Bacterial quantity on biofilm coupons through heterotrophic plate count (HPC)
The microbial characterization of drinking water mostly relies on conventional culture-based methods such as heterotrophic plate count (HPC) (Bertelli et al., 2018).
The extracted Biofilm coupons reported the development of biofilm on both PVC and RCC coupons after 15 and 30 days, for which results were compared. The development and growth of biofilms can take days to weeks depending on environmental conditions and nutrient availability. Upon reaching the maturation stage, biofilms experience a dispersal phase in which single cells are released from the biofilm into the bulk water to form colonies elsewhere to complete the biofilm lifecycle. In DWDS, biofilms are relatively thin and can reach a thickness of up to a few hundred micrometers (Momba et al., 2000; Bitton, 2014). The bacterial quantity of biofilms on both coupons of overhead storage tanks of Z-I and Z-II and randomly selected underground storage tanks after 15 and 30 days are shown in Fig. 2.
At Z-I, greater bacterial quantity was observed on RCC coupons than on PVC coupons after both 15 and 30 days, as shown in Fig. 2. Moreover, it was also observed that RCC coupon supported more bacterial growth after 15 days compared to 30 days at both Z-I and Z-II. This is justified on the basis of rationale that layer of biofilms gets thicker and denser over time, and it may detach from the surface of material. However, less bacterial quantity was observed on RCC as compared to PVC after 30 days as shown in Fig. 2. This anomaly is attributed to the reason that polymeric material could be a source of biodegradable organic compounds which serves as a food for bacteria. The results are in line with a previous study in which leaching of growth promoting organic compounds from the increasingly popular use of polymer-based pipes was found. Besides, less growth and microbial diversity on polymeric materials compared to those formed on corrosion-prone materials was reported (Liu et al., 2016). A subsequent study revealed that water stored in plastic containers is more prone to bacterial incorporation into biofilms compared to other rough surface containers. These biofilms act as reservoirs for pathogens, making untreated water unsafe for consumption (Ezenobi et al., 2018). For underground tank, more growth of bacterial biomass was observed on RCC coupons compared to PVC both after 15 and 30 days. Moreover, more growth of bacterial biomass was observed on RCC coupons of underground water storage tank after 30 days compared to 15 as shown in Fig. 2. Similar study was conducted by Douterelo et al., 2014 in which an investigation of early stages of biofilm formation in an experimental, chlorinated DWDS depicted the occurrence of significant changes in biofilms composition, with a gradual increase in species richness over 28 days. There revealed no difference between bacterial quantity in biofilms of overhead storage tanks of Z-I, II and randomly selected underground storage tank. Most of the biofilm organisms arise from raw water which has passed through the treatment process, in quite few numbers and attach themselves to solid surfaces where they multiply depending upon the availability of nutrients (Flemming et al., 2002). However, for treatment of raw water chlorine is being added in overhead water storage tank before distribution of water but the amount of this added chlorine is far less compared to that prescribed by World Health Organization (WHO) and Pakistan Standards for Drinking Water Quality (PSDWQ). Numerous studies have concluded that material used for DWDS is directly related to the quality of water being transported. It has been reported that the selection and organization of biomass depends upon the nature of the material. The growth of biofilm is supported on those surfaces which provide appropriate nutrients for microbial growth (Momba et al., 2000; Batté et al., 2003). Research has found that materials having rough surfaces promote more bacterial attachment and growth (like RCC in current study). According to a similar study, the number of heterotrophic plate count and total bacteria were found 36–93 times and 5–30 times greater in iron pipe material as compared to biofilm in PVC pipes due to surface roughness. In addition, the density of grown biofilm varies depending on different types of materials (Ren et al., 2015). It has been discovered that some materials when in contact with the flowing drinking water can release biodegradable compounds in drinking water which promotes biofilm growth and multiplication (like PVC in the current study). However, in terms of comparing materials, studies depicted that plastic materials (like PVC or polyethylene) encourage less bacterial attachment, growth and multiplication because of their smoother surfaces as compared to iron materials (e.g., gray iron), stainless steel-based materials and cement materials (like cemented cast iron and asbestos-cement etc.). Hence, material characteristics such as roughness and composition impact microbial communities and morphology in grown biofilms (Niquette et al., 2000; Douterelo et al., 2019)
