Extracellular enzymatic activity of two hydrolases in

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Jun 21, 2017 - Zinc sulfate heptahydrate (ZnSO4·7H2O). 0.036. 0.036. Cobalt chloride hexahydrate (CoCl2·6H2O). 0.045. 0.045 a The synthetic wastewater ...

Appl Microbiol Biotechnol DOI 10.1007/s00253-017-8423-1


Extracellular enzymatic activity of two hydrolases in wastewater treatment for biological nutrient removal Jorge Mario Berrio-Restrepo 1 & Julio César Saldarriaga 1 & Mauricio Andrés Correa 1 & Néstor Jaime Aguirre 2

Received: 3 March 2017 / Revised: 21 June 2017 / Accepted: 2 July 2017 # Springer-Verlag GmbH Germany 2017

Abstract Due to the complex nature of the wastewater (both domestic and non-domestic) composition, biological processes are widely used to remove nutrients, such as carbon (C), nitrogen (N), and phosphorous (P), which cause instability and hence contribute to the damage of water bodies. Systems with different configurations have been developed (including anaerobic, anoxic, and aerobic conditions) for the joint removal of carbon, nitrogen, and phosphorus. The goal of this research is to evaluate the extracellular activity of βglucosidase and phosphatase enzymes in a University of Cape Town (UCT) system fed with two synthetic wastewaters of different molecular complexity. Both types of waters have medium strength characteristics similar to those of domestic wastewater with a mean C/N/P ratio of 100:13:1. The operation parameters were hydraulic retention time (HRT) of 10 h, solid retention time (SRT) of 12 days, mean concentration of the influent in terms of chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorus (TP) of 600, 80, and 6 mg/L, respectively. According to the results obtained, statistically significant differences have been found in the extracellular enzyme activities with the evaluated wastewaters and in the units comprising the treatment system in some of the cases. An analysis of principal components showed that the extracellular enzymatic activity has been correlated to

* Jorge Mario Berrio-Restrepo [email protected]


Grupo de Ingeniería y Gestión Ambiental – GIGA, School of Engineering, Universidad de Antioquia, Cl. 67 No. 53-108 Of, 20-250 Medellín, Colombia


Grupo de Geografía, Limnología y Modelación Ambiental – GEOLIMNA, School of Engineering, Universidad de Antioquia, Cl. 67 No. 53-108 Of, 20-403 Medellín, Colombia

nutrient concentration in wastewater, biomass concentration in the system, and metabolic conditions of treatment phases. Additionally, this research has allowed determining an inverse relationship between wastewater biodegradability and the extracellular enzyme activity of β-glucosidase and phosphatase. These results highlight the importance of including the analysis of biomass biochemical characteristics as control methods in wastewater treatment systems for the nutrient removal. Keywords UCT system . Extracellular enzyme activity . Biological nutrient removal . Glucosidase . Phosphatase

Introduction Wastewaters are a complex combination of organic and inorganic pollutants in a liquid matrix, which can be present at several concentrations and result from all kinds of domestic and industrial activities (Henry et al. 2008). The composition of domestic wastewater varies according to different factors such as the population size producing it, the degree of urbanization of the area, the socioeconomic levels, and its geographic position (Friedler et al. 2013; Mara 2004). The level of industrialization and the nature of the industrial activities developed (Orhon et al. 2009) may also impact the nondomestic or industrial wastewaters. Discharge of wastewaters without an appropriate treatment adjusted to the environmental regulations result in accumulation of nutrients in water bodies, thus affecting the balance of aquatic ecosystems. These problems have been most frequently associated to eutrophication, excessive growth of water plants, and proliferation of algae (UN-Water 2015). An increasing number of plant species and algae in water bodies deplete the dissolved oxygen in water, thus affecting the life sustainability for other species of the ecosystems (Wetzel 2001).

