman microscopy for simultaneous and quantitative evaluation of multiple intracellular polymers dynamics functionally relevant to enhanced biological phosphorus removal processes

Polyphosphate (poly-P), polyhydroxyalkanoates (PHAs), and glycogen are the key functionally relevant intracellular polymers involved in the enhanced biological phosphorus removal (EBPR) process. Further understanding of the mechanisms of EBPR has been hampered by the lack of cellular level quantification tools to accurately measure the dynamics of these polymers during the EBPR process. In this study, we developed a novel Raman microscopy method for simultaneous identification and quantification of poly-P, PHB, and glycogen abundance in each individual cell and their distribution among the populations in EBPR. Validation of the method was demonstrated via a batch phosphorus uptake and release test, in which the total intracellular polymers abundance determined via Raman approach correlated well with those measured via conventional bulk chemical analysis (correlation coefficient r = 0.8 for poly-P, r = 0.94 for PHB, and r = 0.7 for glycogen). Raman results, for the first time, clearly showed the distributions of microbial cells containing different abundance levels of the three intracellular polymers under the same environmental conditions (at a given time point), indicating population heterogeneity exists. The results revealed the intracellular distribution and dynamics of the functionally relevant polymers in different metabolic stages of the EBPR process and elucidated the association of cellular metabolic state with the fate of these polymers during various substrates availability conditions.     

Bacterial phosphate metabolism and its application to phosphorus recovery and industrial bioprocesses

Enhanced biological phosphorus removal (EBPR) has become a well-established process and is currently applied in many full-scale wastewater treatment processes. Phosphorus recovered from EBPR waste sludge can be used as a raw material for the fertilizer industry, if a sound recycling strategy is developed and applied. In this review, we summarize our current knowledge on phosphate metabolism in bacteria, focusing on molecular mechanisms of bacterial polyphosphate (polyP) accumulation. A simple method for releasing polyP from EBPR waste sludge and recovering phosphorus in a reusable form for the fertilizer industry is presented. We also describe a recent development of bioprocesses for the expanded use of polyP in the production of value-added chemicals.     

Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI)

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.     

Heterogeneity of intracellular polymer storage states in enhanced biological phosphorus removal (EBPR)--observation and modeling

A number of agent-based models (ABMs) for biological wastewater treatment processes have been developed, but their skill in predicting heterogeneity of intracellular storage states has not been tested against observations due to the lack of analytical methods for measuring single-cell intracellular properties. Further, several mechanisms can produce and maintain heterogeneity (e.g., different histories, uneven division) and their relative importance has not been explored. This article presents an ABM for the enhanced biological phosphorus removal (EBPR) treatment process that resolves heterogeneity in three intracellular polymer storage compounds (i.e., polyphosphate, polyhydroxybutyrate, and glycogen) in three functional microbial populations (i.e., polyphosphate-accumulating, glycogen-accumulating, and ordinary heterotrophic organisms). Model predicted distributions were compared to those based on single-cell estimates obtained using a Raman microscopy method for a laboratory-scale sequencing batch reactor (SBR) system. The model can reproduce many features of the observed heterogeneity. Two methods for introducing heterogeneity were evaluated. First, biological variability in individual cell behavior was simulated by randomizing model parameters (e.g., maximum acetate uptake rate) at division. This method produced the best fit to the data. An optimization algorithm was used to determine the best variability (i.e., coefficient of variance) for each parameter, which suggests large variability in acetate uptake. Second, biological variability in individual cell states was simulated by randomizing state variables (e.g., internal nutrient) at division, which was not able to maintain heterogeneity because the memory in the internal states is too short. These results demonstrate the ability of ABM to predict heterogeneity and provide insights into the factors that contribute to it. Comparison of the ABM with an equivalent population-level model illustrates the effect of accounting for the heterogeneity in models.     

