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Research Note

Understanding carbon regulation in aquatic systems - Bacteriophages as a model

[version 1; peer review: 2 approved]
PUBLISHED 01 Jun 2015
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Abstract

The bacteria and their phages are the most abundant constituents of the aquatic environment, and so represent an ideal model for studying carbon regulation in an aquatic system. The microbe-mediated interconversion of bioavailable organic carbon (OC) into dissolved organic carbon (DOC) by the microbial carbon pump (MCP) has been suggested to have the potential to revolutionize our view of carbon sequestration. It is estimated that DOC is the largest pool of organic matter in the ocean and, though a major component of the global carbon cycle, its source is not yet well understood. A key element of the carbon cycle is the microbial conversion of DOC into inedible forms. The primary aim of this study is to understand the phage conversion from organic to inorganic carbon during phage-host interactions.
Time studies of phage-host interactions under controlled conditions reveal their impact on the total carbon content of the samples and their interconversion of organic and inorganic carbon compared to control samples. A total organic carbon (TOC) analysis showed an increase in inorganic carbon content by 15-25 percent in samples with bacteria and phage compared to samples with bacteria alone. Compared to control samples, the increase in inorganic carbon content was 60-70-fold in samples with bacteria and phage, and 50-55-fold for samples with bacteria alone. This study indicates the potential impact of phages in regulating the carbon cycle of aquatic systems.

Keywords

interconversion, microbial carbon pump, carbon sequestration, refractory carbon, global carbon cycle

Introduction

The regulation of carbon in aquatic systems is a major biogeochemical process. The oceans’ surface takes up about 2% more CO2 gas than they release, a proportion of which dissolves into the water, forming carbonic acid. The increase in CO2 levels in oceans decreases the pH, resulting in acidification which affects the oceanic ecosystem1. Carbon also enters the seas through the food web via photosynthesis, but does not last for long periods and is either released into the atmosphere as CO2 or sinks to the ocean depths as dead organic matter. However, a significant amount of carbon is present in the water in the form of DOC2,4,5. The roles that ocean viruses play are very important in shaping microbial population sizes as well as in regenerating carbon and other nutrients68. It is estimated that every second, approximately 1023 viral infections occur in the ocean. Therefore, it should not be surprising that viruses are major influential forces behind biogeochemical cycles58.

A key element of the carbon cycle is the microbial conversion of dissolved organic carbon into inedible forms. Microbes play a dominant role in “pumping” bioavailable carbon into a pool of relatively inert compounds. The microbial carbon pump (MCP) “may act as one of the conveyor belts that transports and stores carbon in oceans.” The MCP also appears to function in deep waters, where bacteria adapted to the high-pressure environment may be able to degrade refractory DOC. Hiroshi Ogawa et al., showed that marine microbes are able to convert bioavailable DOC to refractory DOC2,4,5.

The present communication represents time studies of phage-host interactions under controlled conditions, in order to analyze their impact on the total carbon content of the source (nutrient broth) and their interconversion between organic and inorganic forms of carbon with respect to control samples. The control sample is just the nutrient broth without the inoculation of bacterium and their respective phage.

Materials and methods

The experiment was designed to measure the inorganic carbon levels in three conditions: control (nutrient broth only), bacteria alone and bacteria with their specific phage. The bacterium used during our study was E. coli (ATCC, strain 13706) and the bacteriophage used was phi X174 (ATCC, strain 13706 B1). They represent a good model for carbon conversion and interconversion through phage-host interactions and their interaction can be easily determined by the instruments like TOC analyzer3,6,7.

All three experimental conditions were conducted in 1L of sterilized nutrient broth each as to have a defined composition of the nutrients available for our study (HiMedia Pvt. Ltd.). For the bacteria without phage condition, sterilized nutrient broth media was inoculated with 100 cfu/ml of E. coli (ATCC 13706) previously enriched and incubated at 37°C; for the bacteria with phage condition approximately 1 ml of 1000 pfu/ml of phage were added. All flasks were sealed and incubated at 37°C for 18 hours. For control condition, sterile uninoculated nutrient broth was kept at 4°C throughout the experiment.

