Evidence of microscopic correlation between biofilm kinetics and divalent cations for enhanced wastewater treatment efficiency

Abstract

Biofilm-bacterial communities have been exploited in various biological wastewater treatment processes. The present work is aimed at exploring the possible factors for tailoring biofilm mechanics by introducing divalent cations (Ca²⁺) in the constructed wetland soil. The in vitro investigation of soil samples collected at various retention times revealed that the addition of Ca²⁺ facilitates the formation of well organized patterns of biofilm compared to calcium depleted conditions. Afterwards, Ca²⁺ ions were intentionally introduced into the soil of lab-scale constructed wetland to examine its superior treatment efficiency towards the removal of various chemical and microbiological contaminants. The current work therefore may form a basis to implement theoretical predictions experimentally in a real time existing wastewater treatment technology. Hence, the same approach can effectively be adopted in pilot scale wetland systems as well as other biofilm mediated wastewater treatment technologies.

---

1. Introduction

Constructed wetlands (CW) are becoming increasingly popular as economical and environmentally friendly solutions for wastewater treatment. The type and size of microbial populations contained in biofilms are key for efficient wastewater clean-up processes in CW systems. The role of microbial biofilm formation in subsurface surroundings for removing pollutants from wastewater has been exploited in many practices. The development of biofilm is believed to occur in several distinct steps including transportation of microorganisms to surfaces, initial reversible/irreversible adhesion, cell to cell communication, microcolony formation, production of extracellular polymeric substances (EPS) and biofilm maturation.^1–4 Numerous factors can influence the growth and development of biofilm including; temperature, the availability of nutrients, surface topography, the presence of particles and microbial community composition, osmolarity, hydrodynamics and shear forces, turbulence and the amount of EPS produced respectively.^5–7 Microbial biofilms attached to the wetland matrix (solid particles and/or plant roots), are responsible for most of the essential transformations and the decomposition of contaminants in wastewater.

It is widely believed that the efficiency of constructed wetland (CW) is greatly influenced by complex interactions among bacteria within biofilms, thus affecting the quality of wetland effluent.^8,9 Although, as an engineered system, constructed wetlands (CWs) are regarded as one of the highly cost-efficient systems for wastewater treatment. However, their performance is still unpredictable owing to the presence of diverse microorganisms coupled with the existence of various physical, chemical, and biological processes, some of which are yet to be understood. To obtain high performance CW systems, it is highly desirable to put considerable effort into (1) the engineered controlled design of CWs and (2) fundamental and applied research on this technology.¹⁰ This in turn will increase their acceptance as an alternative low cost conventional wastewater treatment technology. Even for the most adaptive wastewater treatment plants, there are severe deficiencies in understanding the fundamental knowledge about the biofilm characteristics for the participating microorganisms. Understanding of the underpinning mechanisms to manipulate biofilm development in various stages, such as initial adhesion, aggregation and maturation, can somehow help to attain high efficiencies for biofilm related wastewater treatment processes.

Very recently, we successfully demonstrated the high performance of a hybrid constructed wetland (HCW) in terms of pollutant removal efficiencies for domestic wastewater through simply regulating hydraulic retention times and the appropriate selection of macrophytes.¹¹ Also, the simultaneous role of divalent cations (Ca²⁺) and extracellular DNA (eDNA) in mediating bacterial aggregation and biofilm formation was well established in another report.¹² However, to the best of our knowledge there are hardly any reports which provide microscopic evidence of biofilm kinetics in the absence or presence of divalent cations (Ca²⁺). Herein, for the first time, we were able to identify the influential role of calcium in biofilm formation and its propagation with time. To study the various stages of biofilm development systematically, in vitro studies were carried out at various retention times. Furthermore, the established concept was successfully implemented on a lab-scale constructed wetland to investigate its treatment efficiencies towards wastewater treatment through studying various physicochemical and microbial parameters. The present work could be a gateway for future biofilm dependant environmental and microbiological applications.

