Bioremediation of Metal-Polluted Industrial Wastewater with Algal-Bacterial Consortia: A Sustainable Strategy

Abstract

Aquatic pollution is a burning issue nowadays due to urbanization and industrialization. Industrial wastewater (IWW) contains pollutants that pose a great risk to the environment and human beings and is a big challenge for industries. The remediation of IWW by microorganisms is an environmentally friendly technique. This study was carried out to evaluate the pollution of IWW and to use consortia of Bacillus pakistanensis, Lysinibacillus composti, and Cladophora glomerata for bioremediation. The IWW was obtained from the Hayatabad Industrial Estate and was evaluated for physicochemical parameters and metal concentration. A pot experiment was carried out for two weeks to assess the efficiency of the developed consortia. The IWW and tap water (control) were treated with three different consortia (Bacillus pakistanensis-Cladophora glomerata (CT1, E1), Lysinibacillus composti-Cladophora glomerata (CT2, E2), and Bacillus pakistanensis-Lysinibacillus composti-Cladophora glomerata (CT3, E3). The three pots (CT1, CT2, and CT3) serving as the control were provided with tap water, and the three experimental pots (E1, E2, and E3) were provided with IWW. After treatment, substantial reductions were obtained in the following parameters and percentages: colour 85.7%, electrical conductivity (EC) 40.8%, turbidity 69.6%, sulphide 78.5%, fluoride 38.8%, chloride 62.9%, biological oxygen demand (BOD) 66%, chemical oxygen demand (COD) 81.8%, total suspended solids (TSSs) 82.7%, total dissolved solids (TDSs) 24.6%, Ca hardness 37.2%, Mg hardness 50%, and total hardness 39%. The samples of water were also examined for metal concentrations using atomic absorption spectrophotometry. The selected species removed 98.2% of Mn, 94% of Cu, 97.7% of Cr, 91.6% of Cd, 92.8% of Co, 79.6% of Ag, 82.6% of Ni, 98% of Ca, 90% of Mg, and 82.1% of Pb. The BCF values showed by the consortia for Mn, Cu, Cr, Cd, Co, Ag, Ni, Ca, Mg, and Pb were 91.8, 67, 97.5, 83.3, 85.7, 48.1, 80.4, 84.3, 82.5, and 80.3%, respectively. The t-test analysis showed that the treatment with the selected species significantly decreased the metal concentrations in the IWW (p ≤ 0.05). Overall, the study concludes that metal concentration in the water was decreased significantly by the consortia of algae-bacteria.

1. Introduction

Contamination of the water ecosystem with heavy metals (HMs) is nowadays a burning issue due to fast-growing urbanization and industrialization [1]. Different industries such as leather/tanning, electroplating, steel, textile, storage batteries, and mining release metals like cadmium (Cd), manganese (Mn), nickel (Ni), copper (Cu), cobalt (Co), silver (Ag), chromium (Cr), and lead (Pb) [2]. These metals are very toxic even if present in low quantities and are the main causes of water pollution. Water pollution produces enormous problems, including shortages of water for drinking and other basic activities or services in industries and households. The IWW containing metals also affects the groundwater quality as well as the fauna and flora of the ecosystem [3,4]. In elemental or combined forms, certain metals like Ni, Hg, Cd, Cr, and Pb are more lethal [5], as these contaminants leach into the water bodies and accumulate rapidly in human cells or tissues. The metals enter the human body through food ingestion, inhalation, and drinking water [6]. Due to the high degree of toxicity, accumulation, persistence in living cells, and easy transference in the aquatic environment, they result in serious toxicological effects on human health and aquatic life [7]. In previous research studies, HMs have been observed in the gills, muscles, and liver tissues of various fish in contaminated marine ecosystems [8], and they can accumulate in various organs of the human body when they enter the food chain [9]. The HMs above permissible limits may have adverse effects on living organisms and ecosystems [10]. The permissible limit of HMs in food items has been related to fewer human health menaces [11]. Therefore, metal removal from IWW is indispensable due to its disease-triggering nature, its alteration of the ecosystem, and to guarantee a good quality of ecological life and food [12].

Various biological, chemical, and physical techniques have been used to eliminate harmful contaminants from aquatic environments. These techniques include ion exchange, reverse osmosis, membrane filtration, flotation, flocculation and electrochemical treatment [13], adsorption, chemical precipitation [14], biosorption, solvent extraction, and activated sludge processes [15]. Most of these methods are not capable of removing metals completely, and they are very expensive, inefficient, and need energy for the metal removal [16]. This has led to the need for further research studies on the efficient remediation of IWW using resources that are environmentally friendly and economic [17].

Using algae-bacteria consortia for wastewater (WW) remediation is a green technology that has been utilized effectively to eliminate contaminants from the aquatic environment and has exhibited many benefits for the economy, energy, and ecosystem [18]. Recently, these algae-bacteria consortia have been widely studied in WW remediation, especially their mechanisms of biosorption and biodegradation, and they have invited great attention due to their capability to reduce inorganic pollutants. Bacteria existing in aquatic environments have a great tendency to co-occur in bioremediation [19,20]. Bacteria use oxygen released by the photosynthesis of the algal species, while the algae use carbon dioxide released by the bacterial species, leading to a symbiotic relationship [21]. This beneficial relationship between bacteria and algae makes these species the basic pillars of aquatic environments. The huge numbers of bacteria in water bodies build a bond with algal species and serve as a natural system of purification. The bond among algae and bacteria encloses the whole scope of beneficial relationships that are regarded as conceivable [22]. The consortium of microalgae and bacteria would appear to be a new perspective to pave the way not only for the bioremediation process but other techniques like pharmaceutical and biofuel production.

Previous studies on the Hayatabad Industrial Estate (HIE) showed higher levels of HMs in the groundwater, plants, and soil. The physiochemical parameters of, for example, nitrate, total suspended solids (TSSs), sulphate, total dissolved solids (TDSs), chloride, chemical oxygen demand (COD), hardness, and alkalinity were found to exceed their permissible limits. The pollution load of HIE wastewater has been researched intensively, but research studies on the potential techniques for HM removal in Pakistan have information gaps and are scant. Tariq et al. [23] evaluated the different industrial effluents of HIE but did not suggest any effective method for WW treatment. Industrial effluents of HIE used for irrigation purposes in vegetable growing areas were tested for their HM contents using AAS (atomic absorption spectrophotometry) [24]. Khan et al. [25] studied the impact of organic and inorganic amendments on the HM content of soil and wheat crop irrigated with the WW of HIE. Amin and Ahmad [26] investigated the pollution of soil with HMs from industrial effluents of HIE and their translocation in vegetables of Peshawar. Later, Khan et al. [27] conducted research on the remediation of IWW of HIE containing HMs with four freshwater algae. Similarly, Ayaz et al. [12] investigated the remediation of IWW using hydrophytes and compared the effect of mix (constructed wetland) and individual plants (pot experiments). Recently, Khan et al. [2] explored the pollution load of HIE and investigated the phycoremediation of WW using Cladophora glomerata and Vaucheria debaryana. To date, mainly the bioremediation studies employed for the management and remediation of IWW use monoculture and are contaminant specific, targeting one or two particular contaminants. However, IWW is complex in nature, comprising different organic and metals pollutants. Therefore, it is perceptible that a scheme which includes a number of microbes would be more appropriate for bioremediation of such WW, as it is almost impossible to find a single microbe that can remediate a mixture of different contaminants competently. Furthermore, the remediation of contaminants based on a mixture or consortium of microbes naturally present at the polluted sites could be more efficient, since the microorganisms adapt to those specific conditions. Therefore, the recent study aimed to develop novel consortia of algae-bacteria using native species of Pakistan and employed it for the remediation of IWW containing a mixture of pollutants including a large number of HMs. In the study, the locally available species Bacillus pakistnanesis was employed for the first time for bioremediation purposes.

