<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Publishing DTD v1.2 20190208//EN" "http://jats.nlm.nih.gov/publishing/1.2/JATS-journalpublishing1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="other" dtd-version="1.2" xml:lang="en">
    <front>
        <journal-meta>
            <journal-id journal-id-type="pmc">F1000Research</journal-id>
            <journal-title-group>
                <journal-title>F1000Research</journal-title>
            </journal-title-group>
            <issn pub-type="epub">2046-1402</issn>
            <publisher>
                <publisher-name>F1000 Research Limited</publisher-name>
                <publisher-loc>London, UK</publisher-loc>
            </publisher>
        </journal-meta>
        <article-meta>
            <article-id pub-id-type="doi">10.12688/f1000research.12438.1</article-id>
            <article-categories>
                <subj-group subj-group-type="heading">
                    <subject>Research Note</subject>
                </subj-group>
                <subj-group>
                    <subject>Articles</subject>
                    <subj-group>
                        <subject>Marine &amp; Freshwater Ecology</subject>
                    </subj-group>
                </subj-group>
            </article-categories>
            <title-group>
                <article-title>Growth performance and feed utilization of African catfish 
                    <italic>Clarias gariepinus</italic> fed a commercial diet and reared in the biofloc system enhanced with probiotic</article-title>
                <fn-group content-type="pub-status">
                    <fn>
                        <p>[version 1; peer review: 2 approved]</p>
                    </fn>
                </fn-group>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Putra</surname>
                        <given-names>Iskandar</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Funding Acquisition</role>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <role content-type="http://credit.niso.org/">Resources</role>
                    <role content-type="http://credit.niso.org/">Validation</role>
                    <uri content-type="orcid">https://orcid.org/0000-0001-5531-2309</uri>
                    <xref ref-type="aff" rid="a1">1</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Rusliadi</surname>
                        <given-names>Rusliadi</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Data Curation</role>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <xref ref-type="aff" rid="a1">1</xref>
                </contrib>
                <contrib contrib-type="author" corresp="yes">
                    <name>
                        <surname>Fauzi</surname>
                        <given-names>Muhammad</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Data Curation</role>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <uri content-type="orcid">https://orcid.org/0000-0002-9159-155X</uri>
                    <xref ref-type="corresp" rid="c1">a</xref>
                    <xref ref-type="aff" rid="a1">1</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Tang</surname>
                        <given-names>Usman M.</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <role content-type="http://credit.niso.org/">Validation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <xref ref-type="aff" rid="a1">1</xref>
                </contrib>
                <contrib contrib-type="author" corresp="no">
                    <name>
                        <surname>Muchlisin</surname>
                        <given-names>Zainal A.</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Supervision</role>
                    <role content-type="http://credit.niso.org/">Validation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <uri content-type="orcid">https://orcid.org/0000-0002-0858-1853</uri>
                    <xref ref-type="aff" rid="a2">2</xref>
                </contrib>
                <aff id="a1">
                    <label>1</label>Faculty of Fisheries and Marine Sciences, Riau University, Pekanbaru, Riau, 28293, Indonesia</aff>
                <aff id="a2">
                    <label>2</label>Faculty of Marine and Fisheries, Syiah Kuala University, Banda Aceh, 23111, Indonesia</aff>
            </contrib-group>
            <author-notes>
                <corresp id="c1">
                    <label>a</label>
                    <email xlink:href="mailto:m.fauzi@lecturer.unri.ac.id">m.fauzi@lecturer.unri.ac.id</email>
                </corresp>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>22</day>
                <month>8</month>
                <year>2017</year>
            </pub-date>
            <pub-date pub-type="collection">
                <year>2017</year>
            </pub-date>
            <volume>6</volume>
            <elocation-id>1545</elocation-id>
            <history>
                <date date-type="accepted">
                    <day>22</day>
                    <month>8</month>
                    <year>2017</year>
                </date>
            </history>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2017 Putra I et al.</copyright-statement>