Average values of residual chlorine content and bacterial quantity
Avoiding microbial regrowth is important because some bacterial pathogens, such as Legionella pneumophilia and Mycobacterium avium, or various free-living pathogens can grow in drinking water distribution systems (DWDSs). Regrowth and the direct risks associated with the presence of pathogenic microorganisms in water are often mitigated by the addition of residual disinfectants (Douterelo et al., 2019). For this purpose, minimal residual disinfectant concentration is usually maintained (Bertelli et al., 2018) because higher residual concentrations lead to water quality concerns aside from taste, odor, or discoloration, and the production of disinfectant by-products, which serve as a carbon source for subsequent microbial growth (Fish et al., 2018). Chlorine is a powerful disinfectant for eliminating viruses and pathogenic bacteria and is used in water for disinfection, as its action lasts even after it is added to water (Abbas, 2011; Madzivhandila & Chirwa, 2017). For optimal disinfection WHO and PSDWQ suggested the concentration of residual chlorine between 0.5 and 1.5 mg/L at source and 0.2 to 0.5 mg/L at consumer end. The amount of residual chlorine may decrease despite the addition of a 1 mg/L dose by university’s concerned authorities (Javed et al., 2022). This reduction in concentration is justified based on rationale that considerable concentration of added chlorine is consumed by the oxidation of organic and inorganic compounds present in water (Vargas et al., 2021), thereby leading to more biofilm formation. Moreover, studies have revealed the impact of different residual chlorine contents on biofilm characteristics, such as composition, structure, microbiome, and water quality. Average value of residual chlorine content of water obtained from overhead storage tank during insertion and extraction of biofilm coupons from overhead storage tank of Z-I was 0.08 and 0.17 mg/L after 15 and 30 days, respectively. Furthermore, average value of residual chlorine content of water obtained from overhead storage tank of Z-II during insertion and extraction of biofilm coupons is 0.07 and 0.08 mg/L after 15 and 30 days, respectively which is far less compared to limits set by World Health Organization (WHO) and Pakistan Standards for Drinking Water Quality (PSDWQ). Similarly, negligible amount of residual chlorine content was also found in water sample obtained from underground storage tank i.e., 0.07 mg/L after 15 days because during sampling period chlorine was used for cleaning purpose of tank and no residual chlorine was found in underground storage tank after 30 days. Ultimately, the highest bacterial quantity was observed in biofilms coupons (both PVC and RCC) of underground storage tank as shown in Fig. 2. Studies showed that chlorine tends to have a diminishing effect on the number of bacteria present within biofilm communities (Inkinen et al., 2018; Zhang et al., 2019). A similar study conducted by Fish et al., (2020) examined the impact of various chlorine doses on water quality and biofilm. The results showed that a high chlorine concentration reduced the biofilm cell concentrations, but the presence of observable inorganic contents also poses unique risks. These findings challenge the assumption that measurable free residual chlorine ensures drinking water safety. Moreover, there was not much difference observed between the bacterial quantities of biofilms formed on coupons (both PVC and RCC) of underground and overhead water storage tanks due to less or negligible amount of added chlorine.
Pearson’s co-relation matrix between average values of residual chlorine content of water and bacterial quantity within the formed biofilm
The correlation coefficient between water parameters offers a systematic approach for efficient water monitoring. A strong relationship is observed when one variable consistently changes with the values of another. The relation is stronger whenever variables are in the range of + 0.8 to + 1.0 and − 0.8 to -1.0 (Khan et al., 2021).