2. Experimental section

2.1 Development of biofilms on glass slides

Two soil samples were collected from a lab-scale HCW planted with T. portulacastrum (150 g, equivalent to 133.5 g dry weight), the details of the HCW have already been reported.¹⁰ After collection and drying, these soils were separately mixed with 300 ml of autoclaved ddH₂O in two large beakers (150 rpm for 30 min followed by gentle agitation at 30 rpm overnight) to form soil slurries. Autoclaved noble agar (1.0%) (Difco, BD, New Jersey, USA) was deposited on glass slides and then put in a rack in the soil slurry. Only the liquid phase of the soil slurry covered the glass slides without disturbing the solid phase. The beakers were incubated for 7 days at room temperature with a shaking speed of 30 rpm after which the rack was removed. After 7 days of incubation, the glass slides were removed and stained with 0.01% crystal violet (CV) solution for 15 min followed by a quick wash in 0.9% NaCl. For Ca²⁺ addition, 10 g of Ca²⁺ in the form of CaCO₃ was introduced into one of the soil samples at the stage of mixing with ddH₂O.

2.2 Biofilm formation on gravel samples

Gravels collected from the lab-scale HCW were generously rinsed three times with DI water followed by autoclaving (15 min at 15 psi pressure at 121 °C) for sterilization and then cooled at room temperature. The gravels were gently placed in the soil slurries (with and without addition of 10 g of Ca²⁺) for the period of three weeks at room temperature, following the same procedures as described before.

2.3 Physico-chemical and microbiological analysis of wastewater

Physico-chemical parameters were determined through following the American Public Health Association (APHA) Standard Methods.¹³ To introduce divalent cations into wetland soil, limestone (CaCO₃) as a source of calcium was deposited on the HCW soil planted with T. portulacastrum and allowed to settle down for one week or so. After one week, domestic wastewater from a residential colony was subjected to treatment through the HCW with an optimized retention time of 20 days both in the absence and presence of added Ca²⁺. The sewage treatment efficiency (considering the concentrations of each physico-chemical parameter) after a retention time of 20 days was determined by using the following formula:
where: C_pi and C_pe represent the concentrations of pollutant at input (influent) and output (effluent) respectively.

Domestic wastewater was subjected to microbiological analysis through the colony forming unit (CFU per ml) and most probable number technique (MPN index/100 ml) of pathogenic indicators i.e. fecal coliforms and Enterococcus faecalis as per the guidelines of Bergey’s Manual of Determinative Bacteriology.¹⁴ The conventional serial dilution method was employed to calculate the CFU per ml of bacterial colonies in the influent and effluent samples (before and after addition of Ca²⁺). Wastewater samples were serially diluted in sterile water up to 10⁻¹⁰ and these dilutions were spread plated on nutrient agar plates and incubated at 37 °C. After 24 h of incubation, the colonies which appeared were enumerated using a colony counter, and the CFU of each colony was then calculated as follows:

CFU per ml = (no. of colonies × dilution factor)/volume of culture plate

For determining the MPN index of pathogen indicators (fecal coliforms, E. coli, Salmonella, Shigella, Klebsiella sp., Enterobacter and Citrobacter), untreated (influent) and treated (effluent) wastewater samples both in the absence and presence of Ca²⁺ were incubated at 42.2 °C for 24–48 h in MacConkey’s broth using a multiple tube technique with inverted Durham tubes. Positive tubes were subcultured on MacConkey’s agar (MacA), nutrient agar (NA) and mannitol salt agar (MSA) plates and incubated at 37 °C for 24–48 h. Positive isolates showing growth of bacterial colonies were confirmed through microscopy and checked for the total count.

2.4 Optical and scanning electron microscopy

The optical images of biofilms on agar coated glass slides at different time intervals ranging from 1–5 weeks were obtained using a light microscope (Zeizz, objective). The surface morphologies of the biofilms on coated glass slides were examined through Scanning Electron Microscopy (Nova SEM 230) coupled with EDX to identify various existing elements on the biofilm coated glass slides.