                <copyright-year>2017</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
                <license>
                    <license-p>The author(s) is/are employees of the US Government and therefore domestic copyright protection in USA does not apply to this work. The work may be protected under the copyright laws of other jurisdictions when used in those jurisdictions.</license-p>
                </license>
            </permissions>
            <self-uri content-type="pdf" xlink:href="https://f1000research.com/articles/6-1545/pdf"/>
            <abstract>
                <p>

                    <bold>Background</bold>&#x00a0;</p>
                <p>The objective of the present study was to evaluate the growth performance and feed utilization of African catfish 
                    <italic toggle="yes">Clarias gariepinus</italic> fed a commercial diet and reared in the biofloc system enhanced with probiotic.</p>
                <p>

                    <bold>Methods</bold>&#x00a0;</p>
                <p>The treatment was the frequency of probiotic application into the cultured system, namely, 5-day interval, 10-day interval, and 15-day interval for 60 days of experiment. Biofloc culture was grown in an experiment tank (vol. 2000 L) by mixing the probiotic (
                    <italic toggle="yes">Bacillus</italic> sp.) 10 mL and molasses 200 mL per liter of water. The fish was stocked into the biofloc system 7 days after cultured at stocking density of 1000 fish tank
                    <sup>-1</sup>. The fish was fed a commercial diet that contains 38% crude protein, twice a day at satiation. The application of probiotic was reperformed after 5 days, 10 days, and 15 days after stocking.</p>
                <p>

                    <bold>Results</bold>&#x00a0;</p>
                <p>The study showed that the growth performance, survival, and feed utilization of African catfish were higher in the treatment at 5-day intervals over 60 days. The ANOVA test showed that the application frequency of probiotic into biofloc system of cultured media had the significant effect on the growth performance, survival rate, and feed utilization of African catfish.</p>
                <p>

                    <bold>Conclusion</bold>&#x00a0;</p>
                <p>The best growth performance and feed utilization were found at the application of probiotic into biofloc system at 5-day intervals over 60 days.</p>
            </abstract>
            <kwd-group kwd-group-type="author">
                <kwd>Biofloc</kwd>
                <kwd>Probiotic Frequency</kwd>
                <kwd>Survival Rate</kwd>
            </kwd-group>
            <funding-group>
                <award-group id="fund-1">
                    <funding-source>Ministry of Research, Technology and Higher Education of the Republic of Indonesia</funding-source>
                    <award-id>430/UN.19.5.1.3/LT/2016</award-id>
                </award-group>
                <funding-statement>This study was supported by the Ministry of Research, Technology and Higher Education (Ristekdikti) of the Republic of Indonesia through the competitive grants scheme (Contract number: 430/UN.19.5.1.3/LT/2016). </funding-statement>
                <funding-statement>
                    <italic>The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</italic>
                </funding-statement>
            </funding-group>
        </article-meta>
    </front>
    <body>
        <sec sec-type="intro">
            <title>Introduction</title>
            <p>Feed is one of the important agro-inputs in aquaculture production system that contributes to approximately 40&#x2013;60% of production cost
                <sup>
                    <xref ref-type="bibr" rid="ref-1">1</xref>,
                    <xref ref-type="bibr" rid="ref-2">2</xref>
                </sup> and it has direct effect on the growth rate of the fish
                <sup>
                    <xref ref-type="bibr" rid="ref-3">3</xref>&#x2013;
                    <xref ref-type="bibr" rid="ref-6">6</xref>
                </sup>. The aquaculture activity is commonly produced waste, for example, feed remains and feces which changes into ammonia and nitrite once the oxygen level is low. In the closed culture system the concentrations of ammonia (NH
                <sub>3</sub>) and nitrite (NO
                <sub>2</sub>) are increasing rapidly and would be toxic to organisms
                <sup>
                    <xref ref-type="bibr" rid="ref-7">7</xref>,
                    <xref ref-type="bibr" rid="ref-8">8</xref>
                </sup>.</p>
            <p>According to Asaduzzaman 
                <italic toggle="yes">et al.</italic>
                <sup>
                    <xref ref-type="bibr" rid="ref-9">9</xref>
                </sup> and De Schryver 
                <italic toggle="yes">et al.</italic>
                <sup>
                    <xref ref-type="bibr" rid="ref-10">10</xref>