2. Materials and Methods

2.1. Species Collection

The bacterial species Lysinibacillus composti (Gram positive) and Bacillus pakistanensis (Gram positive) were obtained from NARC (National Agricultural Research Centre), Islamabad, and Cladophora glomerata was collected from the river in Bajuar, Khyber Pakhtunkhwa. The bacteria were transported using a portable delAgua kit, while Cladophora glomerata was first washed with tap and then with distilled water to remove the clay, dust and sand. Using a microscopic procedure, the algal species was identified as adapted by Khan et al. [27]. The bacteria and algae were cultured in IWW of HIE for 14 days (light/dark 14:10 h and room temperature) [28]. After two weeks, the algal species were again analysed in the laboratory for HM concentration and physiochemical characteristics (presented in Supplementary Figure S1). The motive behind the selection of these algae and bacteria species is their rapid growth at high and low temperatures.

2.2. Industrial Effluent Collection

The study area (HIE) is located on latitudes 33°59′21” north and longitude 71.42252° or 71°25′21” east, which covers an area of about 868 acres. Peshawar is covered with consolidated deposits of gravel, sands and silt of recent geological times. The average temperature is 72.1 °F or 22.3 °C. Precipitation ranges from 817 mm to 32.2 inches per year.

The WW sample was obtained from the source where all industrial effluents fall into the main drain of HIE, Peshawar. The WW of HIE contains organic and inorganic pollutants [12]. The HIE has paper industries, as well as matches, incinerators, pharmaceuticals, plastics, steel, paint, and rubber, generating massive quantities of WW [27]. Before the experiment, the sample was analysed for BOD, total hardness, colour, chloride Mg hardness, pH, Ca hardness, TSS, turbidity, electric conductivity, fluoride, COD and TDS using standard methods [12]. Metals like Mg, Ca, Ag, Cd, Cr, Co, Cu, Ni, Pb and Mn were analysed using Atomic Absorption Spectrophotometer (AAS-700 PerkinElmer: Norwalk, Waltham, MA, USA) in the laboratory of the Environmental Protection Agency (EPA) at Peshawar [2].

2.3. Experimental Design

To investigate the efficiency of the algae-bacteria consortium for pollutants, pot experiments were performed. For this study, six pots, thoroughly washed with distilled water and dilute nitric acid, were utilized [28]. Three pots were serving as control having tap water and three treatment pots were provided with IE.

The pots labelled as CT1 and E1 were control and experimental pots for the Bacillus pakistanensis-Cladophora glomerata consortium, respectively. The pots CT2 and E2 were control and treatment for the Lysinibacillus composti-Cladophora glomerata consortium, respectively. Similarly, CT3 and E3 were control and treatment pots for the consortium of Bacillus pakistanensis-Lysinibacillus composti-Cladophora glomerata, respectively. Each control pot (CT1, CT2 and CT3) was provided with 500 mL tap water, and each experimental pot (E1, E2 and E3) was containing IE (500 mL). In each pot, 5 mL of respective bacterial culture and 5 g of algal specie were added. The consortia of the selected species were transplanted in water samples for 14 days.

2.4. Sample Preparation and Extraction of HMs from Algae-Bacteria Consortium

The biomass was dried in hot air and an oven for 48 h at 70 °C, and the dry weight was stored in polythene bags for further processing. For the extraction of HMs, the powder form of algae-bacteria and consortium were digested with 0.5 mL of concentrated nitric acid (HNO₃) in the digesting chamber as the protocol adapted by Khan et al. [27]. The double deionized distilled water (50 mL) was added to increase the resulting volume for further analysis. The final samples were investigated for metals by AAS in the Central Laboratory of the Environmental Protection Agency [12].

2.5. Water Analysis

Water samples (500 mL) from all six containers (CT1–CT3 and E1–E3) were collected after two weeks for further analysis. The samples collected were analysed for BOD, total hardness, colour, chloride Mg hardness, pH, Ca hardness, TSS, turbidity, electric conductivity, fluoride, COD and TDS using standard methods [12].

2.5.1. pH and Electric Conductivity (EC)

The pH and EC of the water samples were analysed using a pH meter (Model: PH110 Hong Kong, China) and EC meter (InoLab, Mexico City, Mexico), respectively.

2.5.2. Sulphide

The water sample (50 mL) was taken into the titration flask. Then, 2 mL of 6 normal HCl and 10 mL of iodide solution was added to the water sample. A few drops of starch indicator and sodium thiosulfate pentahydrate (H₁₀Na₂O₈S₂) were added to the water sample. Until the colour changed from brown to white, against water samples, the AgNO₃ was titrated [29].

2.5.3. Fluoride

A water sample (10 mL) was taken in a beaker. Then, 2 mL of reagent spadan was added to the water sample and thoroughly mixed for one minute. The prepared sample was then poured into the cell and placed in Spectro Direct, and the reading was noted [29].

2.5.4. Chloride

First, 50 mL of water sample was taken in a titration flask, and a few drops of potassium chromate (indicator) were added to it. Silver nitrate (AgNO₃) was placed in the burette, and it was titrated against water samples until the colour changed from yellow to reddish-brown [29]. The reading on the burette was noted as chloride contents in the water sample.

2.5.5. Calcium, Magnesium and Total Hardness

The total and calcium hardness was determined by a volumetric method known as the EDTA (ethylenediaminetetraacetic acid) titrimetric method. The water sample (10 mL) was taken in the titration flask to which ammonium chloride solution (2 mL) and a pinch of eriochrome black tea indicator were added. The EDTA standard solution was taken in the burette and was titrated against water samples until the colour disappeared [29].

For Ca hardness, a water sample (10 mL) was taken in a titration flask, and then NaOH solution (2 mL) and a pinch of murexide indicator were added. The EDTA standard solution was taken in the burette and was titrated against water samples until the colour disappeared [29]. The magnesium hardness was the difference between the total hardness and calcium hardness.

2.5.6. Colour and Turbidity

Colour and turbidity were measured using a Portable Water Analyzer (Model; WA 1). For calibration of the Portable Water Analyzer, standard solution was used. For the turbidity and coloration, 10 mL of WW from each container was taken. The solution in vials was wiped and shaken properly [29]. The reading on the analyser was noted.