3. Results and discussion

3.1 Optical microscopy

The different stages of biofilm formation (initial adhesion, aggregation and maturation) on agar-coated glass slides both in the presence and absence of added Ca²⁺ were studied through optical microscopy at different time intervals between 1 to 5 weeks. It is an established fact that in the presence of bacterial self-produced eDNA, bacteria interacts with the substratum and forms a conditioning film on surfaces, thus enhancing adhesion and subsequent biofilm development.¹⁵ Optical microscopy results revealed that during the first week of incubation, different microbial communities from soil slurry under the influence of eDNA start adhering to the glass surface (Fig. 1a). This is due to the existence of physico-chemical forces between bacterial cell surfaces and substratum surfaces. However, the presence of Ca²⁺ could facilitate strong electrostatic interactions between positively charged Ca²⁺ and negatively charged eDNA, therefore bacterial communities closely adhere and aggregate with each other in the form of thick clusters as shown in Fig. 2a. By prolonging the biofilm growth time to two weeks, short rods and coco-bacilli-like structures were observed pointing towards the active role of Ca²⁺ addition in stabilizing bacterial cell walls and promoting ionic cross bridging among bacterial cells (Fig. 2b) compared to in the absence of Ca²⁺ (Fig. 1b). At the third week of incubation, an increase in the overall density and complexity of the biofilm was observed leading to a maturation stage (Fig. 2c) which may involve different mechanisms including gene transfer,¹⁶ quorum sensing¹⁷ and/or its persistent development etc. The increased bulk density of the biofilms may also be attributed to the presence of surface-bound organisms which start replicating with the production of extracellular components (EPS). The presence of Ca²⁺ as a divalent cation possibly facilitates bridging among the negatively charged moieties of extracellular polymeric substances (EPS) through electrostatic interactions¹⁸ that results in the formation of thicker, denser, and mechanically more stable biofilms in the form of a mat on the entire surface of the glass slide compared to in the absence of Ca²⁺ (Fig. 1c). No significant difference in biofilm growth and development (with or without Ca²⁺ addition) was observed by further incubating the agar-coated glass slides for up to five weeks as shown in Fig. 1d and 2d. This can be an indication of saturation or a steady state beyond which no further progression in biofilm growth can be observed.

Fig. 1 Optical microscope images of rhizosphere of T. portulacastrum on agar coated glass slides in the absence of added Ca²⁺: (a) initial adhesion of bacterial communities at week 1, (b) aggregation and initial maturation at week 2, (c) biofilm strengthening and stabilization at week 3, and (d) static phase of biofilm growth at week 5 of incubation.

Fig. 2 Optical microscope images of rhizosphere of T. portulacastrum in the presence of added Ca²⁺ on agar coated glass slides: (a) initial aggregation of biofilm in the form of clusters at week 1, (b) biofilm attains initial maturity through forming cationic bridging in the presence of bacterial self-produced eDNA and Ca²⁺ at week 2, (c) a thicker, denser and stabilized biofilm at week 3, and (d) static phase of biofilm at week 5 of incubation.

3.2 Scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS)

The growth and development of biofilm was also analysed through Scanning Electron Microscopy (SEM). Surface examination of agar coated glass slides (1 week of incubation) in the absence of added Ca²⁺ showed the formation of microcolonies in the form of long beads (Fig. 3a). However, upon Ca²⁺ addition, thick aggregates (Fig. 4a) were observed pointing towards the influential role of Ca²⁺ in bringing bacterial communities together due to strong cationic bridging. These aggregates both in the presence and absence of added Ca²⁺ mostly comprised cocci-shaped bacteria showing the dominant E. coli and E. faecalis species residing in the rhizosphere of T. portulacastrum fed with domestic wastewater. Two weeks old biofilm both in the absence and presence of added Ca²⁺ showed more ordered structures through having a relative increase in the number of short rods and cocci shaped bacteria colonizing the glass slides as shown in Fig. 3b and 4b. The presence of homogeneous and abundant microorganisms forming a thick mat on the entire agar-coated glass surface were observed in the presence of added Ca²⁺ when the growth times of biofilms were prolonged to three weeks (Fig. 4c) indicating the maximum growth and stability of biofilms compared to those in the absence of Ca²⁺ (Fig. 3c). Further patterns of biofilm growth both in the absence and presence of added Ca²⁺ were also monitored for 5 weeks of incubation (Fig. 3d and 4d). However, no significant difference in terms of biofilm growth was observed indicating a saturated state/static phase.

Fig. 3 Scanning electron micrographs (30 μm) of rhizosphere of T. portulacastrum on agar coated glass slides in the absence of added Ca²⁺: (a) initial aggregation of biofilm resulting in a bead-like structure at week 1, (b) biofilm growth at week 2, (c) a thicker, denser and stabilized biofilm at week 3, and (d) static phase of the biofilm at week 5 of incubation. EDS spectrum of the highlighted region of the SEM analysis of the agar-coated glass slide (c) in the absence of added Ca²⁺: (e) multipoint spectrum and (f) line spectrum.