                </sup> the intensive application of commercial feed in the aquaculture causes environmental pollution and increases the possibility of the disease outbreak. Therefore, the water quality management is crucial in the aquaculture system. The objective of water quality management is to provide the comfortable environment and meet the optimum requirements for cultured organisms
                <sup>
                    <xref ref-type="bibr" rid="ref-11">11</xref>
                </sup>. According to Gunadi and Hafsaridewi
                <sup>
                    <xref ref-type="bibr" rid="ref-12">12</xref>
                </sup> the microbial activities can be used to improve water quality and reduce the burden of contamination by fish farming waste. Therefore, the heterotrophic bacteria have promising potency to be applied in the utilization of waste ammonia in the fish culture. Beside, these bacteria are formed as a floc (clumps) in the cultures media; hence it can be used as an alternative feed source for cultured fish
                <sup>
                    <xref ref-type="bibr" rid="ref-13">13</xref>
                </sup>. Biofloc has abilities to suppress the toxic compounds such as ammonia and harmful bacteria (pathogenic) so that the cultured organisms grow well
                <sup>
                    <xref ref-type="bibr" rid="ref-14">14</xref>
                </sup>. Application of biofloc in the cultures system has been reported by several researchers, for example, in the culture of channel catfish
                <sup>
                    <xref ref-type="bibr" rid="ref-14">14</xref>,
                    <xref ref-type="bibr" rid="ref-15">15</xref>
                </sup>, in the South American catfish 
                <italic toggle="yes">Rhamdia quelen</italic>
                <sup>
                    <xref ref-type="bibr" rid="ref-16">16</xref>
                </sup>, in Nile tilapia 
                <italic toggle="yes">Oreochromis niloticus</italic>
                <sup>
                    <xref ref-type="bibr" rid="ref-17">17</xref>,
                    <xref ref-type="bibr" rid="ref-18">18</xref>
                </sup>, 
                <italic toggle="yes">Farfantepenaeus brasiliensis</italic>
                <sup>
                    <xref ref-type="bibr" rid="ref-19">19</xref>
                </sup>, and in the cultured system of the shrimps 
                <italic toggle="yes">Litopenaeus vannamei</italic> and 
                <italic toggle="yes">Penaeus monodon</italic>
                <sup>
                    <xref ref-type="bibr" rid="ref-11">11</xref>,
                    <xref ref-type="bibr" rid="ref-20">20</xref>
                </sup>. However, application of biofloc on African catfish 
                <italic toggle="yes">Clarias gariepinus</italic> cultures has never been reported previously.</p>
            <p>	African catfish is the popular species for aquaculture business in Southeast Asian countries
                <sup>
                    <xref ref-type="bibr" rid="ref-21">21</xref>
                </sup>. This species has several advantages, for example, resistance to diseases and handling stress and high growth rate
                <sup>
                    <xref ref-type="bibr" rid="ref-22">22</xref>
                </sup>, thus accounting for its commercial importance worldwide
                <sup>
                    <xref ref-type="bibr" rid="ref-23">23</xref>
                </sup>. Nowadays, the fish farmer fed a commercial diet for African catfish. The protein requirement for African catfish ranges from 25% to 40%, lipid 9.5 to 10%, carbohydrates 15 to 30%, vitamins 0.25 to 0.40%, and minerals 1.0%
                <sup>
                    <xref ref-type="bibr" rid="ref-1">1</xref>
                </sup>, with energy level of 2000 cal/g to 3000 cal/g
                <sup>
                    <xref ref-type="bibr" rid="ref-24">24</xref>
                </sup>. In addition, the application of probiotic into African catfish diet has been reported by several researchers, for example, Al-Dohail 
                <italic toggle="yes">et al.</italic>
                <sup>
                    <xref ref-type="bibr" rid="ref-25">25</xref>
                </sup>, Ige
                <sup>
                    <xref ref-type="bibr" rid="ref-26">26</xref>
                </sup>, and Dennis and Uchenna
                <sup>
                    <xref ref-type="bibr" rid="ref-27">27</xref>
                </sup>. However, application of probiotic combing with biofloc has never been reported previously. Hence, the aim of the study was to evaluate the growth performance and feed utilization of African catfish fed experimental diet reared in the biofloc cultured system and enhanced with probiotic.</p>
        </sec>
        <sec sec-type="materials">
            <title>Methods</title>
            <sec>
                <title>Site and time</title>
                <p>The research was conducted from June 2016 to August 2016 at Aquaculture Technology Laboratory, Faculty of Fishery and Marine Sciences, Riau University, Indonesia. The experiments were carried out within the ethical guidelines provided by the research institution and national or international regulations.</p>
            </sec>
            <sec>