Table 4 shows the Pearson’s correlation matrix for average values of residual chlorine in water obtained from selected sampling stations during insertion and extraction of biofilm coupons and bacterial quantity of biofilms formed on coupons after 15 and 30 days, respectively. A strong negative co-relation with r = -0.8 with P value 0.04 between residual chlorine values and bacterial quantity of biofilms formed on both coupons of underground and overhead storage tanks after 15 days has been observed. Similarly, strong negative co-relation also exists between the residual chlorine values and bacterial quantity of biofilms formed on both coupons of underground and overhead storage tanks after 30 days with r = -0.8 having P value 0.05. Hence, a strong inverse relation between residual chlorine contents and bacterial quantity within the biofilms is evident. The results of current study are in line with that of Fish and Boxall (2018) which concluded that bacterial quantification in biofilms formed within drinking water distribution system (DWDS) was statistically significantly reduced as residual chlorine concentration increased.
Table 4
Pearson’s co-relation matrix of average values of residual chlorine and bacterial quantity within the biofilms after 15 and 30 days
Parameters
|
Residual Chlorine (mg/L)
|
15D
|
30D
|
Residual Chlorine (mg/L)
|
1
|
1
|
Bacterial Quantity (HPC)
|
-0.82
|
-0.80
|
(*Strong correlation having P value < 0.05)
Spatial distribution of bacterial quantity within the formed biofilms and residual chlorine content of water samples of selected sampling stations
Heat maps can be considered as a useful method for spatial data presentation where values are depicted by colors. Sampling stations were taken on the left side of the heat map along the rows while sample of biofilm coupons (PVC and RCC) and values of residual chlorine content (mg/L) present in water obtained from each sampling station were taken along the columns. The results of heat map also show the strong relation between the bacterial quantity and residual chlorine content present in water samples obtained from selected sampling stations. The lowest residual chlorine content was observed in water sample obtained from underground water storage tank after 30 days and ultimately highest bacterial quantity was found in biofilms formed on RCC coupons of underground storage tank. Highest residual chlorine content was observed in water sample obtained from overhead storage tank of Z-I after 30 days and ultimately lowest bacterial quantity was observed as shown in Fig. 4.
Bacterial morphology and diversity in biofilms
Morphological identification
Analysis of microbial communities residing in biofilms grown in different materials highlighted that material of DWDS not only affects the biofilm formation potential but also the diversity and richness of microbial population (Yu et al., 2010). Bacterial diversity was elucidated by observing the morphology of different bacterial colonies grown on nutrient agar plates based on size, shape, margins, form, texture, elevation, opacity, and color. A total of 12 bacterial strains (one from each sample) were selected from 15 and 30 days old biofilms formed on both PVC and RCC coupons of selected sampling stations for cell morphology based on their abundance (HPC results). Results of cell morphology are summarized in Table 5.
Table. 5 Morphology of selected bacterial species obtained from each sample of 15 and 30 days old biofilms obtained from PVC and RCC coupons.
Coupons
|
Isolates
|
Size
|
Shape
|
Elevation
|
Margin
|
Opacity
|
Texture
|
Form
|
Color
|
Surface
|
OhST Z-I
(PVC)
|
15 D
|
Moderate
|
Round
|
Convex
|
Entire
|
Opaque
|
Slimy Moist
|
Circular
|
Creamy white
|
Smooth
|
30D
|
Moderate
|
Irregular
|