Fig. 4 Scanning electron micrographs (30 μm) of rhizosphere of T. portulacastrum in the presence of added Ca²⁺ on agar coated glass slides: (a) formation of thick aggregates as a result of being under the influence of Ca²⁺ at week 2, (b) increased biofilm growth under the influence of Ca²⁺ at week 2, (c) stable and dense biofilm forming a mat on the glass surface at week 3, and (d) static phase of the biofilm at 5 week of incubation. EDS spectrum of the highlighted region of the SEM analysis of the agar-coated glass slide (c) in the presence of added Ca²⁺: (e) multipoint spectrum and (f) line spectrum.

As the SEM analysis of the agar coated glass slides showed development of mature biofilms after an incubation time period of three weeks, we therefore selected only three weeks incubated agar-coated glass slides for further EDS analysis. The highlighted regions of Fig. 3c and 4c were analysed through point and line scans. The multipoint and line spectra confirmed that prior to CaCO₃ addition, the content of carbon (C) and oxygen (O) were much higher than calcium (Ca) as shown in Fig. 3e and f pointing towards overall domination of organic materials. However with the addition of CaCO₃ in the medium, as expected, a strong signal of Ca was detected both in the multipoint and line scan spectra as illustrated in Fig. 4e and f. The strong signal of Ca indicates the excess of calcium on the entire highlighted area which helped to grow biofilm more effectively and efficiently. The reduced levels of phosphorous and other elements as illustrated in the multipoint and line spectra in the same figure may be attributed to the precipitation of phosphorous. Calcium phosphate precipitation takes place as Ca²⁺ cations interact with dissolved ortho-phosphates in the water, thus converting it into particulate form with the production of chemical precipitates having low solubility.¹⁹

In most bacterial species, biofilm formation is mediated via bacterial self-produced exopolymeric substances (EPS) that primarily consist of various proteins, polysaccharides, lipids, RNA and extracellular DNA (eDNA). EPS also play a critical role in various stages of biofilm formation including bacterial adhesion to surfaces, cell-to-cell attachment, and the formation of micro colonies as well as in biofilm maturation. The bacterial cell surface and eDNA are usually negatively charged and their association with each other to form aggregates often requires cations, mostly divalent cations like Ca²⁺, to act as a link between the components. Fig. 5 shows a comparison of biofilm development in (1) no added calcium conditions (left panel) and added calcium (right panel) conditions. The presence of positively charged Ca²⁺ would facilitate the link between eDNA and Ca²⁺ through electrostatic interactions, which is hard to imagine for the case of calcium depleted conditions. This strong linkage may bring bacterial cells close to each other through forming aggregates and micro colonies, thus promoting the growth and development of biofilm as evidenced by the aforementioned microscopic results (Fig. 2 and 4). Besides, free calcium is essential for the structural integrity of biofilm as it helps to maintain the tertiary structure of polymeric substances so that interactions between adjacent sugars on different chains are promoted.

Fig. 5 Schematic illustrations of biofilm growth and development in the presence and absence of added Ca²⁺.

3.3 Assessment of biofilm growth on gravel samples

Further investigations on the effectiveness of Ca²⁺ addition in promoting biofilm formation on a different substrate/medium were carried out. In order to do this, we deliberately chose the same gravels which were used in the fill media of the HCW. The in vitro studies of biofilm formation on gravel surfaces were carried out by considering the fact that the gravel surface is rough and it may provide additional benefits for bacterial attachment and biofilm formation. The importance of substratum is usually considered as very crucial in microbial attachment or adhesion which mainly depends on surface roughness, hydrophobicity and surface conditioning film. The extent of microbial colonization increases with increased surface roughness due to diminished shear forces and increased surface area on rougher surfaces.²⁰