                <title>Experimental design</title>
                <p>The completely random design (CRD) method was used in this study. The tested treatment was the frequency of probiotics application (bacteria inoculation), namely, at 5-day interval (treatment A), 10-day interval (treatment B), and 15-day interval (treatment C). The treatment was conducted at three replications. The experimental fish was maintained in the canvas tank (vol. 2000 L) at stocking density of 1000 fishes and reared for 60 days.</p>
            </sec>
            <sec>
                <title>Biofloc culture and feeding</title>
                <p>The biofloc was cultured in the nine canvas tanks with a volume of 2000 L. Each tank was filled with water up to a water level of 100 cm or equivalent to 2000 L. Biofloc culture was done by mixing the probiotic (
                    <italic toggle="yes">Bacillus</italic> sp.) 10 mL and molasses 200 mL L
                    <sup>-1</sup> of water and then mixed into the cultures fish tanks and aerated continuously for 7 days to grow the floc.</p>
                <p>The catfish larvae were stocked at the density of 1000 fish tank
                    <sup>-1</sup> with average weight 1.12&#x00b1;0.05 g and average total length 4.42&#x00b1;0.09 cm. The application of 10 mL inoculants bacteria with density of 
                    <italic toggle="yes">Bacillus</italic> sp. about 5&#x00d7;10
                    <sup>10</sup> colony forming units (CFU) was performed according to respective treatment, that is, 5-day, 10-day, and 15-day intervals. The experimental catfish feed was a commercial diet with 38% crude protein, crude lipid 5%, and crude fiber 6%, mineral mix 13%, and 13% moisture contents. The fish were fed twice a day at satiation. The weight gain of fish was measured every 12 -days for 60 days.</p>
            </sec>
            <sec>
                <title>Measured parameters</title>
                <p>The weight gain was calculated as follows: W = Wt &#x2013; Wo, where W is weight gain (g), Wt is the weight of the fish at the end of experiment (g), and Wo is the weight of fish at the start of experiment (g). The daily growth rate, survival rates, and feed utilization were calculated based on Muchlisin 
                    <italic toggle="yes">et al.</italic>
                    <sup>
                        <xref ref-type="bibr" rid="ref-28">28</xref>,
                        <xref ref-type="bibr" rid="ref-29">29</xref>
                    </sup> The main water quality parameters such as dissolved oxygen (DO), pH, and temperature were measured using a digital water checker (YSI-550 A, ASTM, Alla, France) at 6-day intervals, while total ammonia nitrogen (TAN) was measured every 6 days using spectrophotometric method
                    <sup>
                        <xref ref-type="bibr" rid="ref-30">30</xref>
                    </sup>.</p>
            </sec>
            <sec>
                <title>Data analysis</title>
                <p>The data were subjective to one-way analysis of variant (ANOVA) test to determine the effect of treatment on the tested parameters and followed by Newman-Keuls multiple range test with a confidence level of 95%, while the water quality of the data was analyzed descriptively.</p>
            </sec>
        </sec>
        <sec sec-type="results">
            <title>Results</title>
            <p>The ANOVA test showed that the treatment had a significant effect on the weight gain (WG), specific growth rate and survival rate (SGR), feed efficiency (FE), and feed conversion ratio (FCR) (P&lt;0.05). The study showed that the highest weight gain and specific growth rate were recorded at treatment A; these values were different significantly from other treatments. A similar trend was also found in the survival rate (SR) where the highest survival rate was recorded in treatment A, but this value was not different significantly from treatment C (
                <xref ref-type="table" rid="T1">Table 1</xref>). The highest feed efficiency and lower feed conversion ratio were also found in fish with application of probiotic into biofloc system at 5-day intervals (treatment A). However, these values were not different significantly from treatment C (probiotic application at 15-day intervals). In addition, the water temperature ranges from 29.50&#x00b0;C to 29.62&#x00b0;C, dissolved oxygen rages from 3.64 mg L
                <sup>-1</sup> to 3.88 mg L
                <sup>-1</sup>, and pH ranges from 6.93 to 7.02. In addition, the ammonia (NH
                <sub>3</sub>) content ranged from 0.292 mg L
                <sup>-1</sup> to 0.411 mg L
                <sup>-1</sup> and nitrite content ranged from 0.08 mg L
                <sup>-1</sup> to 0.09 mg L
                <sup>-1</sup>. Therefore, there were no significant differences regarding water quality among the treatments; however, the quality in treatment A was slightly better compared to two other treatments (
                <xref ref-type="table" rid="T2">Table 2</xref>). The data showing the total length, body weight and total feed consumed by fish at every experiment can be found in 