2.5.7. Biological Oxygen Demand (BOD)

For the initial DO (dissolved oxygen), a water sample (200 mL) was taken in a titration flask, and 1 mL each of concentrated H₂SO₄, alkali iodide and MnSO₄ was added. Then, a few drops of the indicator (starch) were added. In the burette, the solution of Na₂S₂O₃ was taken. The Na₂S₂O₃ in the water sample was titrated until disappearance of the colour. In BOD bottles, 20 mL water samples were taken to store the samples for 5 days in an incubator at 20 °C. The same titration process was performed after 5 days for the final DO value. The difference between the initial and final DO is the BOD of the water sample [29].

BOD₅ (mg/L) = DO_i − DO_f

DO_i = initial DO., DO_f = final DO at 20 °C after 5 days of incubation.

2.5.8. Chemical Oxygen Demand (COD)

COD is the measure of water and WW quality. First of all, the sample blanks were prepared, and 2 mL of each sample was taken through a micropipette and poured in the COD reagent vial available in the market. The vial was inverted several times to mix it properly. The vial became hot during mixing. For blank preparation, the 2 mL of deionized water was poured into another COD reagent vial and mixed properly. The samples were then ready for digestion. Both the vials were placed into the COD reactor for digestion. The temperature was set at 150 °C for 2 h. After digestion, the samples were cooled. The blank vial was inserted into the adapter slowly with minimum shaking and placed into the spectrophotometer. The blank vial reading was noted as zero. The blank vial was removed from the adaptor, and the sample vial was inserted into the adaptor. The reading that appeared on the spectrophotometer was noted [30].

2.5.9. Total Suspended Solids (TSS)

Whatman No. 42 filter paper was dried in oven at 101 °C and then was cooled and weighed. On this filter paper, a 10 mL water sample was filtered and kept again in an oven for drying at 101 °C and then was cooled and weighed. The difference in initial weight (W_i) and final weight (W_f) of the filter paper was the value of TSS [31].

$$\mathrm{TSS}=\frac{\mathrm{Wf}-\mathrm{Wi} \times 1000}{\mathrm{water} \mathrm{sample} \left(\mathrm{mL}\right)}$$

Wi = filter paper’s initial weight, Wf = filter paper’s final weight, TSS = Total Dissolved Solids (TDS).

In the oven, a clean crucible was placed at 105 °C. Using a desiccator, the crucible was cooled and weighed. For evaporation, the filtered 10 mL water sample in the crucible was kept at 105 °C in the oven. Then, after cooling, the crucible was weighed in the desiccator. The difference in initial (W_i) and final crucible weight (W_f) was TDS [31].

$$\mathrm{TDS}=\frac{\mathrm{Wf}-\mathrm{Wi} \times 1000}{\mathrm{water} \mathrm{sample} \left(\mathrm{mL}\right)}$$

Wi = Crucible’s initial weight, Wf = Crucible’s final weight

2.5.10. HM Analysis

The samples were prepared for HM analysis. For this purpose, 25 mL of nitric acid and 75 mL of HCl were added to 50 mL of every sample, and wet digestion was performed for 24 h; after, that the makeup was completed with distilled water to 250 mL [29]. Through ASS, the samples were quantified for Mg, Ca, Ag, Cd, Cr, Co, Cu, Ni, Pb and Mn concentration [32].

2.6. Formula

2.6.1. Bioconcentration Factor (BCF) (%)

For metals accumulation from the water, the bacterial and algal efficiency was determined using the BCF formula [33].

$$BCF (\%)=\frac{C consortium }{C water}\times 100$$

(1)

where C consortium shows the metal concentration in algae-bacteria cells, and C water refers to the metals in water.

2.6.2. Bioremoval Efficiency (%)

Bioremoval efficiency was calculated using the equation mentioned below [2].

$$R=\frac{Ci-Cf}{Ci}\times 100$$

(2)

where R refers to the percentage of removal, Ci is the initial metal concentration in water, and C_f is the final metal concentration in water samples.

2.7. Statistical Analysis

The data were statistically analysed using various software like Sigma plot (version 14), Microsoft Excel (version 2013) and SPSS (Statistical Package for Social Science 16.0). The t-test was applied for significance difference (p) between the variables of the parameters.

3. Results and Discussion

3.1. Physiochemical Parameters of Water Sample

To assess the pollution load in the IE of HIE and in tap water, the samples were investigated for various physicochemical parameters, and the findings were compared with National Environmental Quality Standards (NEQS, 2008) [34] set by Pak-EPA. The results achieved on characteristics of IE and tap water are presented in

Table 1. Physiochemical parameters of water samples at initial and final stages.

Physiochemical Parameters		CT1		E1		CT2		E2		CT3		E3		PAK-EPA NEQS
		Mean	Ef. %	Mean	Ef. %	Mean	Ef. %	Mean	Ef. %	Mean	Ef. %	Mean	Ef. %
pH	I	7.3	−6.2	6.83	−14.0	7.3	−5.2	6.83	−13.9	7.3	−7.5	6.83	−11.4	6–10
	F	7.74		7.79		7.68		7.78		7.85		7.61
EC (mS/cm)	I	0.436	14.9	1.195	14.3	0.436	40.8	1.195	10.1	0.436	49.5	1.195	14.5	----
	F	0.371		1.023		0.258		1.074		0.22		1.021
Temperature	I	26.6	48.1	26.8	47.3	26.6	49.2	26.8	44	26.6	46.6	26.8	43.6	----
	F	13.8		14.1		13.5		15.0		14.2		15.1
BOD (mg/L)	I	04	25	85.5	66.0	04	50	85.5	57.8	04	50	85.5	54.3	80
	F	03		29		02		36		02		39
COD (mg/L)	I	05	20	921	81.8	05	40	921	76.0	05	40	921	78.3	150
	F	04		167		03		221		03		199
TSS (mg/L)	I	02	20	232	82.7	02	10.5	232	75.8	02	31.5	232	66.3	150
	F	1.6		40		1.79		56		1.53		78
TDS (mg/L)	I	288	5.2	794	24.3	288	24.6	794	10.5	288	17.7	794	14.3	3500
	F	273		601		217		610		237		680
Sulphide (mg/L)	I	0.4	50	5.6	78.5	0.4	35	5.6	75	0.4	35	5.6	78.5	1
	F	0.20		1.2		0.26		1.4		0.26		0.9
Colour (TCU)	I	0.345	85.5	440	84.3	0.345	15.9	440	85.7	0.34	70.5	440	85.6	<15
	F	0.05		68.91		0.29		62.72		0.10		63.10
Turbidity (NTU)	I	1.080	5.5	54.65	69.6	1.080	2.7	54.65	61.5	1.080	13	54.65	40.2	<5 NTU
	F	1.02		16.59		1.05		21.01		1.04		32.68
Fluoride (mg/L)	I	0.13	23	3.6	38.8	0.13	30.7	3.6	30.5	0.13	23.0	3.6	8.3	10
	F	0.10		2.2		0.09		2.5		0.10		3.3
Chloride (mg/L)	I	54	25.9	162	32.0	54	27.7	162	19.7	54	62.9	162	1.2	1000
	F	40		110		39		130		20		160
Calcium Hardness (mg/L)	I	280	14.2	590	11.8	280	17.8	590	37.2	280	7.1	590	28.8	----
	F	240		520		230		370		260		420
Magnesium Hardness (mg/L)	I	180	33.3	395	48.3	180	27.7	395	41.7	180	50	395	34.1	----
	F	120		110		130		230		90		260
Total Hardness (mg/L)	I	460	21.7	985	36.0	460	21.7	985	39.0	460	23.9	985	30.9	<500
	F	360		630		360		600		350		680