Three weeks old biofilms developed on gravel media without and with Ca²⁺ addition were subjected to SEM analysis. Interestingly, a pronounced increase in biofilm growth was observed in the presence of added Ca²⁺ forming a dense mat on the entire gravel surface (Fig. 6b) compared to the biofilm formed in the absence of added Ca²⁺ (Fig. 6a). This enhanced biofilm growth may be attributed to the electrostatic interaction of Ca²⁺ with cell wall-embedded biopolymers (polysaccharides, proteins, eDNA etc.) and the surfaces to which they attach.²¹ Also, the outer surface of bacteria is generally considered to possess net negative charge due to abundantly found negatively charged functional groups. Therefore, the presence of these biopolymers and cell appendages (fimbrae, pilli etc.) that extend hundreds of nanometres from the cell surface contributes to cell surface hydrophobicity by repelling water and attracting adjacent bacteria that eventually promote biofilm formation.²² These biopolymers and cell appendages further intermingle with Ca²⁺ ions due to electrostatic interactions and reduce the energy barriers, thus bringing bacterial cells closer which results in more aggregation and enhanced biofilm growth. Moreover, with Ca²⁺ addition, bacterial communities may tend to adjust their physiology and metabolism and show transition from a planktonic stage to an irreversibly attached biofilm formation stage.²³

Fig. 6 Scanning electron microscopic images of biofilm formed on gravel samples (a) without added Ca²⁺ and (b) with added Ca²⁺.

3.4 Water quality analysis through the hybrid constructed wetland

The interaction of Ca²⁺ ions with naturally produced eDNA in soil (released through cell lysis) through cationic bridging not only promotes biofilm formation but also makes it thicker and denser with the passage of time as evidenced by the microscopic analysis (Fig. 3 to 5). Therefore, it is expected to play a critical role in contaminants removal from wastewater after passing through subsequent units of a HCW. It is also mandatory to mention here that the Ca²⁺ has no influence on the release of naturally produced eDNA, as the amount of eDNA measured before and after calcium addition remained static (1.8 μg eDNA per gram of soil). The comparative variations in physico-chemical and bacteriological parameters and their details with and without the addition of Ca²⁺ at 20 days HRT are discussed in the following section.

3.5 Physico-chemical analysis of wastewater before and after the addition of Ca²⁺

The pH values of domestic wastewater prior to treatment were recorded as 7.9, while after treatment through the HCW planted with T. portulacastrum, the pH value declined to 7.57, however upon addition of CaCO₃, it increased to 8.17. This increase in pH may be attributed to the dissolution of calcium carbonate that can increase the alkalinity (HCO₃⁻ + OH⁻) of water as follows;

CaCO₃ + H₂O → Ca²⁺ + OH⁻ + HCO₃

It is generally accepted that constructed wetlands are proficient in removing organic matter and nitrogen (N) compounds but are less efficient in removing phosphorous (P) compounds.²⁴ Generally, removal of phosphate in constructed wetlands depends upon various factors including the composition of the wastewater, peat/soil accretion (mostly in natural and free flow systems), soil adsorption, precipitation, plant uptake, microbial activities, the type of root media and the calcium, iron and calcium content of the substrate. The material used as a substrate (gravel, sand etc.) in the present study does not contain high concentrations of these elements; therefore the removal of phosphate is generally low. So, fill media with higher phosphorus-adsorption capacities are preferred as substrates in wetlands²⁵ so keeping in view the above-mentioned reason, commercially available limestone (CaCO₃) was added in the existing HCW. The concentration of calcium carbonate was varied from 50 to 120 mg L⁻¹ to obtain optimized treatment efficiencies for domestic wastewater as shown in Fig. S1 of the ESI.† The optimized concentration of calcium carbonate to achieve a pronounced/superior contaminant removal efficiency was 80 mg L⁻¹ as can be depicted from Fig. S1.† Therefore, from now onwards, we only discuss and compare results obtained without any added calcium and with an added calcium concentration of 80 mg L⁻¹. The concentration of phosphates in domestic wastewater before being subjected to HCW was measured as 2.3 mg L⁻¹. The concentration of phosphates decreased significantly (0.60 mg L⁻¹) in effluent with a removal efficiency of 73.69% after processing through a HCW planted with T. portulacastrum at 20 days hydraulic retention time. Further decrease in the phosphate concentration to 0.25 mg L⁻¹ was observed with the removal efficiency of 88.84% of its original value in raw wastewater through adding calcium in the HCW system (Tables 1 and 2). This may be attributed to chemical precipitation of phosphates with calcium upon the addition of limestone in the wetland system. Depending on pH, phosphates may be present in different forms in neutral wastewater such as H₃PO₄⁻, H₂PO₄⁻, HPO₄²⁻ and PO₄³⁻ in neutral wastewater. Calcium carbonate has an ability to serve as a phosphate binder under the following reaction mechanism:

2H₃PO₄ + 3CaCO₃ → (Ca₃PO₄)₂ + 3CO₃²⁻ + H⁺

2H⁺ + CO₃²⁻ → CO₂ + H₂O

Table 1 Comparative variation in the physico-chemical and microbiological analysis of domestic wastewater; before and after treatment [with and without the addition of Ca²⁺] through a HCW planted with T. portulacastrum at 20 days HRT. ± values represent standard deviations from multiple readings (n = 5)

Parameters	Concentration (influent/untreated domestic wastewater)	Concentration (effluent/after treatment)
		Without added (Ca^2+)^a	With added Ca^2+
a Ref. 11.
DO (mg l^−1)	1.55 ± 0.12	6.25 ± 0.9	8.0 ± 0.4
TDS (mg l^−1)	580.5 ± 3.41	150.93 ± 1.5	62.93 ± 2.1
TSS (mg l^−1)	480.7 ± 4.42	144.06 ± 2.3	83.06 ± 2.1
BOD5 (mg l^−1)	145.3 ± 0.68	40.18 ± 0.98	7.93 ± 1.2
COD (mg l^−1)	208.75 ± 1.32	38.62 ± 1.5	9.81 ± 1.4
EC (μS cm^−1)	614.5 ± 2.27	196.18 ± 2.1	98.66 ± 1.5
PO4^3− (mg l^−1)	2.3 ± 0.29	0.60 ± 0.2	0.25 ± 0.1
SO4^2− (mg l^−1)	2.5 ± 0.05	0.59 ± 0.3	0.23 ± 0.1
NO3^2− (mg l^−1)	2.87 ± 0.11	0.56 ± 0.5	0.21 ± 0.1
NO3^2− (mg l^−1)	2.59 ± 0.11	0.48 ± 0.8	0.13 ± 0.1
CFU per ml	1.54 × 10^10	5.10 × 10^3	52.5
MPN/100 ml	1630 MPN ± 1.15	265.5 ± 1.2	64.25 ± 1.5

---

Table 2 Comparison of the treatment efficiency of domestic wastewater in the absence and presence of added Ca²⁺ through a HCW planted with T. portulacastrum at 20 days HRT

Parameters	Treatment efficiency (%) of effluent
	Without added (Ca^2+)^a	With added Ca^2+
a Ref. 11.
DO (mg l^−1)	75.2	80.62
TDS (mg l^−1)	74	89.15
TSS (mg l^−1)	70.03	82.72
BOD5 (mg l^−1)	72.34	94.53
COD (mg l^−1)	80.67	95.29
EC (μS cm^−1)	68.07	83.93
PO4^3− (mg l^−1)	73.69	88.84
SO4^2− (mg l^−1)	76.59	90.65
NO3^2− (mg l^−1)	80.14	92.40
NO3^2− (mg l^−1)	81.46	94.88
CFU per ml	80.5	94.5
MPN/100 ml	83.71	96.05

---

The precipitation of calcium phosphate is an effective method to remove excess phosphates. The interaction of Ca²⁺ cations with dissolved phosphates in the water converts dissolved phosphate into a particulate form with the production of chemical precipitates having low solubility. These unwanted precipitates can settled down in the HCW due to gravitational effects followed by their flotation and finally filtration. Similar findings were reported previously in which calcium carbonate served as a potential phosphate binder to decrease the phosphate concentration in water from 1.49 to 0.4 mg L⁻¹ with an efficiency of 70%.²⁶