A laboratory-scale test of anaerobic digestion and methane production after phosphorus recovery from waste activated sludge

In enhanced biological phosphorus removal (EBPR) processes, activated sludge microorganisms accumulate large quantities of polyphosphate (polyP) intracellularly. We previously discovered that nearly all of polyP could be released from waste activated sludge simply by heating it at 70 degrees C for about 1 h. We also demonstrated that this simple method was applicable to phosphorus (P) recovery from waste activated sludge in a pilot plant-scale EBPR process. In the present study, we evaluated the effect of this sludge processing (heat treatment followed by calcium phosphate precipitation) on anaerobic digestion in laboratory-scale experiments. The results suggested that the sludge processing for P recovery could improve digestive efficiency and methane productivity at both mesophilic (37 degrees C) and thermophilic (53 degrees C) temperatures. In addition, heat-treated waste sludge released far less P into the digested sludge liquor than did untreated waste sludge. It is likely that the P recovery step prior to anaerobic digestion has a potential advantage for controlling struvite (magnesium ammonium phosphate) deposit problems in sludge handling processes.     

Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon

NADPH, a major reducing power in microorganisms, is mostly generated from the pentose phosphate (PP) pathway by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) expressed by the zwf and gnd genes, respectively. The characteristics of these two genes in Escherichia coli were compared after their re-introduction into the parent strain for over-expression. zwf encoding G6PDH increased the level of NADPH 3 folds compared to gnd encoding 6PGDH. An excess of NADPH depressed cell growth mainly due to the inhibition of citrate synthase in the TCA cycle. Recombinant plasmids containing zwf or gnd co-integrated with the phbCAB operon from Ralstonia eutropha were constructed, and introduced into E. coli for the oddball biosynthesis of PHB. The amount of PHB increased after enforcing the genes; especially the zwf gene an increase of around 41%, due to the rise in NADPH and the depressed TCA cycle, leading to the metabolic flux of intermediates to the pathway for the biosynthesis of PHB.     

黄曲霉积累L-苹果酸代谢机制初探

L-苹果酸是生物体内三羧酸循环的成员之一,在食品、医药、日用化工等部门具有广泛的用途.文中从限氧发酵、碳酸钙的添加量、乙醛酸循环和TCA循环相应酶的抑制剂几个方面初步探讨黄曲霉积累L-苹果酸的代谢机制,得出CO2固定途径是积累L-苹果酸的主要途径.     

Multi-scale individual-based model of microbial and bioconversion dynamics in aerobic granular sludge

Aerobic granular sludge is a novel compact biological wastewater treatment technology for integrated removal of COD (chemical oxygen demand), nitrogen, and phosphate charges. We present here a multiscale model of aerobic granular sludge sequencing batch reactors (GSBR) describing the complex dynamics of populations and nutrient removal. The macro scale describes bulk concentrations and effluent composition in six solutes (oxygen, acetate, ammonium, nitrite, nitrate, and phosphate). A finer scale, the scale of one granule (1.1 mm of diameter), describes the two-dimensional spatial arrangement of four bacterial groups--heterotrophs, ammonium oxidizers, nitrite oxidizers, and phosphate accumulating organisms (PAO)--using individual based modeling (IbM) with species-specific kinetic models. The model for PAO includes three internal storage compounds: polyhydroxyalkanoates (PHA), poly phosphate, and glycogen. Simulations of long-term reactor operation show how the microbial population and activity depends on the operating conditions. Short-term dynamics of solute bulk concentrations are also generated with results comparable to experimental data from lab scale reactors. Our results suggest that N-removal in GSBR occurs mostly via alternating nitrification/denitrification rather than simultaneous nitrification/denitrification, supporting an alternative strategy to improve N-removal in this promising wastewater treatment process.     