CT1: control for Bacillus pakistanensis-Cladophora glomerata consortium; E1: Experimental pot for Bacillus pakistanensis-Cladophora glomerata consortium CT2: control for Lysinibacillus composti-Cladophora glomerata consortium; E2: Experimental pot for Lysinibacillus composti-Cladophora glomerata consortium; CT3: control for Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium; E3: Experimental pot for Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium; I: initial; F: final; % ef.: % efficiency.

.

Temperature is a significant indicator of water quality with respect to the survival of aquatic organisms. The IE temperature depends on the progression of production in the industrial units. The temperature values of IE and tap water ranged from 26.6 to 26.8 °C. The temperature in all the samples was within the permissible limits of NEQS. The parameters analysed for IE such as COD (921 mg/L), TSS (232 mg/L), sulphide (5.6 mg/L), colour (440 TCU), turbidity (54.65 NTU) and total hardness (985 mg/L) were exceeding the maximum permissible limits set by Pak-EPA, while all other parameters were within permissible limits. Similarly, all the physiochemical parameters determined for tap water were also within permissible limits, as shown in Table 1. The results found on HM contents (Mg, Ca, Ag, Cd, Cr, Co, Cu, Ni, Pb and Mn) in IE and tap water are presented in

Table 2. Metal concentrations in water samples at initial and final stage.

Heavy Metals		CT1		E1		CT2		E2		CT3		E3		NEQS
		Mean	Ef. %	Mean	Ef. %	Mean	Ef. %	Mean	Ef. %	Mean	Ef. %	Mean	Ef. %
Mn	I	0.013	46.1	1.23	94.5	0.013	38.4	1.23	85.6	0.013	7.6	1.23	98.2	1.5
	F	0.007		0.067		0.008		0.176		0.012		0.021
Cu	I	0.010	20	0.81	94.0	0.010	60	0.81	92.0	0.010	40	0.81	52.3	1.0
	F	0.008		0.048		0.004		0.064		0.006		0.386
Cr	I	0.016	87.5	2.12	93.2	0.016	31.2	2.12	91.6	0.016	56.2	2.12	97.7	0.1
	F	0.002		0.144		0.011		0.177		0.007		0.047
Cd	I	0.012	75	0.18	77.7	0.012	66.6	0.18	75.5	0.012	91.6	0.18	76.6	0.1
	F	0.003		0.040		0.004		0.044		0.001		0.042
Co	I	0.014	92.8	0.151	26.4	0.014	28.5	0.151	47.6	0.014	50	0.151	28.4	---
	F	0.001		0.111		0.010		0.079		0.007		0.108
Ag	I	0.054	74.0	0.24	41.6	0.054	79.6	0.24	54.1	0.054	70.3	0.24	33.3	1.0
	F	0.014		0.14		0.011		0.11		0.016		0.16
Pb	I	0.002	50	1.12	82.1	0.002	50	1.12	22.3	0.002	50	1.12	79.4	0.5
	F	0.001		0.2		0.001		0.87		0.001		0.23
Ni	I	0.046	43.4	0.113	41.5	0.046	82.6	0.113	30.9	0.046	43.4	0.113	41.5	1.0
	F	0.026		0.066		0.008		0.078		0.026		0.066
Ca	I	1.72	98.0	14.5	79.0	1.72	52.3	14.5	59.8	1.72	76.3	14.5	82.7	---
	F	0.034		3.036		0.819		5.819		0.406		2.504
Mg	I	0.040	90	0.19	82.1	0.040	67.5	0.19	45.7	0.040	55	0.19	48.9	---
	F	0.004		0.034		0.013		0.103		0.018		0.097

CT1: control for Bacillus pakistanensis-Cladophora glomerata consortium; E1: Experimental pot for Bacillus pakistanensis-Cladophora glomerata consortium CT2: control for Lysinibacillus composti-Cladophora glomerata consortium; E2: Experimental pot for Lysinibacillus composti-Cladophora glomerata consortium; CT3: control for Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium; E3: Experimental pot for Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium; I: initial; F: final; % ef.: % efficiency.

. Results revealed that the concentrations of Cr (2.12), Cd (0.18) and Pb (1.12) were exceeding the permissible limits compared with the NEQS, while all other HMs were within permissible limits. The metal concentration analysed in tap water was very low and within permissible limits.

In the results, Table 1 revealed that the values of EC were significantly (p ≤ 0.05) decreased (10.1–40.8%), which could be related to precipitation and metal uptake of the consortia. TSS decreased, ranging from 10.5 to 82.7%. Similarly, the value of TDS was also reduced, ranging 5.2–24.6%. BOD and COD values were decreased, ranging 25–66% and 20–81.8%. Similarly, sulphide, fluoride and chloride were reduced in the ranges of 35–78.5%, 8.3–38.8% and 1.2–32%, respectively. Meanwhile, the removal efficiency of colour, turbidity, Ca hardness, Mg hardness and total hardness were decreased, ranging 15.9–85.6%, 2.7–69.6%, 7.1–28.8%, 27.7–5% and 21.7–39%, respectively. Mahmood et al. [35] evaluated the bioremediation of textile effluents by bacterial consortia and showed higher decreases in TSS (52.58%), pH (11.85%), COD (61.35%), EC (52.98%), chloride (46.42%), BOD (59.49%) and TDS (44.93%). All the values were in agreement with the present results, except the values of EC, chloride and TDS removal efficiencies were higher than the present findings. Similarly, Mubashar et al. [36] experimentally investigated the consortium of Enterobacter sp and Chlorella vulgaris for HM removal and decolorisation from WW and found similar results for colour (70%) and COD (74%) removal efficiency. Raza et al. [37] investigated the bioremediation of textile WW with a microalgal-bacterial consortium and found 91.5% chemical oxygen demand (COD) and 41.54% of colour removal. Similarly, Das et al. [38] investigated the efficient WW remediation by consortium of Phormidium sp. and Chlorella sp. and strain of these species and found a reduction in BOD and COD by ≥90% and TDS by >50% in the consortium and also reported that the efficiency of consortium was higher compared to individual species. The variation in the results can be attributed to the difference in consortium used in both the studies.

