The accumulation of the heavy metals in wastewater depends on many local factors, such as the type of industries in the region, way of life and awareness of the impact on the environment through the careless disposal of wastes
[23, 33]. Being a country with extensive industrialisation, water pollution by metal ions has emerged as one of the serious challenges currently faced by water service authorities in South Africa. Hence, this study focused on the chemical characteristics of South African industrial wastewater samples collected from one mining area at Witbank, Mpumalanga, and assessed their effect on the growth of selected bacterial and protozoan species that are among the dynamic population of wastewater and reported to be tolerant to heavy metals
[21, 34, 35].
The finding of the present study revealed that the industrial wastewater had COD concentrations above the South African permissible limit of 75 mg/l. The pH, Mn, Pb, Cu, Zn and Cd values were also found to be beyond the South African permissible limits of 5.5 to 9.5, 0.1 mg/l, 0.01 mg/l, 0.01 mg/l, 0.1 mg/l and 0.005 mg/l, respectively. Although previous reports revealed that metals such as Co, Ni, V, Ti, Al are also toxic when present in high concentrations
[4, 36], no existing limits for industrial effluent discharge of these metals were found in the South African National Act of 1998
. For this study, the limits set by the UN-Food and Agriculture Organization
 and the South African National Standards (SANS, 241) for drinking water
 were considered for these metals. Results indicated that these metals (Co, Ni, V) were present in industrial wastewater at concentrations higher than the UN-FAO permissible limits of 0.05 mg/l, 0.2 mg/l, 0.1 mg/l, respectively
 and also at concentrations higher than the maximum limits of 1.00 mg/l, 0.35 mg/l and 0.5 mg/l, set by SANS 241, respectively. Furthermore, Al concentrations in industrial wastewaters exceeded the national standard limit of 0.5 mg/l; however, none of the regulations
[37–39] has established the limit of Ti in the industrial wastewater effluent.
Although the toxicity of heavy metals to both bacteria and protozoa, previous studies reported that some microorganisms can develop detoxifying mechanisms even in water containing high concentrations of heavy metals
[6, 12, 16]. As a result, they are used for the bioremediation of heavy metals in polluted wastewater. Intensive studies have been carried out with bacteria and their role in the bioremediation of heavy metals
[6, 33], whereas, few studies report on the role of protozoan species in the bioremediation of heavy metals in polluted wastewater
[14, 40]. The present study compared the effect of heavy metals from industrial wastewater on the growth performance of protozoan species (Peranema sp., Trachelophyllum sp. and Aspidisca sp.) to those of bacterial species (Bacillus licheniformis, Pseudomonas putida and Brevibacillus laterosporus); they also assessed their uptake ability of heavy metals from the highly polluted industrial wastewater.
The results revealed that the exposure of both groups of test organisms in highly polluted industrial wastewater containing various heavy metals (Table
2) resulted in the inhibition of some bacterial and protozoan growth on the first day of incubation, followed by a recovery of bacterial as well as protozoan isolates between the second and third days of incubation. However, Pseudomonas putida, Bacillus licheniformis and Peranema sp. were able to tolerate the co-occurrence of several metals in the culture media and did not show any growth inhibition up to the fourth, third and third day of incubation, respectively (Figure
1). For Brevibacillus laterosporus, Trachelophyllum sp. and Aspidisca sp., the inhibition and slow growth response occurred after the second day of incubation, which could be due to the antimicrobial/toxicity effects of heavy metals as reported by Kamika and Momba
. As the tolerance and bioaccumulation of heavy metals by microorganisms depends on the microbial species, the culture media, the number of cells, the type of heavy metal and the presence of other metals in the samples
, this study revealed that the industrial wastewater did not exert any major effect on the growth of Pseudomonas putida when compared to other bacterial isolates. Moreover, no major effect was found in the media innoculated with Peranema sp., which appeared to be the most tolerant protozoan isolate and the second most tolerant isolate when compared to bacterial isolates. The results of the present study are in agreement with Nilsson
, who reported that heavy metals can affect the survival of microbial isolates in many ways such as the reduction of food uptake, growth inhibition, and reduction in the rate of endocytosis, which may influence their survival. A study conducted by Cabrera et al.