Denitrifying polyphosphate accumulating organisms population and nitrite reductase gene diversity shift in a DEPHANOX-type activated sludge system fed with municipal wastewater

Enhanced biological phosphorus removal (EBPR) is a widely applied method for nutrients removal, although little is known about the key genes regulating the complex biochemical transformations occurring in activated sludge during phosphorus removal. In the present study, the nitrite reductase gene (nirS) diversity and the denitrifying polyphosphate accumulating organisms (DPAOs) population, grown in a bench scale, two-sludge, continuous flow plant, operating for biological anoxic phosphorus removal (DEPHANOX-type), fed with municipal wastewater, were examined by means of physicochemical analyses and the application of molecular techniques. The DEPHANOX configuration highly influenced biomass phosphorus as well as polyhydroxyalkanoates content and facilitated the enrichment of the DPAOs population. The application of double probe fluorescent in situ hybridization (double probe FISH) technique revealed that DPAOs comprised 20% of the total bacterial population. Based on clone libraries construction and nirS gene sequencing analysis, a pronounced shift in denitrifying bacteria diversity was identified during activated sludge acclimatization. Moreover, nirS gene sequences distinct from those detected in any known bacterial strain or environmental clone were identified. This is the first report studying the microbial properties of activated sludge in a DEPHANOX-type system using molecular techniques.     

巴氏醋杆菌TCA循环代谢对醋酸发酵的影响

巴氏醋杆菌是醋酸发酵常用微生物,通过添加醋酸、三羧酸(tricarboxylic acid,TCA)循环抑制剂和中间产物干扰TCA循环能量代谢,研究其对菌体生长和醋酸发酵的影响。研究结果发现,添加质量分数1%的醋酸,胞内TCA代谢关键酶的表达量出现上调,胞内ATP含量增加了125%,表明能够强化TCA循环能量代谢,促进菌体生长。添加TCA循环抑制剂抑制TCA循环能量代谢,巴氏醋杆菌菌体生长和产酸受到显著抑制,菌体生物量分别降低了90%和87%,产酸量分别降低了90%和94%。添加0.05%草酰乙酸、琥珀酸、苹果酸等TCA中间产物,巴氏醋杆菌胞内ATP含量分别提高了202%、185%和165%,表明添加草酰乙酸等中间物质能够显著提高TCA循环偶联呼吸链产能,发酵72 h时,菌体生物量分别提高了92%、106%和104%,菌体产酸量分别提高了30%、33%和31%。实验结果表明,TCA循环能量代谢对巴氏醋杆菌菌体生长和产酸产生显著影响,TCA能量代谢的强化对醋酸发酵产生正向作用。     

Effect of modifying metabolic network on poly-3-hydroxybutyrate biosynthesis in recombinant Escherichia coli

The regulatory mechanism for poly-3-hydroxybutyrate (PHB) biosynthesis in recombinant Escherichia coli is markedly different from that of Ralstonia eutropha (formerly, Alcaligenes eutrophus) since the former efficiently synthesizes PHB during growth without any nutrient limitation. To analyze how the central metabolic pathways should be balanced with pathways necessary for cell growth and PHB formation, a stoichiometric model was developed to predict the theoretical maximum PHB production capacity for different metabolic variants. Flux analysis results illustrated the importance of the availability of acetyl-CoA and NADPH for achieving the maximum yield of PHB. In order to examine whether the increased availability of the above substances can enhance PHB synthesis in recombinant E. coli, both genetic and environmental perturbations were attempted. Several E. coli K12 derivatives, namely, HMS174, TA3516 (pta-/ack-), and DF11 (pgi-), were transformed with a plasmid which contains the native phb operon. The fermentation characteristics of these recombinant strains were studied and compared. In this study we examined the effects of intracellular acetyl-CoA accumulation, which may promote PHB synthesis in vivo, by perturbations induced from attenuation of acetate kinase and phosphotransacetylase (TA3516, blocked in the acetate pathway) and by cultivation of E. coli HMS174 on gluconate; it can convert gluconate to acetyl-CoA at a higher rate. The effects of intracellular accumulation of NADPH were investigated by introducing a perturbation induced from attenuation of phosphoglucose isomerase, which redirects the carbon flow to the pentose-phosphate (PP) pathway. Results from the analyses of these perturbations indicate that intracellular buildup of acetyl-CoA may not be able to promote PHB synthesis in vivo. On the other hand, since the biosynthesis of PHB in the pgi- mutant strain can utilize the NADPH overproduced through the PP pathway, the growth of the pgi- mutant on glucose was recovered, indicating that the overproduction of NADPH might be able to enhance PHB synthesis.     