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Problems associated to the accumulation of nutrients have brought the need for setting mechanisms where nutrient removal may be possible, with carbon, nitrogen, and phosphorous being the most critical nutrients (Saldarriaga et al. 2010). Since the development of the activated sludge processes over 100 years ago, several treatment configurations known as biological nutrient removal (BNR) systems have been designed under specific operation conditions to the elimination of carbon, nitrogen, and phosphorus, according to the specific characteristics of the composition of the wastewater to be treated (Crittenden et al. 2012). The developed systems to the biological nutrient removal are usually integrated by anaerobic, anoxic, and aerobic phases adjusted in series, whose number and arrangement can vary according to the configuration type to be used. Phosphorus removal is achieved through the sequencing of the polyphosphate-accumulating organisms (PAOs) in anaerobic/ aerobic (or anoxic) phases, such that phosphorus is first released and later uptaken. In the anaerobic phases, the polyphosphate-accumulating organisms accumulate high energetic material as Bpolyhydroxialkanoates (polyhydroxybutirate and polyhydroxyvalerate, mainly) inducing the polyphosphate (poly-P) release due to the absence of an external electron acceptor. Subsequently, in the aerobic (or anoxic) phases, the polyphosphate-accumulating organisms use their energetic reserves and take up the phosphorus that is initially released in anaerobic phases to store it as intracellular polyphosphate (Wentzel et al. 2008). On the other hand, the nitrogen elimination involves more complex mechanisms mediated by autotrophic and heterotrophic microorganisms, usually under aerobic and anoxic phases. In the aerobic phase, autotrophic populations oxidize in two sequential steps the ammonium (NH4+) to nitrite (NO2−) and nitrate (NO3−) in the nitrification process. Then, in the anoxic phase, nitrite and nitrate are reduced to N2 and escape to the atmosphere due to the denitrifying heterotrophic bacteria (Ekama and Wentzel 2008). Related to the removal of nitrogen, several researches have evidenced that the growth rate of the nitrifying bacteria is too low compared to other microbial populations in wastewater treatment processes (Henze et al. 1997). Recent studies have shown that the attached growth may optimize the efficiency of nitrogen removal (Ekama 2015; Saldarriaga et al. 2010). Concerning its molecular structure, wastewater is composed of complex combinations of dissolved substances and particles which may vary in size. Its components include carbohydrates, amino acids, alcohols, proteins, polysaccharides, volatile fatty acids, and lipids (Szilveszter et al. 2010; Frølund et al. 1995). The molecular complexity of wastewater influences the degradation processes, since the small size and low molecular weight particles can be directly transported to the internal side of the cell membrane from the surrounding environment (Arnosti 2003; Wetzel 2001).

Additionally, between 30 and 85% of wastewaters contain particles with sizes over 1 μm (Levine et al. 1991), and most of the dissolved organic matter (about 95%) is composed of high molecular weight polymeric compounds (Wetzel 2001). This complexity of the wastewaters produces high competition for the acquisition of easily assimilated resources by microbial communities (Kirchman 2003). According to this, Chróst (1991) affirms that depolymerization and hydrolysis of macromolecules outside of the cell are necessary for obtaining microbial energy. The mechanisms for the nutrient assimilation by the cell are regulated by extracellular enzymes, which mineralize the organic matter. These enzymes are usually classified in two categories, ectoenzymes and exoenzymes. Ectoenzymes remain adhered to the cell surface, while exoenzymes are released into the surrounding environment (Szilveszter et al. 2010). Accordingly, the extracellular enzymatic activity has particular relevance in the improvement of the wastewater treatment process and eventually could be applied to realscale systems (Bitton 2011). However, the high specificity of exoenzymes requires new researches in the different treatment conditions to determine their influence on the system performance (Demarche et al. 2012). In the biological processes for wastewater treatment, βglucosidase and phosphatase enzymes play a fundamental role (Szilveszter et al. 2010). β-Glucosidase, produced by heterotrophic microorganisms such as bacteria and fungi, catalyzes the hydrolysis of β bonds of glucose, hexose, and carboxymethyl cellulose disaccharides (Chróst 1991). The phosphatase is synthesized by phytoplankton, zooplankton, bacteria, and protozoa, and catalyzes hydrolysis of a wide variety of phosphate esters, including diester bonds in the presence of phosphate and polyphosphate acceptors (Giraldo et al. 2014; Chróst 1991). Usually, most of the relevant studies in microbial ecology applied in wastewater treatment systems have been focused on the identification of microbial populations (Seviour 2009), although the extracellular enzymatic activity determination has gained relevance for a better understanding of the biochemical mechanisms developed for the microbial communities in nutrient removal processes. Related to the extracellular enzymatic activity evaluation in wastewater treatment systems, the observations carried out by Kreutz et al. (2016) in a municipal wastewater treatment plant (WWTP), Gómez-Silván et al. (2013) in a membrane bioreactor (MBR) of a wastewater treatment plant, and Szilveszter et al. (2010) and Goel et al. (1998), both using sequencing batch reactors (SBRs) in a lab scale, can be highlighted. Despite of this, no relevant information about extracellular enzymatic activity in continuous processes with anaerobic, anoxic, and aerobic phases is currently available. The aim of this study was to determine the extracellular enzymatic activity of the β-glucosidase and phosphatase enzymes in a University of Cape Town (UCT)-type system, for the biological nutrient removal of carbon, nitrogen, and phosphorus. In addition, the