Flat
|
Undulate
|
Opaque
|
Slimy moist
|
Circular
|
Creamy white
|
Rough
|
OhST Z-II (RCC)
|
15D
|
Small
|
Round
|
Convex
|
Entire
|
Opaque
|
Slimy moist
|
Circular
|
Creamy white
|
Smooth
|
30D
|
Large
|
Irregular
|
Raised
|
Lobate
|
Opaque
|
Slimy moist
|
Irregular
|
Off-white
|
Rough
|
OhST Z-II (PVC)
|
15D
|
Moderate
|
Irregular
|
Raised
|
Entire
|
Opaque
|
Slimy moist
|
Irregular
|
Yellow
|
Rough
|
30D
|
Large
|
Round
|
Convex
|
Entire
|
Opaque
|
Slimy moist
|
Circular
|
Pale yellow
|
Smooth
|
OhST Z-II (RCC)
|
15D
|
Punctiform
|
Round
|
Convex
|
Entire
|
Opaque
|
Slimy moist
|
Circular
|
White
|
Smooth
|
30D
|
Small
|
Round
|
Raised
|
Entire
|
Opaque
|
Slimy moist
|
Circular
|
Pale yellow
|
Smooth
|
UST
(PVC)
|
15D
|
Moderate
|
Round
|
Raised
|
Entire
|
Opaque
|
Slimy moist
|
Irregular
|
Creamy white
|
Smooth
|
30D
|
Large
|
Round
|
Raised
|
Entire
|
Opacity
|
Slimy moist
|
Circular
|
Pale yellow
|
Smooth
|
UST
(RCC)
|
15D
|
Moderate
|
Round
|
Entire
|
Entire
|
Opaque
|
Slimy moist
|
Circular
|
Pale yellow
|
Smooth
|
30D
|
Moderate
|
Round
|
Flat
|
Entire
|
Opaque
|
Slimy moist
|
Circular
|
Pale yellow
|
Smooth
|
Bacterial diversity
For identification of bacterial diversity within the biofilms formed on both PVC and RCC coupons of selected sampling stations after 15 and 30 days, heat maps were generated. These heat maps were intended to provide a spatial overview of bacterial diversity which helped to identify the hotspots of microbial communities in selected sampling stations. In the case of PVC coupons, the highest bacterial diversity was observed on PVC coupon of Z-II both after 15 and 30 days having values 6 and 10, respectively. Similarly, in the case of RCC coupons highest bacterial diversity was also observed on RCC coupon of Z-II both after 15 and 30 days having values 7 and 11, respectively as shown in Fig. 5 (a-d). This implies that Z-II has more bacterial diversity on both coupons after 15 and 30 days as compared to other selected sampling stations as depicted by heat maps. Moreover, at Z-I, PVC coupons exhibited greater bacterial diversity compared to RCC after 30 days and equal bacterial diversity was observed on both RCC and PVC coupons after 15 days as shown in Fig. 6. At Z-II higher bacterial diversity was observed on RCC coupons compared to PVC coupons after both 15 and 30 days. Additionally, both PVC and RCC coupons have depicted greater bacterial diversity after 30 days as compared to 15 days as shown in Fig. 6. In the case of underground storage tank, higher bacterial diversity was observed on RCC coupons compared to PVC coupons after 30 days. PVC coupons have almost equal bacterial diversity both after 15 and 30 days. Whereas, PVC coupons after 15 days held more bacterial diversity compared to RCC as shown in Fig. 6. It has been found that organic additives leach out of plastic pipes which in turn affect the accumulation of biofilm and these are reported to enhance the growth of different opportunistic pathogens (Batté et al., 2003). In general, more bacterial diversity was observed on RCC coupons compared to PVC coupons.
Shape, gram staining, catalase, and oxidase test
A single bacterial species was selected from each coupon of the selected sampling stations both after 15 and 30 days for conducting the shape, gram staining, catalase and oxidase tests. Table 7 shows the shape and cell morphology of selected bacterial strains being bacillus, and strain being gram positive at each 15 and 30 days old biofilms of both the coupons (PVC & RCC). These results are supported by the study conducted by Yousaf et al., 2014 in which survey of gram-negative and gram-positive bacteria in drinking water supplies of Karachi, Pakistan was conducted. The results revealed that among 50 collected samples of drinking water, 38 samples had gram positive bacteria while only 7 samples had gram negative bacteria. Similarly, biochemical identification demonstrated the majority of selected bacterial strains being both catalase and oxidase positive, regardless of the age of the biofilm (15 or 30 days old) and the type of tank material used (PVC & RCC). These findings indicate an imperative role of certain enzymatic activities within the bacterial strains in sustaining their metabolic processes and environmental adaptation
