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Isolation and characterization of bacteriophages infecting nocardioforms in wastewater treatment plant.

1. Introduction

Activated sludge process (ASP) is the most commonly used process to reduce the toxicity of waste water by processing it microbiologically. In activated sludge system, treatment is given to every type of wastewater using microbial communities for the degradation of organic matter present in water [1]. Microbes use the organic matters as their energy source and degrade them into a less toxic form, but most of the system suffers from the excessive growth of unwanted mycolic acid containing filamentous bacteria or mycolata [2], which lead to the formation of brown and sticky foam [3]. Mycolata like Sphaerotilus spp., Leptothrix spp., Microthrix parvicella, Corynebacterium spp., Dietzia spp., Nostocoida limicola, Gordonia spp., Skermania spp., Mycobacterium spp., Nocardia spp., Rhodococcus spp., Tsukamurella spp., Type 021N, and Type 0041 play a role in foaming. However, among all mycolata the Eikelboom species are mainly responsible for foaming [4-7].

The foam covers a large area in the aeration tank which create interference in the treatment process and ultimately leads to the major environmental, operational, cosmetic, and health problems [3, 8, 9]. The outer surface of foam forming bacteria has hydrophobic properties similar to that of fats, oils, and grease which enables the bacterial bulk to float on the surface of liquid in aeration tank. The wastewater containing slowly degradable organic material like lipids, proteins, and fats may favor the growth of filamentous microorganisms like M. parvicella and G. amarae thereby leading to increased foaming [3, 6, 10]. The presence of unfavorable conditions in the activated sludge system, such as toxic conditions (pH below 6.5 or above 9.0), insufficient dissolved oxygen (DO), nutrient deficiencies, or seasonal temperatures may contribute to the foaming. It has been reported that the formation of scum and stable foam in aeration tank (AT), secondary clarifier (SC), and activated sludge (AS) is a global problem [9,11,12].

Various studies suggest that a range of physical, chemical, and biological [11, 13-15] methods is available for the control of foaming in activated sludge process [12], but they need a higher cost of maintenance. Current approaches for controlling foam includes decreasing mean cell retention time [16], use of classifying selectors, and nonspecific measures such as water sprays, steam application [17], polymer addition [18], and chlorination [19]. Dosing with cationic polymer [18] and controlling dissolved oxygen levels in the preoxidation reactor have been reported as useful methods for foam reduction [20].

There are only limited reports related to biological foam control methods especially the application of bacteriophages in AS systems [11, 14, 15, 21-23]. Bacteriophage therapy for the treatment of infectious diseases has shown promise [24]; on the similar lines lytic bacteriophages has the potential to be exploited as an biocontrol for filamentous bacteria which may lead to foam reduction in the AS treatment plant [11, 25]. Efforts have been made by Thomas et al. [23] and Petrovski et al. [13-15] for the isolation of bacteriophages against filamentous bacteria responsible for the stable foam formation in ASP. The successful application of phage for effective bacterial control relies on the population density which must be sufficient to support phage replication 26].

Bacteriophage based approach may have the potential as an environmentally safe option for tackling worldwide ASP operational problem of foaming. Nevertheless, phage-based foam control approach has certain limitations: (1) high concentration of phages must be applied for the successful application; (2) due to the polyvalent phages, broader host range could lead to the degradation of useful bacteria; (3) specific phage must be identified by the operator to counter specifically the foam forming bacteria without affecting other bacteria; (4) the microbial analysis of the system is a prerequisite to phage application as the bacterial population may vary between wastewater treatment plants (WWTP) [11].

The present research has been done with the objective of developing a biocontrol approach to manage foaming within AS systems in Nagpur city of India. In this study, we report the characterization of three Nocardia phages NOC1, NOC2, and NOC3 isolated from effluent treatment plant (ETP) and dairy ETP Nagpur, India. To the best of our knowledge to date, no work has been published from India related to biological approach for the control of foaming in WWTP.

