Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach

ABSTRACT

This addendum to “Novel application of bacteriophage for controlling foaming in wastewater treatment plant- an eco-friendly approach “ includes characteristics of the phages NOC1, NOC2 and NOC3 not discussed in the previous paper. The phage adsorption and host interaction properties, their sensitivity to pH and temperature are inferred. NOC2 is seen to be more temperature resistant while others are not. All the phages show pH sensitivity. There is a variance observed in the behavior of these phages. Also, applicability of the phage based system to large scale reactors is studied and discussed here.

Introduction

We have reported previously, the use of bacteriophages in biocontrol of foaming by Nocardioforms in activated sludge process of wastewater treatment.^1,2 In the previous study, we had isolated and tested specific bacteriophages against Nocardioforms – NOC1, NOC2 and NOC3 and showed that they can be used effectively to curb foaming. Their host range was determined and genome sequences studied. Submitted to GenBank under accession numbers – KF879861, KF879862, KF879863 for NOC1, NOC2 and NOC3 respectively. They were demonstrated to be potential inhibitors of nocardioform growth.¹ In the present study, we have discussed the nature of these bacteriophages with respect to temperature and pH. Also, the applicability of this process was studied using lab scale reactors as a preliminary experiment.

The main objective of this work was to get a deeper understanding of phage characteristics and test the extendibility of this method from in vitro level to lab scale reactors. Study of specific properties and interaction of phage with bacteria would help fine tune the reaction conditions for maximum effect. Biocontrol of foaming bacteria by use of specific phages^3,4,5 would be most ideal if efficiency of this technique can be maximized by detailed study of the system.

Characterization of lytic bacteriophages of nocardioforms

i) Phage adsorption and host interaction - single-step growth curve analysis

The method for this experiment has been followed as described in Petrovski et al, 2011.^6,7 Phage stock and host cultures FB4, FB6, FB7 were infected with NOC1, NOC2, and NOC3 at a multiplicity of infection (MOI) of 1. After an adsorption period of 5 min at 30°C, these were used as a sample to calculate the single step growth curve using pour plate method. This culture was diluted 1 in 100 to minimize the possibility of non-adsorbed phages infecting cells. After 24 hours incubation at 30°C a graph was plotted on the basis of number of plaques observed (Fig. 1). The data is shown in Table 1 which is used for plotting the graph of time versus number of phages adsorbed.

Figure 1.

Single step growth curve.

Table 1.

Number of phage adsorbed within 30 minutes of phage and host interaction.

		No. of plaques observed after 24 hrs incubation
S.No.	Time (min)	NOC1	NOC2	NOC3
1.	0	50	36	20
2.	5	95	50	30
3.	10	250	65	60
4.	15	275	90	100
5.	20	300	200	305
6.	25	405	350	300
7.	30	750	800	300

ii) pH and thermal sensitivity

The method for this experiment has been followed as described in Yang et al, 2010.⁸ Phages NOC1, NOC2 and NOC3 were incubated under different pH values for one hour before determining the number of infectious phage particles. Results of pH stability tests are summarized in Table 2a and Fig. 2a. Experiments showed that all the 3 phages are stable at pH 6, 8 10 only; they are non-tolerant to highly acidic or basic environment. At neutral or nearly neutral pH, phages are able to grow well and many plaques can be seen on the plates. Phage NOC1 is more stable than NOC2 and NOC3, as it can be clearly seen in table that NOC1 is giving more number of plaques at pH 8. A representative plate of plaque assay of pH stability is given in Fig. 2b where plaque count is about 200.

Table 2(a).

pH sensitivity of isolated filamentous bacteria bacteriophages.

Sr. No.	pH maintained	Plaque count (NOC1)	Plaque count (NOC2)	Plaque count (NOC3)
1	2	0	0	0
2	4	0	0	0
3	6	98	200	35
4	8	300	250	210
5	10	60	99	52
6	12	0	0	0
7	14	0	0	0

Figure 2.

(a) pH sensitivity curve of NOC1, NOC2 and NOC3.