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results will support the understanding of the biochemical mechanisms in a wastewater treatment process. To the achievement of this goal and its possible application in a full-scale wastewater treatment process, the following research question was posed: How is the extracellular enzymatic activity of the βglucosidase and phosphatase enzymes related to the performance of a UCT system fed with two synthetic wastewaters of different molecular complexity? We also wanted to determine how the extracellular enzymatic activity of both enzymes is associated with the operation system parameters.

Experimental methodology University of Cape Town (UCT) system For the experimentation, a UCT system of 150 L with three phases was used. These phases consist of a 25-L anaerobic tank, a 50-L anoxic tank, and a 75-L aerobic tank, keeping a volume ratio between the phases of 1:2:3 (Chuang et al. 1997). In order to facilitate the biomass adherence and increase its contact with wastewater, 25% of the volume of the aerobic tank (18.8 L approximately) was a carrier material. The selected carrier material, AnoxKaldness K1 and K3 (Veolia Water Technologies AB, AnoxKaldness, Lund, Sweden), had a specific gravity of 0.96 g/cm3, specific surface of 500 m2/m3, and diameters of 9.1 and 25 mm. The total useful volume of the UCT system was 131.3 L. The system was inoculated with facultative activated sludge from a municipal wastewater treatment plant located in Medellin (Colombia) and operated for 307 days with a mean temperature of 26 °C. The system operation was divided in two stages (209 days of operation in the first stage, 98 days of operation in the second stage) according to the synthetic wastewater with which it was fed. In the experimentation stages, the adaptation period to the specific operation conditions were included (90 days approximately). The experimentation stages were developed in series operation: At the end of the first stage with SWW1, the system was fed with SWW2 and started a new acclimatization phase. The operational conditions of the system were as follows: food/microorganism (F/M) ratio of 0.3; mean mixed liquor volatile suspended solids (MLVSS) of 2291 mg/L (first stage) and 1530 mg/L (second stage); hydraulic retention time (HRT) of 10 h (1.66 h in anaerobic phase, 3.34 h in anoxic phase, and 5.00 h in aerobic phase approximately); and solid retention time (SRT) of 12 days. Figure 1 shows the UCT system configuration and recirculation ratios. Synthetic wastewater Two synthetic wastewaters with different molecular complexity and high contents of nitrogen were employed. In the first