2. Materials and Method

2.1. Sampling Sites. The sampling was done from effluent treatment plant (ETP) and dairy ETP around Nagpur, India, considering the foaming status of the treatment plants. As filamentous bacteria, for example, Nocardia, grows well on the slowly degradable organic matters like lipids, oil, and grease, fatty matters [3, 5, 10] and is found almost in every WWTP. So, in expectation of getting phages against Nocardia spp. the samples were collected from the above mentioned sites. Samples were collected from different points, namely, AT, SC, and AS, in sterile plastic bottles of 200 mL and processed within two days of sampling.

2.2. Bacterial Strains and Growth Conditions. The bacterial strains used in this study are American type culture collection which is listed in Table 1. All the ATCC Nocardia bacterial strains were grown on peptone-yeast-calcium (PYCa) agar [13], R2A agar [27], and Tryptone Yeast Glucose Agar (TYGA) [28]. Three growth media were used to comparatively see the best growth of Nocardia species. The PYCa agar was found to be the best media for supporting the growth of Nocardia species on the basis of plate count assay. Henceforth, PYCa medium was preferred for maintenance of Nocardia cultures at 4[degrees]C and was routinely maintained by subculturing every two weeks. After incubation, all the bacteria were enriched in the PYCa broth and stored at 4[degrees]C as per the method described by Petrovski et al. [13].

2.3. Isolation and Purification of Nocardiophage. Nocardiophages were isolated from wastewater sample collected around Nagpur, India. In brief, 20 mL wastewater sample from each different sampling sites was subjected to centrifugation at 3000 xg for 20 mins. After centrifugation the supernatant was filtered through cellulose acetate membrane filters (0.22 [micro]m) to remove bacterial cells. For enrichment of bacteriophages, 1 mL of each filtered sample was inoculated with the 50 mL of PYCa broth of mixed bacterial culture and incubated at 30[degrees]C [13]. After enrichment the bacterial cells were first centrifuged at 5,000 xg for 10 min, the supernatant was filtrated through 0.22 [micro]m cellulose acetate filter paper. For plaque assay method, 1mL filtered sample was mixed with 1 mL of host culture of each species and poured onto autoclaved petriplates after mixing the melted PYCa agar [29, 30]. Plates were then incubated at 30[degrees] C for 2 days as per the method of Petrovski et al. [13].

2.4. Nocardiophage DNA Isolation. DNA of all the phages NOC1, NOC2, and NOC3 were isolated using SDS-proteinase K method as described by Petrovski et al. [13].

2.5. Phage Host Range Determination. All three Nocardia species were subjected to host range determination by challenging against nocardiophages as per the method described by Petrovski et al. [13]. Phage recovery and purification was achieved with their respective host, namely, N. rhodochrous, N. amarae, and N. pinensis, as described by Petrovski et al. [13]. The phage characterization was achieved by nucleotide sequencing and analysis.

2.6. Phage Nucleotide Sequence Analysis. Nocardiophage DNA was genome sequenced using the Roche GS FLX genome sequencer and titanium chemistry by Genoseq (CA). The pyrosequencing reads were assembled using the gsAssembler (Roche Applied Science, Indianapolis, IN). All resulting single contigs obtained for each phage had a minimum of 50 times read coverage. After getting the genome sequences, the nucleotide sequences were subjected to analysis like NCBI-BLAST and Open Reading Frame (ORF) prediction using NCBI's ORF Finder tool available at The G+C (%) content for the sequences was evaluated using BitGene genetic analysis software available at http://

2.7. Nucleotide Sequence Accession Number. The partial genome sequences for Nocardia bacteriophages NOC1, NOC2, and NOC3 have been deposited in GenBank under accession numbers KF879861, KF879862, and KF879863, respectively.