The initial reading were analyzed by a total organic carbon (TOC) analyzer (Shimadzu, Japan Model: TOC-Vcph) after 18 hours of incubation for all three sets of samples were recorded as “0” hours reading and before inoculation of bacteria and phages (see Table 1 and Table 2). TOC analysis was further carried out after every 2 hours until a stationary state was achieved. The stationary phase for inorganic carbon was defined by no further increase or decrease in the reading of inorganic carbon.

Table 1. TOC analysis results of control and bacterial samples (with and without phage).

Experiment No. 1Control 1 (ppm)Sample without phage 1 (ppm)Sample with phage 1 (ppm)
Time (hours)TOCTCICTOCTCICTOCTCIC
0291529160.71182740276928.912780281131.53
2283428340.91822818284728.912788281829.72
4250725080.94322162219329.862209223931.38
6243624370.84392301232724.772517254325.34
8215221531.0641921194622.271906192925.89
10192919300.89171530156222.241372139431.51
12188718880.96371757179831.271496152831.93
14182718280.92171415145843.091759180950.66
16190319570.99261658178755.471844205066.94
18216922591.04591931204363.192078227974.41
20239124381.09372179230579.542367239989.23
22261326951.18532444251787.9225742583102.11
24288028821.2382689278494.7626482764116.4
26274127421.7512726281185.8326842789105.5
28333333321.5573047312679.5930913196105.5

Table 2. TOC analysis results of control and bacterial samples (with and without phage).

Experiment No. 2Control 2 (ppm)Sample without phage 2 (ppm)Sample with phage 2 (ppm)
Time (hours)TOCTCICTOCTCICTOCTCIC
0304130420.79922789281828.962844287127.47
2287128720.94592922295128.612756279437.72
4257325740.88082360238929.132365239631.26
6216721680.84492345237024.772286231933.11
8218421851.0391935195723.161953198330.04
10145614571.0041574160025.941536157033.44
12190719080.96371819185234.151592163037.37
14163116320.90142032211564.522023208882.56
16187519171.00132197228373.792113219390.15
18204721321.10212367237886.212281228497.58
20229423531.20082429254192.3423352409104.91
22245525061.35022609276697.8824492523111.63
24267926811.42127522853100.925382657119
26277327751.5332779287798.7727012818116.8
28324432451.653157325092.2230053113107.2

Please refer Figure 1 and Figure 2 for understanding the principle of TOC analysis and different types of carbon compounds. The overall experiment was repeated for 10 times and their averages are represented in the Table 1 and Table 2.

5ab50a97-4297-4fe7-b680-b8a731342329_figure1.gif

Figure 1. Principle of TOC analysis.

5ab50a97-4297-4fe7-b680-b8a731342329_figure2.gif

Figure 2. Flow chart showing ingredient components of total carbon.

5ab50a97-4297-4fe7-b680-b8a731342329_figure3.gif

Figure 3. Variation in inorganic carbon content (in ppm) with respect to time (in hours).

5ab50a97-4297-4fe7-b680-b8a731342329_figure4.gif

Figure 4. Variations in inorganic carbon content (in ppm) with respect to time (in hours).

Results

The average results of the three sets are represented in Table 1 and Table 2, which show that the inorganic carbon content of the samples increased over time (except control) in both sets. The sample set with host-phage inoculation showed a increased reading of inorganic carbon levels compared to bacteria-only. There was an average 15–25 percent increase in inorganic carbon composition of sample set with host-phage inoculation. The result indicates that the phages may have role in regulation of carbon in aquatic systems through carbon sequestration or conversion in different biologically unavailable forms and can elevate inorganic carbon content levels in aqueous environments.

Discussion

The increase in inorganic carbon content may be due to lysis of the host cell releasing its refractory carbon compounds and respiration produced CO2 during utilization of carbon constituent for phage assembly and development. These controlled experiment mimics the continuous viral infections occurring in the different aquatic environments2,4,5. The consistent rise in the inorganic content is an indicator that, viruses somehow, seems to regulate carbon cycle to a greater extent as observed from the increase in IC level. The analytical results as indicated from the TOC analyzer are sole representation of phage lyses event and are worth analyzing further. If we are able to understand the biochemical mechanism and the byproducts generated during this whole process we may be able to determine the carbon sequestration in a better way. Considerable research activity needs to be initiated involving different environments conditions, parameters, sources, etc to facilitate better understanding of viral life cycle involving carbon cycle as an important area of future research. It can be proposed that carbon conversation during these studies gives us the clear ideas of the possible fate of carbon cycle and the role of phages. Similarly, we can also try to elucidate the role of phages (viruses) influencing other biogeochemical cycles including Nitrogen and Sulphur by using CHNS analyzer for better understanding of this process. It is also known that the infection of microbes also alters host metabolism significantly. Carbon sequestering algae like cyanobacteria are infected by cyanophages, which complicates our understanding further and demanding further in-depth studies. Lysogenic condition established by viruses under nutrient depleted condition or harsh environment can regulate the carbon utilization processes differently. Hence, the effect of viral infection on host metabolism remains unknown58.