The effluent concentrations of TSS (83.06 mg L⁻¹) and TDS (62.93 mg L⁻¹) decreased with removal efficiencies of 82.72 and 89.15% respectively, when calcium carbonate was added in the HCW system. However, in the absence of calcium carbonate, the effluent concentrations for TSS and TDS were recorded as 144.06 mg L⁻¹ (70.03%) and 150.93 mg L⁻¹ (74%), respectively, as shown in Tables 1 and 2 Electrical conductivity (EC) is strongly dependent on the amount of dissolved and suspended solids with different ionic constituents in water. Therefore, a decisive trend (from 196.18 μS cm⁻¹ to 98.75 μS cm⁻¹) in EC values was also found for the case of calcium addition in the wetland system compared to its absence. The concentration of dissolved oxygen (DO) in untreated domestic wastewater was as low as 1.55 mg L⁻¹. However, a significant increment in its concentration was observed (6.25 mg L⁻¹) in the HCW planted with T. portulacastrum after 20 days HRT. By introducing calcium into CW, its concentration was further enhanced to 8 mg L⁻¹ with an efficiency of 80.62% compared to untreated wastewater. The extensive network of roots and root hairs of T. portulacastrum into the rhizosphere may induce more aeration to support the aerobic processes that could be a possible reason for the enhanced DO levels in the constructed wetland system. Besides, increased photosynthetic activity from utilization of carbon dioxide released due to calcium–phosphorous precipitation may be another possible reason for the enhanced DO levels. Similar trends were also reported in previous reports in which the dissolved oxygen content was increased significantly up to 7.2 mg L⁻¹ by using Caulerpa verticillata, during calcium carbonate and phosphate reduction in an aquatic ecosystem.²⁶ The increase in dissolved oxygen promotes enhanced biodegradation of organic compounds, as plant roots play an important role in de-aggregation of the soil by increasing microbial activation. Therefore a significant reduction in BOD₅ (7.93 mg L⁻¹) and COD (9.81 mg L⁻¹) was observed with the addition of calcium in the wetland system having removal efficiencies of 94.53% and 95.29%, respectively (Tables 1 and 2). The enhancement of biofilm in the presence of calcium (a large amount of the biofilm is not actively exposed to the organic substrate) could create favourable anoxic conditions to produce facultative micro-organisms which are generally considered to be responsible for COD degradation. The addition of calcium carbonate in wetland systems can serve as a source of carbon for biological denitrification.^27,28 This organic carbon could be utilized for heterotrophic denitrification and sulphate reduction within the wetland matrix resulting in the enhanced removal efficiency of BOD₅ and COD with a corresponding decrease in NO₃–N. This explains the decreases in the levels of NO₃⁻ (0.21 mg L⁻¹), NO₂⁻ (0.132 mg L⁻¹) and SO₄²⁻ (0.23 mg L⁻¹) which were observed in the wetland planted with T. portulacastrum with removal efficiencies of 92.4, 94.88% and 90.65%, respectively (Tables 1 and 2).

3.6 Microbial content of wastewater before and after the addition of Ca²⁺

The concentrations of microbial content in the untreated wastewater were 1.53 × 10¹⁰ CFU per ml and 1630 MPN index/100 ml. A decrease in the average number of bacteria (5.10 × 10³ CFU per ml) and faecal coliforms (265.5 MPN index/100 ml) was observed in wastewater after being subjected to the HCW planted with T. portulacastrum for 20 days HRT.¹¹ However upon the addition of calcium into the wetland system, further reduction in the average number of bacteria and faecal coliforms was observed i.e. 5.25 CFU per ml and 64.25 MPN index/100 ml after passing through subsequent units and the final polishing step of sand bed filtration (Table 1). This reduction in bacterial contaminants may be attributed to the adsorption of calcium.²⁹ The increase in the pH value of wastewater upon calcium addition in the HCW may have also resulted in the decrease in the bacterial content. Besides this, other mechanisms involved in bacterial removal in wetlands include filtration, sedimentation, aggregation, biofilm formation in the filter media of beds, oxidation, solar irradiation, antibiosis, competition, natural die-off and protozoa predation.³⁰

4. Conclusions

Herein, the role of a divalent cation (Ca²⁺) in promoting initial aggregation and biofilm progression with time was systematically investigated at the microscopic level. Light and scanning electron micrographs revealed the influential role of calcium in the growth and development of biofilms. A possible mechanism in which improved cationic bridging between Ca²⁺ and negatively charged eDNA form the basis of progressive biofilm growth was schematically proposed. Furthermore, as a proof of concept, calcium carbonate (as a source of calcium) was deliberately introduced in the lab-scale constructed wetland system to investigate its treatment (contaminant removal) efficiency and was compared with a control system (no extra added calcium). The presence of calcium in a wetland system significantly improved its efficiency compared to the control system in terms of the removal of various contaminants, which further supports our microscopic results. Hence, a similar approach could have potential applications in pilot scale wetland systems as well as other biofilm mediated wastewater treatment technologies.

Conflict of interest

The authors declare no financial or any other conflict of interest.

Acknowledgements

The authors would like to greatly acknowledge Dr Adnan Younis of the University of New South Wales (UNSW), Australia, for his contribution in the SEM-XDS analysis of agar coated glass slides for the detection of biofilm.