Table. 7 Shape, Gram staining, catalase and oxidase tests of bacterial species
Sampling
Stations
|
Coupon material
|
Bacterial species
|
Shape
|
Catalase test
|
Oxidase test
|
Gram staining
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
OhST Z-I
|
PVC
|
1
|
Bacillus
|
Cocci
|
-
|
+
|
+
|
+
|
+
|
-
|
RCC
|
1
|
Bacillus
|
Bacillus
|
-
|
-
|
+
|
-
|
+
|
+
|
OhST Z-II
|
PVC
|
1
|
Cocci
|
Cocci
|
+
|
-
|
+
|
+
|
-
|
-
|
RCC
|
1
|
Bacillus
|
Cocci
|
+
|
+
|
+
|
+
|
+
|
-
|
UST
|
PVC
|
1
|
Bacillus
|
Bacillus
|
+
|
+
|
+
|
+
|
+
|
+
|
RCC
|
1
|
Cocci
|
Bacillus
|
+
|
-
|
+
|
+
|
+
|
+
|
Legend: 15D and 30D= 15 and 30 days, + stands for Positive, - stands for Negative
Biofilm characterization
Elemental composition of biofilms
Elemental composition of biofilms formed on surface of PVC and RCC coupons was determined through SEM-EDX. Higher contents of carbon were observed in most of the biofilm samples of both PVC and RCC coupons as shown in Table 7. The highest percentage of carbon was observed in 30 days old biofilm formed on PVC coupons of randomly selected underground storage tank of Z-III, which indicates slightly higher organic contents in the water. Results of this study corroborate the findings reported by Chowdhary (2012). The ubiquitous use of more economical plastic pipes compared to metal pipes is evident. These plastic pipes release some biodegradable organic compounds which in turn add to the carbon contents of water. Hence, the growth of biofilms may be regulated by reducing the organic carbon in the water as this organic carbon enhances the nutrients availability for the bacterial colonies forming biofilms (Krzeminski et al., 2019). Similarly, higher contents of oxygen were also observed compared to all the other elements in almost all the biofilm samples formed on PVC and RCC coupons of underground and overhead storage tank of Z-I and II. This higher content of oxygen implies that bacteria in the biofilms were good oxidizing agents and aerobic in nature. The highest percentage of oxygen was observed in 15 and 30 days old biofilm formed on RCC coupons of overhead storage tank of Z-I which was 51.1 and 49.2% respectively. Calcium contents were also higher in a few samples of biofilms formed on RCC coupons as shown in Table 7.
Table.7 Elemental composition of formed biofilms on PVC and RCC coupons
Sampling
Stations
|
Carbon
(%)
|
Oxygen
(%)
|
Zinc
(%)
|
Potassium
(%)
|
Calcium
(%)
|
Copper
(%)
|
Cadmium
(%)
|
Lead
(%)
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
15D
|
30D
|
OhST Z-I
PVC
|
6.5
|
7.4
|
30
|
34.0
|
1.7
|
1.3
|
0.3
|
0.2
|
8.7
|
9.1
|
0.6
|
0.3
|
N.D
|
N.D
|
7.1
|
4.2
|
OhST Z-I
RCC
|
2.0
|
1.1
|
51.1
|
49.2
|
1.0
|
0.9
|
0.3
|
0.4
|
35.5
|
36.2
|
0.5
|
0.2
|
0.1
|
0.2
|
2.0
|
2.1
|
OhST Z-II
PVC
|
21.9
|
20.4
|
32.6
|
34.1
|
2.0
|
2.0
|
0.7
|
0.5
|
9.2
|
8.1
|
1.0
|
N.D
|
0.2
|
0.1
|
4.7
|
3.6
|
OhST Z-II
RCC
|
21.4
|
20.0
|
43.0
|
50.7
|
1.1
|
1.8
|
0.5
|
1.0
|
24.1
|
27.9
|
0.4
|
0.3
|
0.1
|
N.D
|
2.0
|
2.1
|
UST PVC
|
35.1
|
37.1
|
38.1
|
33.4
|
1.2
|
1.2
|
0.8
|
0.7
|
4.4
|
4.5
|
0.1
|
N.D
|
N.D
|
0.1
|
4.9
|
5.1
|
UST RCC
|
1.8
|
1.6
|
40.1
|
46.3
|
1.1
|
1.2
|
0.2
|
0.3
|
37.4
|
39.9
|
0.1
|
N.D
|
N.D
|
0.2
|
2.9
|
2.7
|
*N.D= Not detected, %= Percentage composition