3. Results and Discussion

3.1. Isolation and Purification of Nocardiophages. Petrovski's method was followed for the isolation and purification of bacteriophages. Single plaques were observed after 2 days incubation at 30[degrees]C. The quantitative results of plaque assay are summarized in Table 2 indicating phage growth. A representative culture plate of plaque assay is presented, wherein the plaques were picked and subjected to further isolation and purification of bacteriophages (Figure 1). On the basis of growth of bacteriophages on N. rhodochrous, N. amarae, and N. pinensis (reclassified as Skermania piniformis) phages were named as NOCI, NOC2, and NOC3, respectively. Further, plaques were purified by four rounds of dilution and reisolation to ensure that each plaque resulted from a single type of bacteriophage. The plates which were showing the excellent visual growth were taken for the purification of bacteriophages.

3.2. Phage Host Range Determination. Phage host range was determined by challenging each of the three Nocardia species against individual phage on PYCa media. The determination of host range of bacteriophages against nocardioforms is summarized in Table 3. Briefly, NOC2 (isolated from ETP, Nagpur) was able to grow on the two species of Nocardia, namely, N. amarae and N. pinensis; notably few plaques of NOC2 were seen against the N. pinensis. NOC1 and NOC3 were able to propagate only on their host, that is, N. rhodochrous and N. pinensis (Skermania piniformis), respectively. NOC2 had relatively broader host range when compared to NOC1 and NOC3. Although NOC3 was able to show slight growth on N. rhodochrous but clear lytic plaques were not observed, the NOC3 phage did induce a turbid lysis on N. rhodochrous so it was not consider as NOC3's host.

3.3. Phage DNA Sequence Analysis. The nucleotide sequencing of Nocardia bacteriophages NOC1, NOC2, and NOC3 resulted in generating the partial sequence of 34189 bp, 28456 bp, and 28465 bp, respectively. The nucleotide sequences of Nocardia bacteriophages NOC1, NOC2, NOC3 have been submitted to GenBank under accession number [GenBank:KF879861], [GenBank:KF879862], and [GenBank:KF879863], respectively. The results of nucleotide sequence analysis done using various bioinformatics tools like NCBI-BlAsT, NCBI's ORF Finder, and BitGene genetic analysis software are summarized in Table 4. The table summarizes the G+C content in mol%, number of ORFs over 100 bp and percent identity for bacteriophages NOC1, NOC2, and NOC3 with other Genbank submissions.

The major aim of this study was to isolate and characterize the nocardiophages which may reduce the foaming problem in WWTP in India. In the present research bacteriophages NOC1 and NOC3 were isolated from ETP of dairy industry and NOC2 phage was isolated from ETP, Nagpur, India. At the DNA level, NOC1 [GenBank:KF879861] was found to have 99% identity with Gordonia phage GTE7 [GenBank:JN035618] which was reported to reduce the stable foam formation by mycolata strains G. terrae, Gordonia amictia (Ben607), and Nocardia asteroides (Nast23) under laboratory conditions [15]. NOC2 [GenBank:KF879862] was found to have 99% identity with Gordonia phage GTE2 [GenBank:HQ403646] which was reported as lytic for Gordonia terrae, Rhodococcus globerulus, Rhodococcus erythropolis, Nocardia otitidiscaviarum, and Nocardia brasiliensis [14]. NOC3 [GenBank: KF879862] was found to have 99% identity with Nocardia phage NBR1 [GenBank:JN116828] which was reported to have N. otitidiscaviarum and N. brasiliensis as its host [31]. 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, namely, NOC1, NOC2, and NOC3, may be helpful in reducing the stable foaming problem in WWTP.

4. Conclusion

In the present study, isolates (NOC1, NOC2, and NOC3) bacteriophages were identified and characterized. All three phages had the ability to inhibit the growth of filamentous bacteria. The host range determination showed that NOC1and NOC3 were inhibitory against their respective host while NOC2 phage has slightly broader host range. Further, characterization and analysis showed that these phages showed similarity and identity with the previously reported phages against filamentous bacteria. Thus, the result obtained suggests that the isolated bacteriophages are potent inhibitor of foam forming bacteria. Isolated bacteriophages could be used potentially as a biocontrol agent for foaming problems in activated sludge treatment plants. Potential application of bacteriophage need much attention for the control of foaming as it is the ecofriendly and cost effective approach. Further work is in progress for isolation and characterization of other phages against other foam forming filamentous bacteria.