Notes and references

1. R. J. Doyle, Microbes Infect., 2000, 2, 391–400.
2. K. Sauer and A. K. Camper, J. Bacteriol., 2001, 183, 6579–6589.
3. J. D. Bryers and J. P. Ratner, Am. Soc. Microbiol. News, 2004, 70, 232–237.
4. S. Dobretsov, M. Teplitski and V. Paul, Biofouling, 2009, 25, 413–427.
5. M. E. Davey and G. A. O’ Tooley, MMBR, 2000, 64, 847–867.
6. E. D. L. Pulcini, Néphrologie, 2001, 22, 439–441.
7. M. J. Lehtola, M. Laxender, I. T. Miettinen, A. Hirvonen, T. Vartiainen and P. J. Martikainen, Water Res., 2006, 40, 2151–2160.
8. E. Larsen and M. Greenway, Water Sci. Technol., 2004, 49, 115–122.
9. J. L. Faulwetter, G. Vincent, S. Carina, C. Florent, D. B. Mark, B. Jacques, K. C. Anne and R. S. Otto, Ecol. Chem. Eng. A, 2009, 35, 987–1004.
10. L. Fan, H. Reti, W. Wang, Z. Lu and Z. Yang, J. Environ. Sci., 2008, 20, 1415–1422.
11. S. Sehar, Sumera, S. Naeem, I. Perveen, N. Ali and S. Ahmed, Ecol. Chem. Eng. A, 2015, 81, 62–69.
12. T. Das, S. Sehar, L. Koop, Y. K. Wong, S. Ahmed, K. S. Siddiqui and M. Manefield, PLoS One, 2014, 9(3), e91935.
13. American Public Health Association and A. D. Eaton, Water Environment Federation and American Water Works Association, Standard Methods for the Examination of Water and Wastewater, ed. A. D. Eaton, L. S. Clesceri, E. W. Rice, A. E. Greenberg and M. A. H. Franson, APHA-AWWA-WEF, Washington, DC, 21^st edn, 2005.
14. J. G. Holt, N. R. Krieg, P. H. A. Sneath, J. T. Ataley and S. T. Williams, Bergey’s Manual of Determinative Bacteriology, Lippincott, Williams and Wilkins Co., Baltimore, 9th edn, 1994, pp. 1860–1937.
15. S. Vilain, J. M. Pretorius, J. Theron and V. S. Brozel, AEM, 2009, 75, 2861–2868.
16. S. Molin and T. Tolker-Nielsen, Curr. Opin. Biotechnol., 2003, 14, 255–261.
17. C. D. Nadell, J. B. Xavier, S. A. Levin and K. R. Foster, PLoS Biol., 2008, 6(1), e14,  DOI:10.1371/journal.pbio.0060014.
18. H. C. Flemming and J. Wingender, Nat. Rev. Microbiol., 2010, 8, 623–633.
19. M. Maurer and M. Boller, Water Sci. Technol., 1999, 39, 147–163.
20. B. Parakash, B. M. Veeregowda and G. Krishnappa, Curr. Sci., 2003, 85, 1299–1307.
21. M. Hermansson, Colloids Surf., B, 1999, 14, 105–119.
22. N. P. Boks, W. Norde, H. C. Van der Mei and H. J. Busscher, Microbiology, 2008, 154, 3122–3133.
23. L. F. Cruz, P. A. Cobine and L. De La Fuente, Appl. Environ. Microbiol., 2012, 78, 1321–1331.
24. R. H. Kadlec and S. D. Wallace, Treatment wetlands, CRC Press, Boca Raton, Florida, 3rd edn, 2009.
25. J. Vymazal, Environ. Sci. Technol., 2011, 45, 61–69.
26. V. Yanamadala, Water Environ. Res., 2005, 77, 3003–3012.
27. J. Kern, in Constructed wetlands for wastewater treatment in cold climates, ed. U. Mander and P. D. Jenssen, 2003, pp. 197–214.
28. O. Dilly, J. Plant Nutr. Soil Sci., 2004, 167, 261–266.
29. W. D. Bellamy, D. W. Hendricks and G. S. Logsdon, J.–Am. Water Works Assoc., 1985, 77, 62–66.
30. J. Vymazal, Sci. Total Environ., 2007, 380, 48–65.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra21076c

---