Figure 2.

(b) Representative ph phage plate of pH stability test (pH = 6).

The preliminary experiments showed that phage NOC1, NOC2, NOC3 stock solution retained almost 100% infection activity after incubation at 30°C for one month (not shown), so temperatures chosen to test thermal stability of phage NOC1, NOC2 and NOC3 were 20°C, 40°C, 60 °C, and 80°C, data is given in Table 2b. Phage count taken for incubation was 10³phages. The results showed phage NOC2 was heat stable, 80% and 50% phages still remained alive after 30 minutes incubation at 60°C and 80°C, respectively; only 3% phages NOC1 were alive after 20 minutes incubation at 80°C; while more than 99% phages lost their infection ability in 30 minutes at 80°C. Similarly NOC3 phages also lost their infectivity after incubation at 80°C for 30 min while all the phages were growing well at incubation temperatures 40°C and 60°C.

Table 2(b).

Heat stability of bacteriophages NOC1, NOC2 and NOC3.

Temp.	Incubation Time(min)	NOC1	NOC2	NOC3
20	10	140	750	530
	20	200	800	210
	30	600	850	600
40	10	580	700	150
	20	210	730	600
	30	300	450	350
60	10	500	852	425
	20	305	650	320
	30	12	800	800
80	10	65	600	55
	20	35	550	24
	30	0	520	9

Applicability of phage to lab scale reactor

The method for this experiment has been followed as described in Petrovski 2011.² Bacteriophages were grown in lab and a sufficient amount of bacteriophages were applied to their respective hosts. The data showed CFU/ml decreased markedly in the presence of NOC1, NOC2, and NOC3 when collectively applied to the lab scale reactor. Hence, it is considered that the foaming ability of bacteria is reduced if a specific formulation of bacteriophages is applied to the reactor (Table 3). Thus, the phage mediated cell number reduction eliminates foam under laboratory conditions (Fig. 3) and encourages its application in the control of foaming-activated sludge mixed liquor in full-scale plants.

Table 3.

Bacterial count reduction in the reactor by applying bacteriophages.

Sample collected	Dilution in 1 ml	Time interval for sample collection (mins.)	Bacterial count after 24 hrs. incubation at 30°C (2 µl)	% reduction
	5 ml	10−6	0	800	0
		5	750	6.25
		10	500	37.50
		15	430	46.25
		20	380	52.50
		25	200	75.00
		30	100	87.50

Figure 3.

Bacterial count reduction and % reduction after applying bacteriophage.

Conclusions

In the present research bacteriophage NOC1, NOC2 and NOC3 were isolated from waste water of a lab scale reactor where foam was observed during aeration process. Significant reduction in the foam was observed when the isolated phages were applied to the reactor at lab scale. Various tests were performed on isolated bacteria and bacteriophages. Experiments showed that phage NOC2 was considerably stable after exposure to high temperature and pH, emphasizing that phage can withstand the seasonal/operational fluctuations. The isolated phage showed no cross infectivity with other bacteria most commonly found in activated sludge systems, thus validating its suitability for biocontrol of filamentous foaming caused by filamentous bacteria. The phage-based biocontrol, therefore, holds a great potentiality for large-scale applications as an economic agent in the mitigation of several water, wastewater and environmental problems.

The isolated and characterized phages in this study were showing similarity to those phages reported earlier as active against the stable foam forming bacteria under laboratory conditions. This observation implies that the isolated bacteriophages viz. NOC1, NOC2, NOC3 may be helpful in reducing the stable foaming problem in wastewater treatment plant. Bacterial count shows significant reduction in less time when exposed to these phages in lab scale reactors. Scale-up experiments and their standardization might prove to be beneficial to establish this technique in practicality in activated sludge process of waste water treatment.

Disclosure of potential conflicts of interest

The authors declare that they have no conflict of interest.

Funding

We are thankful to Department of Science and Technology, New Delhi, India for providing funding for completion of this study through project (Project No. GAP-1-2128).