Energy and greenhouse gas profiles of polyhydroxybutyrates derived from corn grain: a life cycle perspective

Polyhydroxybutyrates (PHB) are well-known biopolymers derived from sugars orvegetable oils. Cradle-to-gate environmental performance of PHB derived from corn grain is evaluated through life cycle assessment (LCA), particularly nonrenewable energy consumption and greenhouse gas emissions. Site-specific process information on the corn wet milling and PHB fermentation and recovery processes was obtained from Telles. Most of energy used in the corn wet milling and PHB fermentation and recovery processes is generated in a cogeneration power plant in which corn stover, assumed to be representative of a variety of biomass sources that could be used, is burned to generate electricity and steam. County level agricultural information is used in estimating the environmental burdens associated with both corn grain and corn stover production. Results show that PHB derived from corn grain offers environmental advantages over petroleum-derived polymers in terms of nonrenewable energy consumption and greenhouse gas emissions. Furthermore, PHB provides greenhouse gas credits, and thus PHB use reduces greenhouse gas emissions compared to petroleum-derived polymers. Corn cultivation is one of the environmentally sensitive areas in the PHB production system. More sustainable practices in corn cultivation (e.g., using no-tillage and winter cover crops) could reduce the environmental impacts of PHB by up to 72%.     

Causes of variable biomass density and its effects on settleability in full-scale biological wastewater treatment systems

Both variable biomass density and floc structure were determined to affect the settleability of microbial biomass produced during biological wastewater treatment (activated sludge). Average biomass density varied from 1.022 to 1.056 g/mL in a survey of 17 full-scale biological wastewater treatment systems with a variety of configurations. Biomass settleability was correlated with density in samples with higher filament contents and/or more open floc structures, but settleability was independent of density in biomass with lower filament contents and more rounded floc structures. Biomass density increased with polyphosphate content, and enhanced biological phosphorus removal (EBPR) plants had higher density and better settleability than non-EBPR plants, including two systems that converted to EBPR during the course of this study. Density also increased with increasing nonvolatile suspended solids content, which was linked both to polyphosphate and to increasing solids residence times. Both density and floc structure should be considered in future analyses of activated sludge settleability, and it may be possible to improve system performance by adopting a new set of operational and design strategies to increase density.     

Analysis of cellular metabolism of hybridoma cells at distinct physiological states

Hybridoma cells were cultivated in a chemically defined medium in continuous cultures. These cultures reached different steady states marked by distinctive cell metabolism depending on the culture conditions leading to the steady state. Those steady states with different metabolism are characterized by different stoichiometric ratios of lactate production to glucose consumption (deltaL/deltaG). The specific consumption rates of glucose, glutamine and other amino acids are reduced when DeltaL DeltaG reduces. Those steady states do not have a few discrete values of deltaL/deltaGs , rather they span from a high deltaL/deltaG state (> 1.0) to an intermediate state (0.1 < or = deltaL/deltaG < or = 1.0), and reduces even further at a low deltaL/deltaG state (< 0.1). Metabolic flux analysis was performed to compare energy metabolism of cells in cultures representing these three distinct metabolic states. The material balance on carbon and nitrogen was facilitated by the use of chemically defined medium. The formation of biomass was systematically estimated. It was revealed that all glycolysis and TCA cycle fluxes are reduced as deltaL/deltaG decreases. At the low deltaL/deltaG state, a reduction in amino acid specific consumption rate is accompanied by a reduction in all the fluxes around pyruvate. The analysis also shows that the outflux from the TCA cycle to form pyruvate, which contributes to lactate formation, is possibly linked to the higher consumption rate of amino acids at the high deltaL/deltaG state. Taken together the results suggest the amino acid metabolism plays an important role in reducing lactate production in mammalian cell culture.     