3.2. Heavy Metal Analysis

3.2.1. Manganese (Mn)

Concentrations of Mn tested at the initial point for tap and WW samples were 0.013 and 1.23 mg/L, respectively. At final point, the Mn concentrations were ranging from 0.007 to 0.012 mg/L for the control pots and from 0.021 to 0.176 mg/L for the experimental water samples, as presented in Table 2. The study found 46.1, 94.5, 38.4, 85.6, 7.6, and 98.2% decreases in the final samples of water obtained from CT1, E1, CT2, E2, CT3, and E3, respectively. The Mn highest removal efficiency was revealed by E3, while the lowest was for CT3 (Figure 1). Mahmood et al. [35] evaluated the bioremediation of textile effluents by bacterial consortia and found similar results (88.3%) for Mn removal efficiency by bacterial consortia. Orandi et al. [39] conducted a study on the biofilm establishment and removal capacity of HM by an algal-microbial consortium and determined much lower results for Mn removal (40–45%).

The Mn concentrations detected in the biomass collected from CT1, E1, CT2, E2, CT3, and E3 were 0.0081, 0.74, 0.0062, 0.93, 0.0015 and 1.13 mg/g respectively (

Table 3. Metal concentrations in consortium.

Species	Mn	Cu	Cr	Cd	Co	Ag	Pb	Ni	Ca	Mg
CT1	0.0081	0.0023	0.012	0.0080	0.012	0.023	0.0009	0.019	1.451	0.033
E1	0.74	0.543	1.756	0.142	0.039	0.062	0.90	0.044	9.77	0.151
CT2	0.0062	0.0053	0.005	0.0071	0.0039	0.026	0.0009	0.037	0.901	0.025
E2	0.93	0.470	1.613	0.131	0.071	0.084	0.21	0.030	8.01	0.084
CT3	0.0015	0.0036	0.008	0.010	0.0069	0.005	0.0008	0.018	1.093	0.020
E3	1.13	0.297	2.068	0.135	0.041	0.044	0.85	0.039	7.41	0.086

). The maximum Mg uptake in control pots was recorded in CT1, and in experimental pots, the highest Mg concentration was noted in E3. The BCF of Mn were determined as 62.3, 60.1, 47.6, 75.6, 11.5 and 91.8%, respectively, for CT1, E1, CT2, E2, CT3, and E3. E3 had the highest BCF, and CT3 had the lowest BCF (Figure 1).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that Bacillus pakistanensis-Cladophora glomerata (CT1) was the most efficient consortium. The results confirmed that at lower Mn concentration (tap water), the consortium of Bacillus pakistanensis-Cladophora glomerata can efficiently remove Mn from water samples. Meanwhile, E3 (Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium) was efficient for higher Mn concentrations (wastewater) from the comparison of experimental pots (E1, E2 and E3). Overall results revealed that the experimental group consortia are more effective than the control groups, suggesting that all consortia are more effective at higher concentrations of Mn (Figure 1).

3.2.2. Copper (Cu)

The Cu levels found at the start point of the experiment for control and experimental samples of water were 0.010 and 0.81 mg/L, respectively. At the end of the experiment, the Cu level was ranging from 0.004 to 0.008 mg/L for the control samples and 0.048 to 0.386 mg/L for the experimental samples. The Cu highest values were noted in E3 for experimental water and in CT1 for the control samples. The study found a 20, 94, 60, 92, 40, and 52.3% decrease in the final samples in CT1, E1, CT2, E2, CT3, and E3, respectively. The highest Cu removal efficiency was detected in E1, whereas the lowest was detected in CT1 (Figure 2). Orandi et al. [39] conducted a study on the biofilm establishment and HM removal capacity of an indigenous algal-microbial consortium and determined much lower results for Cu removal (50%). The results of the Cu removal determined in the present study are in agreement with the results (92.3%) of Mahmood et al. [35]. Mubashar et al. [36] experimentally investigated the consortium of Enterobacter spp. and Chlorella vulgaris for HM removal from WW and found similar results for Cu removal (72% decreases). However, Allam [40] found similar removal efficiency results for Cu (95%) while conducting a comparative study on the bioremediation efficiency of HMs and dyes by an individual or consortium of bacteria (Bacillus subtilis, Rhizobium radiobacter and Sphingomonas paucimobilis).

The Cu concentration detected in CT1, E1, CT2, E2, CT3, and E3 biomass were 0.0023, 0.543, 0.0053, 0.470, 0.0036 and 0.297 mg/g, respectively (Table 3). The Cu highest uptake in control pots was tested in CT2, and in experimental pots, the highest Cu uptake was investigated in E1. The BCF values of Cu determined for CT1, E1, CT2, E2, CT3 and E3 were 23, 67, 53, 58, 36 and 36.6%, respectively. E1 recorded the highest value, and CT1 recorded the lowest value (Figure 2).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) revealed that Lysinibacillus composti-Cladophora glomerata (CT2) was the most efficient consortium. The results confirmed that at lower Cu concentration (tap water), the consortium of Lysinibacillus composti-Cladophora glomerata can efficiently remove Cu from water samples. Meanwhile, E1 (Bacillus pakistanensis-Cladophora glomerata) is efficient for Cu removal from the comparison of experimental pots (E1, E2 and E3). The overall results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Cu (Figure 2).

3.2.3. Chromium (Cr)

The Cr levels, tested at the start point of the experiment for the control and experimental water samples, were 0.016 and 2.12 mg/L, respectively. At final points, the Cr levels were ranging from 0.002 to 0.011 mg/L for the control samples and from 0.047 to 1.77 mg/L for the experimental samples. The maximum values were noted in E2 for experimental water, while they were noted in CT2 for the control samples. The research found 87.5, 93.2, 31.2, 91.6, 56.2, and 97.7% decreases in the final samples for CT1, E1, CT2, E2, CT3, and E3, respectively. The highest Cr removal efficiency was determined in E3, and the lowest was in CT2 (Figure 3). Mahmood et al. [35] determined a similar result for Cr removal (83.52%) while evaluating the bioremediation of textile effluents by bacterial consortia. Mubashar et al. [36] experimentally investigated the consortium of Enterobacter sp. and Chlorella vulgaris for HM removal from WW and found similar results for Cr removal (79% decreases). However, Allam [40] found much higher removal efficiency results for Cr (97%) while conducting a comparative study on the bioremediation efficiency of HMs and dyes by an individual or consortium of bacteria (Bacillus subtilis, Rhizobium radiobacter and Sphingomonas paucimobilis). The variation in the findings can be credited to the dissimilarity in the species used for both the studies. Das et al. [38] investigated the efficient remediation of WW by the consortium of Phormidium spp. and Chlorella spp. and determined that the removal efficiency for Cr was ranging from 90.17 to 94.45%.