 reported that at high concentrations, metals could slow microbial population growth. Moreover, the toxicity of these heavy metals on aerobic microorganisms can also affect the consumption of dissolved oxygen
. Shuttleworth and Unz
, when investigating the effects of several heavy metals on the growth of axenic filamentous bacteria (Thiothrix, type 021N and type 1701), found that these organisms could grow in the presence of single toxic metals (Ca, Cu, Ni and Zn); but when mixed together, the latter appeared to act synergistically in suppressing the development of Thiothrix strain A1. Contrary to this, Ni2+ at concentrations of 10/20 mg/l was reported to stimulate the growth of Pseudomonas putida, Bacillus licheniformis and Peranema sp. in a modified mixed liquor medium
. Conversely, in the present study, the stimulating action of Ni2+ was not evident at similar concentrations, which could have been inhibited by the presence of other heavy metals in the industrial wastewater. Besides the pH level, the slow growth/inhibition of the test isolates might also be due to the complexity of the culture media in terms the presence of toxic ions.
Despite slight microbial growth or growth inhibition observed with regards to certain test isolates, the removal/bioaccumulation of heavy metals (Ni, Mn, Mg, V, Pb, Cu, Zn, Al, Cd) occurred in mixed liquor culture media (Figure
2). Statistically significant differences (p < 0.05) observed between the removal efficiency for dead-microbial cells (Figure
3) and living ones (Figure
2) indicated that the selected isolates were also removing heavy metals from the culture media by using active mechanisms. This was confirmed by the presence of certain specific heavy metal-resistance genes in test isolates (Figure
4). Bacterial isolates (Pseudomonas putida, Bacillus licheniformis and Brevibacillus laterosporus) contained the genes copC, chrB, cnrA3 and nccA encoding the resistance to Cu, Cr, Co-Ni and Co-Ni-Cd, respectively, but did not contain the genes copA, copB, cnrC2 and czcD. However, the presence of metal-resistant genes in Brevibacillus laterosporus and its growth inhibition could not be explained in the present study. Furthermore, protozoan isolates (Peranema sp., Trachelophyllum sp. and Aspidisca sp.) contained only the genes copC and chrB encoding the resistance of Cu and Cr, respectively. An exception was found with Peranema sp. that contained the gene cnrA3 encoding the resistance of Co and Ni. This is in agreement with Mohapatra
, who reported that apart from the sensitivity of protozoa to metal toxicants, Peranema is one of the protozoan isolates that are generally resistant. In addition, Ruthven and Cairns
 reported that Peranema could tolerate approximately 1000 mg-Pb/l. The ability of Pseudomonas putida observed in this study to tolerate and remove several heavy metals from polluted industrial wastewater can be explained by the findings of Canovas and co-workers
. These authors reported that the genome of Pseudomonas putida encodes an unexpected capacity to resist heavy metals and metalloids. This species in its different strains has been reported to exhibit high maximal tolerant concentrations of a large spectrum of divalent metals
. Contrary to the present findings, Pseudomonas putida has been previously reported to contain at least four Zn/Cd/Pb efflux transporters and two czc chemiosmotic transporters
. It has also been reported that Bacillus licheniformis produce extracellular polymers with great affinity for metals; these polymers are able to complex with and accumulate metals such as Fe, Ni, Cd, etcetera
This study corroborates the findings reported elsewhere that microorganisms can use several mechanisms to simultaneously remove metals
[11, 20, 33]. In addition, the removal efficiency of test microorganisms mostly depended on the availability and concentrations of heavy metals in industrial wastewaters. No individual isolate showed a high removal rate of all the heavy metals from the polluted industrial wastewaters (Figure
2). High removal efficiency for only certain heavy metals was also observed in the culture media inoculated with protozoan isolates such as Peranema sp. This situation highlights the role of each bacterial species as well as protozoan species in the bioremediation of heavy metals in wastewater systems. Moreover, in a study conducted by Clausen
, it was reported that Bacillus licheniformis CC01 could remove 93% of copper, 8% of Chromium and 45% of Arsenic while Pseudomonas putida could remove 25% of copper from nutrient agar. Ledin and co-workers
 revealed in their report that Pseudomonas putida could remove Sr (80%), Eu (97%), Zn (70%), Cd (70%) and Hg (95%) in media containing 10-8 M of the respective metals.