微生物中苹果酸脱氢酶研究现状及展望

苹果酸脱氢酶（MDH）EC 1.1.1.37是一类广泛存在于自然界生物体内的氧化还原酶,主要参与三羧酸循环（TCA）、乙醛酸循环、苹果酸-天冬氨酸循环等代谢途径。该酶可催化草酰乙酸和苹果酸之间的可逆转化,是细胞中心代谢通路中的关键酶之一。MDH广泛应用于心肌梗塞、急性实质性肝损伤、肝癌、肺癌的早期诊断,是常用的临床诊断用酶,并在生物制药、化工检测等领域也具有广泛市场需求。该文综述了微生物中MDH的蛋白结构、催化机制以及活性调节,并对其酶学性质、医疗诊断及免疫领域的应用、工业领域的应用以及生产方面,阐述了苹果酸脱氢酶的研究进展,以期为苹果酸脱氢酶的进一步开发和利用提供参考。     

温度对Zymomonas mobilis糖代谢关键酶活力和代谢流量的影响

以运动发酵单胞菌(Zynwmonas mobilis)ATCC31821为模式菌株,研究不同温度条件对其葡萄糖代谢关键酶活力的影响.采用全自动发酵罐,在整个发酵过程中通过充入氮气调节发酵液的溶氧量(DO)=0%,添加0.5mol/LNaOH溶液控制pH=5.5,发酵温度分别控制为25、30、35、40℃,发酵24h,测定其糖代谢网络中ED、HMP、TCA等途径的关键酶活力和代谢物成分.结果表明,在发酵温度为30～35℃时,乙醇脱氢酶(ADH)、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC)、葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素. 、葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、丙酮酸激酶(PK)和甘油醛-3-磷酸脱氢酶(GD-3-PDH)的活力较高,菌体的ED途径代谢活跃,碳素流量增加,乙醇生产量和糖转化率较高,而TCA途径的苹果酸脱氢酶(MDH)和异柠檬酸脱氢酶(ICDH)等活力较低,进入TCA途径的碳素流量明显减少;发酵温度为25、40℃时,TCA途径的酶活力升高,ED途径的酶活力减弱,生成乙醇的代谢流量减少,因此温度是z.mobilis发酵过程中调控菌体细胞生长和糖代谢的一个重要因素.葡萄糖-6-磷酸脱氢酶(G-6-PDH)、丙酮酸脱羧酶(PDC) 葡萄糖激酶(GK)、     

真养产碱杆菌发酵生产聚β-羟基丁酸(PHB)的研究

对真养产碱杆菌（Ａｌｃａｌｉｇｅｎｅｓｅｕｔｒｏｐｈｕｓ）ＮＣＩＭＢ１１５９９在１０Ｌ发酵罐中利用葡萄糖作为唯一碳源发酵生产聚β－羟基丁酸（ＰＨＢ）的发酵过程进行了研究。在以葡萄糖为唯一碳源的发酵中,分别在缺氧、限氧、限磷情况下及限磷和缺氧情况下对菌体生长及ＰＨＢ积累规律进行了研究。实验表明,氧和磷在菌体生长及ＰＨＢ的积累过程中起着重要作用。缺氧不利于菌体生长及积累ＰＨＢ;在一定程度上限制溶解氧则可促进ＰＨＢ的积累;在供氧充足时,限磷有利于ＰＨＢ大量积累;而限磷和缺氧条件下则抑制菌体生长和ＰＨＢ积累。采用限磷法发酵生产ＰＨＢ,经７２．５ｈ后得到菌体细胞干重为１３８．２ｇ／Ｌ,ＰＨＢ含量为１１１．８ｇ／Ｌ,ＰＨＢ占细胞干重８０．９％。     