Future work is essential for understanding the cellular processes especially infected (Lysogenic) host species. It will also prove helpful in deciphering the role of phages in regulating the carbon flow in the aquatic systems like oceans where their concentration outnumbered other species.

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Sanmukh S, Khairnar K, Paunikar W and Lokhande S. Understanding carbon regulation in aquatic systems - Bacteriophages as a model [version 1; peer review: 2 approved]. F1000Research 2015, 4:138 (https://doi.org/10.12688/f1000research.6031.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 01 Jun 2015
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Reviewer Report 15 Jul 2015
Mayur Bharat Kurade, Department of Natural Resources and Environmental Engineering, Hanyang University, Seoul, South Korea 
Approved
VIEWS 19
This paper deals with Bacteriophages as a model for carbon regulation in aquatic systems. The increase in inorganic carbon content was 60-70-fold in samples with bacteria and phage, and 50-55-fold for samples with bacteria alone being reported by the authors. The ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Kurade MB. Reviewer Report For: Understanding carbon regulation in aquatic systems - Bacteriophages as a model [version 1; peer review: 2 approved]. F1000Research 2015, 4:138 (https://doi.org/10.5256/f1000research.6457.r9219)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 15 Jul 2015
    Swapnil Sanmukh, Environmental Virology Cell, National Environmental Engineering Research Institute (NEERI), Nagpur, 440020, India
    15 Jul 2015
    Author Response
    Dear Sir,

    First of all thank you for approving our manuscript.

    Regarding your queries:

    -We will change the abbreviations for the units used in the figures and results.
    -We will modify the reference style ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 15 Jul 2015
    Swapnil Sanmukh, Environmental Virology Cell, National Environmental Engineering Research Institute (NEERI), Nagpur, 440020, India
    15 Jul 2015
    Author Response
    Dear Sir,

    First of all thank you for approving our manuscript.

    Regarding your queries:

    -We will change the abbreviations for the units used in the figures and results.
    -We will modify the reference style ... Continue reading
Views
16
Cite
Reviewer Report 22 Jun 2015
Balendu Shekher Giri, Centre for Biofuels and Biotechnology Division, Council of Scientific and Industrial Research (CSIR), Thiruvananthapuram, India 
Approved
VIEWS 16
This paper deals with an understanding of the use of Bacteriophages as a model for carbon regulation in aquatic systems The results show the increase in inorganic carbon content by 15-25 percent in samples with bacteria and phage compared to samples with ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Giri BS. Reviewer Report For: Understanding carbon regulation in aquatic systems - Bacteriophages as a model [version 1; peer review: 2 approved]. F1000Research 2015, 4:138 (https://doi.org/10.5256/f1000research.6457.r8840)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 23 Jun 2015
    Swapnil Sanmukh, Environmental Virology Cell, National Environmental Engineering Research Institute (NEERI), Nagpur, 440020, India
    23 Jun 2015
    Author Response
    Dear Sir,

    First of all thank you for approving our manuscript.
    Regarding your queries:
    -I think most of them are not quite critical but we will cite figure 3 and figure 4 as ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 23 Jun 2015
    Swapnil Sanmukh, Environmental Virology Cell, National Environmental Engineering Research Institute (NEERI), Nagpur, 440020, India
    23 Jun 2015
    Author Response
    Dear Sir,

    First of all thank you for approving our manuscript.
    Regarding your queries:
    -I think most of them are not quite critical but we will cite figure 3 and figure 4 as ... Continue reading

Comments on this article Comments (0)

Version 1
VERSION 1 PUBLISHED 01 Jun 2015
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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