stage, a powdered skim milk solution (SWW1) was used, while in the second stage, the system was fed with a sodium acetate and acetic acid solution (SWW2). Both synthetic wastewaters were prepared according to the major nutrient characteristics of local wastewater adding external nitrogen (CH4N2O) and phosphorus (K2HPO4) sources. The mean concentration ratio of chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN), and total phosphorous (TP), defined as COD/TKN/TP, was 100:13:1. The synthetic wastewaters were supplemented with minor elements according to Smolders et al. (1994). Detailed information about the composition of synthetic wastewaters and their mean parameters are shown in Table 1. It is important to clarify that the added K2HPO4 to SWW1 was lower than for SWW2 due the high phosphorus content of the skim milk. The synthetic wastewater’s biodegradability was calculated from the ratio between biochemical oxygen demand (BOD5) and COD, which was 0.47 for SWW1 and 0.72 for SWW2. Determination of extracellular enzyme activities The determination of extracellular enzymatic activity of βglucosidase (GLU, EC, according to the Enzyme Commission classification) and phosphatase (PHO, EC, according to the Enzyme Commission classification) was performed by applying a photometric method developed by Marxsen et al. (1998). This method verifies the hydrolysis of a specific substrate for each enzyme, in the presence of 4nitrophenol (C6H5NO3) as colorimetric indicator. The assays were performed by taking 1-L aliquots from the mixed liquor of the UCT system (anaerobic, anoxic, and aerobic tanks) and depositing them into glass beakers. Later, triplicate dilutions from homogenized mixed liquor subsamples of 5 mL in 50 mL of NaCl were prepared. Finally, 2 mL of these solutions was taken and added to the test tubes. Each test tube was added with 2 mL of a substrate solution in NaCl of 4-nitrophenil-β-D-glucopyranose 98% (CAS No. 2492-87-7) Alfa Aesar (Thermo Fisher Scientific, Haverhill, USA), for determining the extracellular enzymatic activity of β-glucosidase. Also, in order to determine the extracellular enzymatic activity of phosphatase, 4-nitrophenil-phosphatase sodium salt hexahydrate 99% (CAS No. 4264-83-9) Alfa Aesar (Thermo Fisher Scientific, Haverhill, USA) was used. The test tubes were incubated at 30 °C during 3 h. After the incubation period, 2 mL of Na2CO3 was added to each test tube to stop hydrolysis. The tubes were then centrifuged at 4500 rpm for 10 min. The amount of enzyme released by the biomass to the medium has been established according to the concentration of 4-nitrophenol (C6H5NO3) in each sample analyzed. The concentrations analyzed were contrasted to a calibration curve of 4-nitrophenol solutions in NaCl and Na2CO3 with a 2:1 volumetric ratio. The extracellular enzymatic activity photometrically measured in function of time

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Fig. 1 UCT system layout used in the experimental runs (P1 to P4 are peristaltic pumps. Q is the influent flow rate. P1: inlet flow to anaerobic phase; P2: anoxic phase flow recycled to anaerobic phase; P3: aerobic phase flow recycled to anoxic phase; P4: settler flow recycled to anoxic phase)

for each sample has been calculated by employing Eq. 1 below (Marxsen et al. 1998). EEAx ¼

Absx  D  F t


where EEAx Absx Table 1

Extracellular enzyme activity of enzyme X (mol/h) Absorbance after incubation measured at λ = 405 nm Synthetic wastewater composition and mean parameters

Synthetic wastewater compositiona Compound Powdered skim milk Sodium acetate (C2H3NaO2) Acetic acid (C2H4O2) Urea (CH4N2O) Potassium diphosphate (K2HPO4) Sodium chloride (NaCl) Calcium chloride dehydrate (CaCl2·2H2O) Magnesium sulfate heptahydrate (Mg2SO4·7H2O) Iron tri-chloride hexahydrate (FeCl3·6H2O) Boric acid (H3BO3) Copper sulfate pentahydrate (CuSO4·5H2O) Potassium iodide (KI)

SWW1 (mg/L) 534.0 – – 161.8 7.6 7.0 4.0 2.0 0.45 0.045 0.009 0.054

SWW2 (mg/L) – 677.0 0.183 176.0 28.2 7.0 4.0 2.0 0.45 0.045 0.009 0.054

Manganese chloride tetrahydrate (MnCl2·4H2O) Sodium molybdate (Na2Mo4·2H2O) Zinc sulfate heptahydrate (ZnSO4·7H2O) Cobalt chloride hexahydrate (CoCl2·6H2O)