产聚β-羟基丁酸酯菌株LY-1的发酵条件优化

塑料制品在人们的日常生活中起到重要作用,然而废弃的化学塑料由于不能被微生物降解,造成了严重的环境污染。作为一类可望替代传统塑料的新型可降解生物高分子材料,聚β-羟基丁酸酯(PHB)日益引起人们的重视。对从活性污泥中筛选出的高产PHB菌株LY-1进行发酵条件优化,采用控制变量法以及正交试验等方法确立其各自培养基培养的最佳温度、最适生长pH、最佳碳源和氮源。该菌株在最佳培养条件下PHB的产量可达0.84g/L。     

基于蛋白质组学分析尿嘧啶对出芽短梗霉产普鲁兰多糖的影响

为了解尿嘧啶对出芽短梗霉生长及合成普鲁兰多糖的内在机制,研究了尿嘧啶在普鲁兰多糖发酵过程中的最适添加量与最适添加时间。利用非标记（Label-free）定量技术和液相色谱-串联质谱技术比较出芽短梗霉发酵后期（88 h）的蛋白质组分,并对其差异蛋白质进行生物信息学分析。结果表明:在48 h添加0.5 g/L的尿嘧啶对普鲁兰多糖的产量提高最为显著,在5 L发酵罐上验证,产量由70.13 g/L提高到86.27 g/L,提高了23%。进行蛋白质组分分析鉴定出80个差异性蛋白质,其中包括40个上调蛋白和40个下调蛋白（差异倍数>2,P<0.05）,对这些差异蛋白进行聚类分析、GO功能富集分析、KEGG通路分析显示上述差异蛋白广泛涉及细胞过程、代谢过程等重要生物过程。差异蛋白质主要参与糖酵解、果糖和甘露糖代谢、乙醛酸和二羧酸代谢、丙酮酸代谢和TCA循环等代谢过程,最终引起普鲁兰多糖产量的变化。为进一步了解出芽短梗霉产普鲁兰多糖的代谢机理提供了分子基础。     

Correlation of community dynamics and process parameters as a tool for the prediction of the stability of wastewater treatment

Wastewater treatment often suffers from instabilities and the failure of specific functions such as biological phosphorus removal by polyphosphate accumulating organisms. Since most of the microorganisms involved in water clarification are unknown it is challenging to operate the process accounting for the permanent varying abiotic parameters and the complex composition and unrevealed metabolic capacity of a wastewater microbial community. Fulfilling the demands for water quality irrespective of substrate inflow conditions may emit severe problems if the limited management resources of municipal wastewater treatment plants are regarded. We used flow cytometric analyses of cellular DNA and polyphosphate to create patterns mirroring dynamics in community structure. These patterns were resolved in up to 15 subclusters, the presence and abundances of which correlated with abiotic data. The study used biostatistics to determine the kind and strength of the correlation. Samples investigated were obtained from a primary clarifier and two activated sludge basins. The stability of microbial community structure was found to be high in the basins and low in the primary clarifier. Despite major abiotic changes certain subcommunities were dominantly present (up to 80% stability), whereas others emerged only sporadically (down to 3% stability, both according to equivalence testing). Additionally, subcommunities of diagnostic value were detected showing positive correlation with substrate influxes. For instance blackwater (r(s) = 0.5) and brewery inflow (both r(s) = 0.6) were mirrored by increases in cell abundances in subclusters 1 and 6 as well as 4 and 8, respectively. Phosphate accumulation was obviously positively correlated with nitrate (r(s) = 0.4) and the presence of denitrifying organisms (Rhodacyclaceae). Various other correlations between community structure and abiotic parameters were apparent. The bacterial composition of certain subcommunities was determined by cell sorting and phylogenetic tools like T-RFLP. In essence, we developed a monitoring tool which is quick, cheap and causal in its interpretation. It will make laborious PCR based technique less obligatory as it allows reliable process monitoring and control in wastewater treatment plants.