The Cr levels obtained in CT1, E1, CT2, E2, CT3 and E3 biomass were 0.012, 1.756, 0.005, 1.613, 0.008 and 2.068 mg/g, respectively (Table 3). The Cr highest uptake in control pots was investigated in CT1, and in experimental pots, the highest Cr uptake was obtained in E3. The BCF values of Cr determined for CT1, E1, CT2, E2, CT3 and E3 were 75, 82.8, 31.5, 76, 36 and 97.5%, respectively. E3 had the highest BCE, and CT2 had the lowest BCF (Figure 3).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that Bacillus pakistanensis-Cladophora glomerata (CT1) was the most efficient consortium. The results confirmed that at lower Cr concentrations (tap water), the consortium of Bacillus pakistanensis-Cladophora glomerata can efficiently remove Cr from water samples. Meanwhile, E3 (Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium) was found to be efficient for Cr removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Cr (Figure 3).

3.2.4. Cadmium (Cd)

The Cd levels determined at start point for the control and experimental water samples were 0.012 and 0.18 mg/L, respectively. At final points, the levels were ranging from 0.001 to 0.004 mg/L and 0.040 to 0.044 mg/L for the control and experimental samples, respectively. The highest results were determined in E2 for experimental water and in CT2 for the control samples. The research study detected reductions of 70, 77.7, 66.6, 75.5, 91.6, and 76.6% in the final samples of CT1, E1, CT2, E2, CT3, and E3, respectively. The Cd highest removal efficiency was noted in CT3, whereas the lowest was noted in CT1 (Figure 4). However, Abdel-Razek et al. [41] conducted a study on a consortium of cyanobacteria and microalgae and determined its ability to uptake Pb up to 88% efficacy. Mahmood et al. [35] assessed the bioremediation of industrial effluents using bacterial consortia and found similar results (89.46%) for Cd removal efficiency. Previously, a research study was conducted by Mubashar et al. [36] that experimentally investigated the consortium of Enterobacter spp. and Chlorella vulgaris for Cd removal from WW and found higher results (93% decrease) from the present study. The difference in the findings can be credited to the dissimilarity in the used consortia in both research studies. Allam [40] carried out a comparative study on the bioremediation efficiency of HMs and dyes by individuals or consortia of bacteria (Bacillus subtilis, Rhizobium radiobacter and Sphingomonas paucimobilis) and found much higher removal efficiency results for Cd (96%). Yu et al. [42] investigated the effects of algal-bacterial ratio on the Cd accumulation of Bacillus subtilis-Chlorella salina consortium and determined a lower value of Cd removal (51.66%) than the values in the present study.

The Cd concentrations observed in CT1, E1, CT2, E2, CT3, and E3 biomass were 0.0080, 0.142, 0.0071, 0.131, 0.010 and 0.135 mg/g respectively (Table 3). The maximum Cd uptake in control pots was determined in CT3, and in experimental pots, the highest Cd uptake was obtained in E1. The BCF values investigated for CT1, E1, CT2, E2, CT3, and E3 for Cd were 66.6, 78.8, 59.1, 72.7, 83.3 and 75%, respectively. CT3 was found to have the highest BCF, and CT2 was found to have the lowest BCF (Figure 4).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that the Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium (CT3) was the most efficient consortium. The results confirmed that at lower Cd concentration (tap water), the Bacillus pakistanensis, Lysinibacillus composti and Cladophora glomerata consortium can efficiently remove Cd from water samples. Meanwhile, E1 (Bacillus pakistanensis-Cladophora glomerata consortium) was found to be efficient for Cd removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Cd (Figure 4).

3.2.5. Cobalt (Co)

The Co concentrations tested at the primary point for control and experimental water were 0.014 and 0.151 mg/L, respectively. At final points, the values were ranging from 0.001 to 0.010 mg/L for control and 0.079 to 0.112 mg/L for the experimental samples. The maximum value was obtained in E1 for the experimental water and in CT2 for the control. The study determined 92.8, 26.4, 28.5, 47.6, 50, and 24.8% reductions in final samples of CT1, E1, CT2, E2, CT3, and E3, respectively. The Co highest removal efficiency was noted in CT1, whereas the lowest was in E1 (Figure 5). Water and WW containing cobalt ions have to be cleaned before discharging to the environment. Bioadsorption can be a great choice for this purpose. Previously, the potential of red algae with the species Gracilaria corticata was examined by Raju et al. [43]. The study suggested that the maximum removal efficiency of cobalt could be up to 96.57% at pH 4.87 with an initial cobalt concentration of 19.77 mg/L. A study by Vafajoo et al. [44] assessed the removal of cobalt from solution by either modified or raw algal species.

The Co levels determined in CT1, E1, CT2, E2, CT3 and E3 biomass were 0.012, 0.039, 0.0039, 0.071, 0.0069 and 0.041 mg/g respectively (Table 3). The Co highest uptake in control pots was recorded in CT1, and in experimental pots, the highest uptake was observed in E1. The BCF values obtained for CT1, E1, CT2, E2, CT3 and E3 for Co were 85.7, 25.8, 27.8, 47, 49.2 and 27.1%, respectively. CT1 was found to have the highest BCF, and E1 was found to have the lowest BCF (Figure 5).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that the Bacillus pakistanensis-Cladophora glomerata consortium (CT1) was the most efficient consortium. The results confirmed that at lower Co concentration (tap water), the consortium of Bacillus pakistanensis-Cladophora glomerata consortium can efficiently remove Co from water samples. Meanwhile, E2 (Lysinibacillus composti-Cladophora glomerata) is efficient for Co removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Co (Figure 5).

3.2.6. Silver (Ag)

The levels of Ag at the initial point for control and experimental water were 0.054 and 0.24 mg/L, respectively. At final points, the Ag concentrations were ranging from 0.011 to 0.016 mg/L for the control samples and from 0.11 to 0.16 mg/L for the experimental group water samples. The maximum value was noted in E3 for the experimental water, while in CT2, it was noted for the control. The study determined 74, 41.6, 79.6, 54.1, 70.3, and 33.3% decreases in the final samples of CT1, E1, CT2, E2, CT3, and E3, respectively. The Ag highest removal efficiency was determined in CT2, whereas the lowest was in E3 (Figure 6). The present Ag removal efficiency is not in agreement with the results (85.1%) of Ali et al. [45] in a study conducted on HM bioremediation from WW by Lysinibacillus composti and Bacillus pakistanensis. The difference in the results may be attributed to the variation in bioremediators in both studies.

The concentrations of Ag investigated in CT1, E1, CT2, E2, CT3 and E3 biomass were 0.023, 0.062, 0.026, 0.084, 0.005 and 0.044 mg/g, respectively (Table 3). The highest Ag uptake in control containers was recorded in C4, and in treatment, the highest Ag uptake was recorded in T3. The BCF values determined for CT1, E1, CT2, E2, CT3 and E3 for Ag were 42.5, 25.8, 48.1, 35, 9.2 and 18.3%, respectively. CT2 had the highest BCF, and CT3 had the lowest BCF (Figure 6)

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that the Lysinibacillus composti-Cladophora glomerata consortium (CT2) was the most efficient consortium. The results confirmed that at lower Ag concentration (tap water), the Lysinibacillus composti-Cladophora glomerata consortium can efficiently remove Ag from water samples. Meanwhile, E2 (Lysinibacillus composti-Cladophora glomerata) is efficient for Ag removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Ag (Figure 6).