Conflict of Interests

The authors declare that they have no conflict of interests.


The authors are thankful to Council of Scientific and Industrial Research-National Environmental Engineering Research Institute (CSIR-NEERI), Nagpur (India) for providing facilities for carrying out this research activity.


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Krishna Khairnar, (1) Preeti Pal, (2) Rajshree H. Chandekar, (1) and Waman N. Paunikar (1)

(1) Environmental Virology Cell, Council of Scientific and Industrial Research, National Environmental Engineering Research Institute (CSIR-NEERI), Nehru Marg, Nagpur, Maharashtra 440020, India

(2) School of Environment and Earth Science, North Maharashtra University, Jalgaon, Maharashtra 425001, India

Correspondence should be addressed to Krishna Khairnar;

Received 12 June 2014; Accepted 8 July 2014; Published 22 July 2014

Academic Editor: Manuel Canovas

TABLE 1: Bacterial strains used for the isolation of bacteriophage.

Bacterial strains        Deposited as           ATCC number

Nocardia rhodochrous     Nocardia rhodochrous      33278
Nocardia pinensis        Nocardia pinensis         49497
(Skermania piniformis)   Blackall et al.
                         Nocardia amarae
Gordonia amarae          Lechevalier and           27808

TABLE 2: Isolation of bacteriophages against nocardioforms form
various sampling point using plaque assay method.

Sample     Different    Nocardia     Nocardia     Nocardia
sites      points of   rhodochrous    amarae      pinensis
           sampling                              (Skermania

ETP           AT            -          + (b)          -
              AS            -        + + + (b)        -
              SC            -            -            -

Dairy         AT            -            -        + + + (c)
industry      AS        + + + (a)        -            -
              SC            -            -          + (c)

AT: aeration tank, AS: activated sludge, SC: secondary
clarifier,--no visual growth; + appearance of visual
growth; + + + excellent visual growth.

(a) NOC1.

(b) NOC2.

(c) NOC3.

TABLE 3: Determination of host range of isolated bacteriophages
against nocardioforms.

Bacterial species              Bacteriophage

                         NOC1    NOC2     NOC3

Nocardia rhodochrous     + + +     -      +/-
Nocardia amarae            -     + + +     -
Nocardia pinensis          -      + +    + + +
(Skermania piniformis)

- No visual growth; ++ good visual growth; + + + excellent
visual growth. +/- Slight growth.

TABLE 4: Result of nucleotide sequence analysis for isolated
bacteriophages NOC1, NOC2, and NOC3 partial genome.

Isolated                Length of   Total G+C   Number of
bacteriophage partial   sequence     content    ORF over
genome sequences          (bp)       (mol%)      100 bp
(GenBank accession

Nocardia phage            34189       56.70        182
NOC1 (KF879861)
Nocardia phage            28456       61.17        150
NOC2 (KF879862)
Nocardia phage            28465       67.49        147
NOC3 (KF879863)

Isolated                Identity with other    Query     Identity
bacteriophage partial      bacteriophage      coverage     (%)
genome sequences         genome sequences       (%)
(GenBank accession      (GenBank accession
number)                       number)

Nocardia phage            Gordonia phage        100%       99%
NOC1 (KF879861)           GTE7 (JN035618)
Nocardia phage            Gordonia phage        100%       99%
NOC2 (KF879862)           GTE2 (HQ403646)
Nocardia phage            Nocardia phage        100%       99%
NOC3 (KF879863)           NBR1, complete
                         genome (JN116828)
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Title Annotation:Research Article
Author:Khairnar, Krishna; Pal, Preeti; Chandekar, Rajshree H.; Paunikar, Waman N.
Publication:Biotechnology Research International
Date:Jan 1, 2014
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