Besides the interest revealed by several scientists with regards to bacteria for the removal of heavy metals, investigations have been undertaken on certain protozoan species in the bioremediation of and tolerance or resistance to heavy metals
[50–52]. Rehman et al.
 reported that a ciliate Stylonychia mytilus removed Cd (91%), Hg (90%) and Zn (98%) after 96 h of incubation in the culture media containing 10 μg/ml of the respective metal ions. In another study, Rehman and co-workers
 also revealed that Vorticella microstoma can tolerate Cd (22 ug/ml), Cu (22 ug/ml), Ni (17 ug/ml), and Hg (16 ug/ml) and therefore can remove 72%, 82%, 80% and 74% of the above metals, respectively. Leborans et al.
 also stated that certain marine protozoa communities were able to accumulate from 27.02 to 504 μg-Pb/g when they were exposed to 500 and 1000 μg/l of Pb. In addition, El-Sheekh et al.
 reported that Nostoc muscorum and Anabaena subcylindrica were able to grow in sewage and industrial wastewater effluent and removed 12.5%-81.8% Cu, 11.8%-33.7% Co, 26.4%-100% Pb and 32.7%-100% Mn.
Unlike terrestrial environments, in aquatic environments, oxygen is usually a limiting factor and can also influence the toxicity of heavy metals to aquatic life such as aerobic microorganisms
. As an electron acceptor, oxygen uptake by microbial isolates in industrial wastewater could be linked to the growth of aerobic microbial isolates
. However, during the study period, low DO removals were recorded by all test organisms with the exception of Pseudomonas putida and Peranema sp. which showed high DO removal of 84.4 ± 4.02% and 68.83 ± 1.09%, respectively (Table
2). This situation was an indication on the toxic effect of heavy metals resulting in the slow growth of test isolates in the industrial wastewater samples. This is in agreement with Slabbert and Grabow’s finding
, who reported that the oxygen uptake of Pseudomonas putida was stimulated when inoculated in diluted industrial effluent but was inhibited in highly polluted industrial wastewater.
Therefore, the DO depletion during the study could be explained by the growth of the isolates and this had also an impact on the COD which increased in the media, showing a significant microbial growth to enlighten a possible excretion of extracellular polymers involved in the heavy metal resistance
[23, 55]. The highest COD increase (175.86%) was noted with Pseudomonas putida, while Peranema sp. appeared to have the lowest COD increase (12.07%). The results of the present study correspond to the findings of previous investigators who also reported an increase on COD when working on the removal of nutrients
 or on the tolerance of Ni2+/V5+[21, 22] by the same test protozoan species in activated sludge mixed liquor. As opposed to this, Pala and Sponza
 reported an efficient removal of COD in activated sludge with the addition of Pseudomonas sp. Musa and Ahmad
 also reported a reduction on COD of up to 94% in wastewater when using some industrial wastewater bacterial isolates.
Statistical evidence indicated strong and moderate positive correlations consecutively between growth performance and some heavy metal removal regardless of pH, COD increase and DO removal, which could be attributed to combined microbial activities such as the biosorption of metals to cell surfaces
, release of extracellular polymeric substances during the detoxifying process of heavy metals as well as die-off of microbial cells
. The weak correlations between protozoan counts and other parameters could also be attributed to the inhibition of the protozoan isolates throughout the experimental study
It is well known that the pH is also an important and limiting parameter in wastewater treatment systems for the growth and activity of several organisms. In bioremediation processes, acid-tolerant microorganisms are viewed as being beneficial for the treatment of highly polluted wastewater from the mines or industry
[57, 60]. However, by investigating the variations of pH in the polluted industrial wastewaters, which initially had a pH of approximately 4, a slight fluctuation of pH in the inoculated industrial wastewaters was observed throughout the study period (Tables
2). Although the range of pH values for several biological activities is very narrow and ranged between 6 and 9
, this finding revealed that all test isolates except Aspidisca sp. were able to grow in an aqueous solution with a pH value of approximately 4. Akpor et al.
, however, reported an increase in the pH value in activated sludge inoculated with some selected wastewater protozoan isolates.