3.2.7. Lead (Pb)

The concentrations of Pb noted at the initial point for control and experimental water were 0.002 and 1.12 mg/L, respectively. At the final point, the values were ranging 0.001–0.001 mg/L and 0.20–0.87 mg/L for the control and experimental water samples, respectively. The highest results were obtained in E2 for the experimental water. The study determined 50, 82.1, 50, 22.3, 50, and 79.4% decreases in the final samples of CT1, E1, CT2, E2, CT3 and E3, respectively. The Pb highest removal efficiency was noted in E1, whereas the lowest was noted in E2 (Figure 7). Previously, Abdel-Razek et al. [40] conducted a study on the bioremediation of selected HMs in WW from different sources using a consortium of cyanobacteria and microalgae and demonstrated the consortium ability to uptake Pb up to 89% efficacy. However, Mahmood et al. [35] evaluated the bioremediation of WW by bacterial consortia and found different results (93.5%) for Pb removal. Previously, a research study was conducted by Mubashar et al. [36] and experimentally investigated the consortium of Enterobacter sp and Chlorella vulgaris for Cd removal from WW and found similar results (79% decrease). Allam [40] carried out a comparative study on the bioremediation efficiency of HMs and dyes by individual bacteria or a consortium of bacteria (Bacillus subtilis, Rhizobium radiobacter and Sphingomonas paucimobilis) and found much higher removal efficiency results for Pb (98%). The variation in the results can be ascribed to the difference in the species used for both the studies.

The levels of Pb analysed in CT1, E1, CT2, E2, CT3 and E3 biomass were 0.0009, 0.90, 0.0009, 0.21, 0.0008 and 0.85 mg/g, respectively (Table 3). The Pb highest uptake in control pots was observed in CT1 and CT2, and in experimental pots, the Pb highest uptake was observed in E1. The BCF values obtained for CT1, E1, CT2, E2, CT3 and E3 for Pb were 45, 80.3, 45, 18.7, 40 and 75.8%, respectively. T3 had the highest BCF, and T5 had the lowest BCF (Figure 7).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that the Lysinibacillus composti-Cladophora glomerata consortium (CT2) was the most efficient consortium. The results confirmed that at lower Pb concentrations (tap water), the Lysinibacillus composti-Cladophora glomerata consortium can efficiently remove Pb from water samples. Meanwhile, E1 (Bacillus pakistanensis-Cladophora glomerata) is efficient for Pb removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Pb (Figure 7).

3.2.8. Nickel (Ni)

The concentrations of Ni determined at the initial point for control and experimental water were 0.046 and 0.113 mg/L, respectively. At final points, the results were ranging 0.008–0.026 mg/L and 0.066–0.078 mg/L for the control and experimental samples, respectively. The highest findings were recorded in E2 for the experimental water, while they were recorded in CT1 and CT3 for the control samples. The study determined 43.3, 41.5, 82.6, 30.9, 43.4, and 41.5% decreases in the final samples CT1, E1, CT2, E2, CT3 and E3, respectively. The highest Ni removal efficiency was noted in CT2, whereas the lowest was in E2 (Figure 8). The present results are not in agreement with the results of Abdel-Razek et al. [41]. Their study was conducted on the bioremediation of selected HMs in WW from different sources using a consortium of cyanobacteria and microalgae and found that the consortium was able to bioaccumulate Ni up to 95% efficacy. Mahmood et al. [35] analysed the bioremediation of WW by a consortia of bacteria and recorded similar results (80.7%) for Ni removal. Orandi et al. [39] conducted a study on the biofilm and HM removal capacity of algal-microbial consortium and determined much lower results for Ni removal (50%).

The Ni levels assessed in CT1, E1, CT2, E2, CT3 and E3 biomass were 0.019, 0.044, 0.037, 0.030, 0.018 and 0.039 mg/g respectively (Table 3). The highest Ni uptake in control pots was recorded in CT3, and in experimental samples, the highest Ni uptake was obtained in E1. The BCF values calculated for CT1, E1, CT2, E2, CT3 and E3 for Ni were 41.3, 38.9, 80.4, 26.5, 39.1 and 34.5%, respectively. CT2 had the highest BCF, and E2 had the lowest BCF (Figure 8).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that the Lysinibacillus composti-Cladophora glomerata consortium (CT2) was the most efficient consortium. The results confirmed that at lower Ni concentrations (tap water), the Lysinibacillus composti-Cladophora glomerata consortium can efficiently remove Ni from water samples. Meanwhile, E1 (Bacillus pakistanensis-Cladophora glomerata) is efficient for Ni removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Ni (Figure 8).

3.2.9. Calcium (Ca)

The Ca levels investigated at the initial point for the control and experimental water were 1.72 and 14.5 mg/L, respectively. At final points, the values were ranging from 0.034 to 0.819 for the control samples and from 2.504 to 5.819 mg/L for the experimental samples. The highest values were determined in E2 for the experimental samples, while they were in CT2 for the control samples. The study obtained 98, 79, 52.3, 59.8, 76.3, and 82.7% decreases in the final samples of CT1, E1, CT2, E2, CT3 and E3, respectively. The highest Ca removal efficiency was noted in CT1, whereas the lowest was noted in CT2 (Figure 9). The present Ca removal efficiency is not in agreement with the results (98.8%) of Ali et al. [45], which was a study conducted on HM bioremediation from WW by Lysinibacillus composti and Bacillus pakistanensis. The difference in the results may be attributed to the variation in bioremediators in both studies. Kehinde et al. [46] conducted a study on the bioremediation of WW from the pharmaceutical industry using Bacillus subtilis and examined a maximum of 68.75% calcium removal efficiency.

The concentrations of Ca determined in CT1, E1, CT2, E2, CT3 and E3 biomass were 1.451, 9.77, 0.901, 8.01, 1.093 and 7.41 mg/g respectively (Table 3). The highest Ca uptake in control pots was obtained in CT1, and in experimental pots, the highest Ca uptake was recorded in E1. The BCF values calculated for CT1, E1, CT2, E2, CT3 and E3 for Ca were 84.3, 67.3, 52.3, 55.2, 63.5 and 51.1%, respectively. CT1 had the highest BCF, and CT2 had the lowest BCF (Figure 9).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that the Bacillus pakistanensis-Cladophora glomerata consortium (CT1) was the most efficient consortium. The results confirmed that at lower Ca concentrations (tap water), the Bacillus pakistanensis-Cladophora glomerata consortium can efficiently remove Ca from water samples. Meanwhile, E1 (Bacillus pakistanensis-Cladophora glomerata) is efficient for Ca removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Ca (Figure 9).