                <xref ref-type="other" rid="DS0">Dataset 1</xref>.</p>
            <table-wrap id="T1" orientation="portrait" position="anchor">
                <label>Table 1. </label>
                <caption>
                    <title>The growth performance, survival rate, and feed utilization of African catfish, 
                        <italic toggle="yes">Clarias gariepinus</italic>.</title>
                    <p>Mean of values in the same row followed by a different superscript that are significantly different (
                        <italic toggle="yes">p</italic>&lt;0.05).</p>
                </caption>
                <table content-type="article-table" frame="hsides">
                    <thead>
                        <tr>
                            <th align="left" colspan="1" rowspan="1" valign="top">No</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Parameter</th>
                            <th align="center" colspan="3" rowspan="1" valign="top">Application frequency of probiotic</th>
                        </tr>
                        <tr>
                            <th colspan="1" rowspan="1"/>
                            <th colspan="1" rowspan="1"/>
                            <th align="center" colspan="1" rowspan="1">5-day interval</th>
                            <th align="center" colspan="1" rowspan="1">10-day interval</th>
                            <th align="center" colspan="1" rowspan="1">15-day interval</th>
                        </tr>
                    </thead>
                    <tbody>
                        <tr>
                            <td colspan="1" rowspan="1">1.</td>
                            <td colspan="1" rowspan="1">Weight gain
                                <break/>(g)</td>
                            <td align="center" colspan="1" rowspan="1">125.89&#x00b1;1.96
                                <sup>b</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">85.57&#x00b1;5.80
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">94.19&#x00b1;22.81
                                <sup>a</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">2.</td>
                            <td colspan="1" rowspan="1">SGR
                                <break/>(% day
                                <sup>-1</sup>)</td>
                            <td align="center" colspan="1" rowspan="1">7.91&#x00b1;0.06
                                <sup>b</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">7.28&#x00b1;0.06
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">7.34&#x00b1;0.40
                                <sup>a</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">3.</td>
                            <td colspan="1" rowspan="1">Survival rate
                                <break/>(%)</td>
                            <td align="center" colspan="1" rowspan="1">95.77&#x00b1;0.66
                                <sup>b</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">75.23&#x00b1;9.70
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">91.37&#x00b1;4.78
                                <sup>b</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">4.</td>
                            <td colspan="1" rowspan="1">Efficiency of
                                <break/>the feed (%)</td>
                            <td align="center" colspan="1" rowspan="1">110.86&#x00b1;2.60
                                <sup>b</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">88.17&#x00b1;6.89
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">90.98&#x00b1;5.69
                                <sup>a</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">5.</td>
                            <td colspan="1" rowspan="1">Feed
                                <break/>conversion
                                <break/>ratio</td>
                            <td align="center" colspan="1" rowspan="1">0.90&#x00b1;0.02
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">1.14&#x00b1;0.08
                                <sup>b</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">1.10&#x00b1;0.07
                                <sup>b</sup>
                            </td>
                        </tr>
                    </tbody>
                </table>
            </table-wrap>
            <table-wrap id="T2" orientation="portrait" position="anchor">
                <label>Table 2. </label>
                <caption>
                    <title>The main water quality parameter of the cultured media of African catfish (
                        <italic toggle="yes">Clarias gariepinus</italic>).</title>
                </caption>
                <table content-type="article-table" frame="hsides">
                    <thead>
                        <tr>
                            <th align="left" colspan="1" rowspan="2">Parameters</th>
                            <th align="left" colspan="1" rowspan="2">Unit</th>
                            <th align="center" colspan="3" rowspan="1">Application frequency of probiotic</th>
                        </tr>
                        <tr>
                            <th align="center" colspan="1" rowspan="1">5-day