0.036 0.018 0.036 0.045

0.036 0.018 0.036 0.045


The synthetic wastewater composition was adjusted from Smolders et al. (1994)

D F t

Dilution factor Photometric factor resulting from the inverse of calibration curve slope of 4-nitrophenol (mol/L) Incubation time (h)

The absorbance was measured in a Macherey-Nagel NANOCOLOR 500 D (Macherey-Nagel GmbH & Co. KG, Düren, Germany) digital photometer equipped with a 405-nm Macherey-Nagel NANOCOLOR (Macherey-Nagel GmbH & Co. KG, Düren, Germany) filter in a 10-mL glass test tube of 16 mm of thickness. Measurements were performed after calibrating the photometer with a specific blank, prepared from each substrate (2 mL NaCl solution, 2 mL substrate solution, and 2 mL Na2CO3). All the enzymatic activities are expressed as specific extracellular enzymatic activities, dividing the measured extracellular enzymatic activities by the MLVSS concentration (mg MLVSS/L) of the system tanks (Goel et al. 1998).

System operation and analytical methods Different physical-chemical parameters were daily measured during the operation of the system to verify the process stability. These parameters were temperature, pH, electric conductivity, dissolved oxygen, and oxidation-reduction potential. Additionally, performance parameters such as chemical oxygen demand (COD), total organic carbon (TOC), total Kjeldahl nitrogen, ammonium (NH4+), nitrates (NO3−), nitrites (NO2−), total phosphorous, and orthophosphates (PO43−) were measured two times per week in order to establish the treatment performance. The parameter determination was performed based on the Manual on Standard Methods for the Examination of Water and Wastewater (APHA et al. 2012).

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Statistical analysis In the first instance, the system operation was analyzed through descriptive statistical analyses of the extracellular enzymatic activities (β-glucosidase, phosphatase), the operation parameters (temperature, pH, electric conductivity, dissolved oxygen, oxidation-reduction potential), and the performance parameters (COD, total Kjeldahl nitrogen, ammonium, nitrate, nitrite, total phosphorus, orthophosphates). In this descriptive analysis, the mean, standard deviation, and normality test (by the Kolmogorov-Smirnov test) were calculated. Secondly, the homoscedasticity of variance was calculated by applying the Kruskal-Wallis test for the comparative statistical analyses. A significance level of 95% was established as the decision rule for all the hypothesis validations. Additionally, an ordinal correlation between all the variables included in the experimentation was determined by applying the Spearman method. Finally, a

Table 3 Removal performance in the UCT system fed with two synthetic wastewaters (SWW1 and SWW2) of different molecular complexity

Organic matter (COD) Nitrogen (TKN, NO3−, NO2−) Phosphorous (TP, PO43−)

SWW1 Mean (±SD)

SWW2 Mean (±SD)

96.17% (±2.61) 53.45% (±11.72) 87.37% (±4.05)

95.11% (±1.99) 77.09% (±16.37) 85.15% (±11.51)

SD standard deviation

multivariate analysis of principal components was carried out, in order to verify the effect of the measured variables in the experimental stages with SWW1 and SWW2 on the extracellular enzymatic activities. Statistical analyses were developed using the 3.2.3 version of the R free software (R Development Core Team 2015).

Table 2 Median values and standard deviation of the performance parameters in a UCT system fed with two synthetic wastewaters (SWW1 and SWW2) of different molecular complexity IFL





24.70 (±0.65) 7.08 (±0.03) 145.20 (±7.34) – – 700.00 (±103.98) 203.86 90.00 (±6.80) 0.91 (±0.43) 0.21 (±0.17) 0.82 (±0.36) 7.00 (±0.31) 14.99 (±0.32)

25.70 (±0.40) 7.46 (±0.12) 527.00 (±29.78)

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