3.2.10. Magnesium (Mg)

The levels of Mg noted at the initial stage for control and experimental water were 0.040 and 0.19 mg/L, respectively. At final points, the values were ranging from 0.004 to 0.018 mg/L for control and from 0.034 to 0.103 mg/L for the experimental samples. The maximum values were determined in E2 for the experimental water and in CT3 for the control samples. The study found 90, 82.1, 67.5, 45.7, 55, and 48.9% decreases in water samples of CT1, E1, CT2, E2, CT3 and E3, respectively. The highest Mg removal efficiency was found in CT1, and the lowest was found in E2 (Figure 10). The present Mg removal efficiency is not in agreement with the results (91.5%) of Ali et al. [45], which was a study conducted on HM bioremediation from WW by Lysinibacillus composti and Bacillus pakistanensis. The difference in the results may be attributed to the variation in bioremediators in both studies. Kehinde et al. [46] carried out a study on the bioremediation of WW from the pharmaceutical industry using Bacillus subtilis and investigated a maximum of 77.33% magnesium removal efficiency.

The concentrations of Mg analysed in CT1, E1, CT2, E2, CT3 and E3 biomass were 0.033, 0.151, 0.025, 0.084, 0.020 and 0.086 mg/g respectively (Table 3). The highest Mg uptake in control pots was recorded in CT1, and in experimental pots, the Mg highest uptake was determined in E1. The BCF values determined for CT1, E1, CT2, E2, CT3 and E3 for Mg were 82.5, 79.4, 62.5, 44.2, 50 and 45.2% respectively. CT1 had the highest BCF, and E2 had the lowest BCF (Figure 10).

The comparative study of bioconcentration factor and bioremoval efficiency for the control samples (CT1, CT2, CT3) showed that the Bacillus pakistanensis-Cladophora glomerata consortium (CT1) was the most efficient consortium. The results confirmed that at lower Mg concentrations (tap water), the Bacillus pakistanensis-Cladophora glomerata consortium can efficiently remove Mg from water samples. Meanwhile, E1 (Bacillus pakistanensis-Cladophora glomerata) is efficient for Mg removal from the comparison of experimental pots (E1, E2 and E3). Overall, the results revealed that the experimental group consortia are more effective than the control samples, suggesting that all consortia are more effective at higher levels of Mg (Figure 10).

The study showed that the consortium of Bacillus pakistanensis-Cladophora glomerata has higher removal efficiently in tap water (Mn, Cr, Co and Mg) and IWW (Cu, Cd, Pb, Ni and Mg). Therefore, these consortia can be best employed for the bioremediation of water samples contaminated with Mn, Cr, Co, Mg, Cd, Pb and Ni. Similarly, the consortium of Lysinibacillus composti-Cladophora glomerata has the highest Cu, Ag, Pb and Ni removal efficiency from tap water, while in WW, the highest removal efficiency was shown for Co and Ag. So, the consortium can be effectively used for the treatment of water having metals like Cu, Ag, Pb and Ni. The consortium of Bacillus pakistanensis-Lysinibacillus composti-Cladophora glomerata was good for Cd removal from tap water, while it showed high removal efficiency of Mn, Cr and Ca from WW. Therefore, the treatment of water samples polluted with Cd, Mn, Cr and Ca can be carried out with the help of Bacillus pakistanensis-Lysinibacillus composti-Cladophora glomerata consortium. By comparing the efficiency of these three types of consortia, it was observed that for most of the metallic elements, the Bacillus pakistanensis-Cladophora glomerata consortium showed maximum removal efficiency. The previous studies also support and confirm the findings of the current study. Benazir et al. [47] investigated Cr bioremediation and found 99.6 and 97.2% Cr removal efficiency by the consortia of P. aeruginosa-B. subtilis and S. cerevisiae-B. subtilis, respectively. Shashirekhaet al. [48] conducted a study on the bioremediation of tannery WW using a consortium of blue-green algal species and found a considerable decrease in Cr and physicochemical parameters values. In a previous study, the consortium of immobilized bacteria used for bioremediation showed a drastic reduction in the HMs, TDS, TSS and COD after six months of treatment [49]. Sen et al. [50] investigated a bacterial consortium isolated from the hot spring and studied its contribution in Cd and Pb removal. The results of Sen et al. [50] indicated that the consortium of thermophilic isolates removed HMs more effectively than the individual isolates and found 93 and 92% adsorption of Pb and Cd, respectively. Hamid et al. [51] developed an efficient bacterial consortium for the bioremediation of textile effluents and found a significant reduction in colour (90%), COD (70%), Pb (83%), Cu (98%) and Cr (70%). In a previous report by Oaikhena et al. [52], the Cr, Ni and Cd concentrations were 33.14, 73.91 and 100% decreased by a mixed culture consortium (Klebsiella pneumonia, Proteus vulgari, E. coli, Staphylococcus aureus and Pseudomonas aeruginosa) and confirmed that the consortium of the selected species was more effective in metal removal comparing individual species. Similarly, Wan et al. [53] reported a novel Mn-oxidizing bacterial consortium (Sphingobacterium and Bacillus) that presented good performance for Mn(II) removal under different conditions. Naaz et al. [54] explored the HM uptake potential of three algal strains/consortia and observed that the common HMs that were efficiently uptaken by the consortia were Zn, Pb, Cu and Cd (>55%). Chandrashekharaiah et al. [55] observed that the consortium of algae-bacteria (Scenedesmus acutus, Chlorella pyrenoidosa, Bacillus and Micrococcus sp) can improve HM remediation. Such a consortium was constructed which can efficiently remove and tolerate +1.5 ppm Cd²⁺ and 200 ppm Pb²⁺, and it was found that the individual consortia showed a higher bioremediation potential when species were mixed in equal quantities. In another study, Anusha et al. [56] explored the bioremediation of contaminants from polluted sewage WW using a bacterial strain of Bacillus cereus consortium with other bacteria, and after 15 days of treatment, a substantial reduction was observed in hardness (67.55%) and calcium (93.33%).

4. Conclusions

The synergistic relationships of Cladophora glomerata with bacteria Bacillus pakistanensis and Lysinibacillus composti have a key role in the metal removal from water samples. The concentrations of HMs in the WW and tap water samples are reduced significantly. The Bacillus pakistanensis-Cladophora glomerata consortium has the best removal efficiency in the control (CT1) for Cr, Co, and Ca, while in experimental groups (E1), it has the best removal efficiency for Ca, Ni, Pb, Cd, Cu and Mg. The Lysinibacillus composti-Cladophora glomerata has the best removal efficiency in treatment (E2) for Ag and Co. Similarly, control of the consortium (CT2) showed the best removal efficiency for Ni, Pb, Cu and Ag. The consortium of Bacillus pakistanensis-Lysinibacillus composti-Cladophora glomerata has significant removal efficiency in control (CT3) for Cd, while in experimental groups (E3), it has significant removal efficiency for Cr and Mn. Overall, the metals concentration in water has decreased significantly by the consortia of algae-bacteria. The study concludes that algal species C. glomerata and bacterial species Bacillus pakistanensis and Lysinibacillus composti consortia are more competent in metal uptake. These species can survive in conditions of stress triggered by HM concentrations. Therefore, this quality can be encouraging evidence for these algal and bacterial species to be utilized for the treatment of industrially contaminated water in a consortium or individually. Thus, bioremediation is an environmentally friendly and cost-effective technique that can be used for industrially polluted WW remediation.