                                <break/>interval</th>
                            <th align="center" colspan="1" rowspan="1">10-day
                                <break/>interval</th>
                            <th align="center" colspan="1" rowspan="1">15-day
                                <break/>interval</th>
                        </tr>
                    </thead>
                    <tbody>
                        <tr>
                            <td colspan="1" rowspan="1">Temperature</td>
                            <td colspan="1" rowspan="1">C&#x00b0;</td>
                            <td align="center" colspan="1" rowspan="1">29.53&#x00b1;1.72
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">29.62&#x00b1;1.82
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">29.5&#x00b1;1.72
                                <sup>a</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">DO</td>
                            <td colspan="1" rowspan="1">mg L
                                <sup>-1</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">3.88&#x00b1;0.44
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">3.64&#x00b1;0.32
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">3.64&#x00b1;0.35
                                <sup>a</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">pH</td>
                            <td colspan="1" rowspan="1">-</td>
                            <td align="center" colspan="1" rowspan="1">7.02&#x00b1;0.10
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">6.93&#x00b1;0.11
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">6.97&#x00b1;0.09
                                <sup>a</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">(NH
                                <sub>3</sub>)</td>
                            <td colspan="1" rowspan="1">mg L
                                <sup>-1</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">0.292&#x00b1;0.11
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">0.332&#x00b1;0.176
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">0.411&#x00b1;0.195
                                <sup>b</sup>
                            </td>
                        </tr>
                        <tr>
                            <td colspan="1" rowspan="1">NO
                                <sub>2</sub>
                            </td>
                            <td colspan="1" rowspan="1">mg L
                                <sup>-1</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">0.09&#x00b1;0.076
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">0.08&#x00b1;0.036
                                <sup>a</sup>
                            </td>
                            <td align="center" colspan="1" rowspan="1">0.09&#x00b1;0.035
                                <sup>a</sup>
                            </td>
                        </tr>
                    </tbody>
                </table>
            </table-wrap>
            <supplementary-material id="DS0" orientation="portrait" position="float" xlink:href="https://f1000researchdata.s3.amazonaws.com/datasets/12438/0268b804-9820-430e-b5be-8cb7db3077c4_RAW_DATA_OK.xls">
                <label>Study results</label>
                <caption>
                    <p>The total length, body weight and total feed consumed by fish at every experiment</p>
                </caption>
            </supplementary-material>
        </sec>
        <sec sec-type="discussion">
            <title>Discussion</title>
            <p>The study showed that the growth performance, survival rate, and feed utilization of African catfish were the highest in the application of probiotic into the biofloc system at 5-day intervals. This was presumably due to the fact that the applications of probiotics every 5-days can maintain the density of bacteria at suitable forms and effectively decompose organic materials well. This is indicated by lower ammonium (NH
                <sub>3</sub>) content in treatment A. According to Widanarni
                <sup>
                    <xref ref-type="bibr" rid="ref-31">31</xref>
                </sup> the application of biofloc into culture system can improve water quality and reduce the burden of contamination of fish culture waste in the surrounding waters. In addition Irianto
                <sup>
                    <xref ref-type="bibr" rid="ref-32">32</xref>
                </sup> stated that 
                <italic toggle="yes">Bacillus</italic> sp. can improve the quality of the cultured media by decomposing organic materials, suppress the growth of pathogenic, and balance the microbial and had a positive effect on fish health and growth. </p>
            <p>Besides maintaining the water quality, biofloc is also playing an important role as alternative natural feed for cultured fish. This is because the biofloc contains crude protein that reached 48&#x2013;53%
                <sup>
                    <xref ref-type="bibr" rid="ref-33">33</xref>,
                    <xref ref-type="bibr" rid="ref-34">34</xref>
                </sup> and therefore the Feed Conversion Ratio (FCR) in treatment A was 0.90 (below 1.00) and the feed efficiency was higher than 100%. This is because of beside fed on the commercial diet the fish was also fed on floc that contain planktons. This value is better than fish fed on commercial diet without application of biofluc
                <sup>
                    <xref ref-type="bibr" rid="ref-33">33</xref>,
                    <xref ref-type="bibr" rid="ref-35">35</xref>,
                    <xref ref-type="bibr" rid="ref-36">36</xref>
                </sup>. According to Azim
                <sup>
                    <xref ref-type="bibr" rid="ref-34">34</xref>
                </sup> the nutritional quality of biofloc was appropriate at least for herbivorous and omnivorous fish species. In this case, the African catfish is categorized as omnivorous feeding habits
                <sup>
                    <xref ref-type="bibr" rid="ref-35">35</xref>,
                    <xref ref-type="bibr" rid="ref-37">37</xref>
                </sup>. </p>
            <p>It is clear that biofloc contributed to the growth and production of cultured organism as shown in this study. The basic principle of this technology is using the heterotrophic bacteria to manage the C: N ratio in the water media
                <sup>
                    <xref ref-type="bibr" rid="ref-33">33</xref>,
                    <xref ref-type="bibr" rid="ref-38">38</xref>,
                    <xref ref-type="bibr" rid="ref-39">39</xref>
                </sup>. However, biofloc not only contains the bacteria, but also is composed of other microorganisms including microalgae and zooplanktons as food for farmed fish or shrimps
                <sup>
                    <xref ref-type="bibr" rid="ref-33">33</xref>
                </sup>. According to Crab 
                <italic toggle="yes">et al</italic>.
                <sup>
                    <xref ref-type="bibr" rid="ref-40">40</xref>
                </sup> biofloc can be consumed and digested well by the shrimp and therefore possibly substitute for artificial commercial feed. Hence, application of biofloc into cultured system can increase feed efficiency up to 13%
                <sup>
                    <xref ref-type="bibr" rid="ref-39">39</xref>
                </sup>. For example, for feed efficiency of African catfish fed a commercial diet without biofloc was 89.83 %
                <sup>
                    <xref ref-type="bibr" rid="ref-33">33</xref>
                </sup>; it was increased up to 110.86% when the biofloc was applied as shown in this study.</p>
            <p>In addition, according to Avnimelech
                <sup>
                    <xref ref-type="bibr" rid="ref-38">38</xref>
                </sup>, the addition of molasses as a source of carbon in aquaculture system can improve the C/N ratio waters and will further reduce inorganic nitrogen in the waters through increased growth of heterotrophic bacteria, where the heterotrophic bacteria will form a floc which can be fed by fish as feed source. Furthermore the C: N ratio of &gt;10: 1 in the fish farming system is the optimum ratio to enhance the biofloc production and minimize the ammonia regeneration 
                <sup>
                    <xref ref-type="bibr" rid="ref-39">39</xref>
                </sup>.</p>
        </sec>
        <sec sec-type="conclusions">
            <title>Conclusion</title>
            <p>The application of probiotic bacteria with different frequencies in the biofloc system had the significant effect on the growth performance, survival rate, and feed utilization of African catfish (
                <italic toggle="yes">Clarias gariepinus</italic>). The higher growth performance and best feed utilization were recorded in the application of probiotic into biofloc system at 5-day intervals.</p>
        </sec>
        <sec>
            <title>Data availability</title>
            <p>The data referenced by this article are under copyright with the following copyright statement: Copyright: &#x00ef;&#x00bf;&#x00bd; 2017 Putra I et al.</p>
            <p>Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).
                <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/"/>
            </p>
            <p>Dataset 1: The total length, body weight and total feed consumed by fish at every experiment. 
                <ext-link ext-link-type="uri" xlink:href="http://dx.doi.org/10.5256/f1000research.12438.d174980">10.5256/f1000research.12438.d174980</ext-link>
                <sup>
                    <xref ref-type="bibr" rid="ref-41">41</xref>
                </sup>
            </p>
        </sec>
    </body>
    <back>
        <ack>
            <title>Acknowledgments</title>
            <p>The authors thank the Ministry of Research, Technology and Higher Education of the Republic of Indonesia for supporting this study through the competitive grants scheme 2016 Contract number: 430/UN.19.5.1.3/LT/2016. The appreciation goes to all of our students who helped the authors during experiments in the laboratory.</p>
        </ack>
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    </back>
    <sub-article article-type="reviewer-report" id="report25289">
        <front-stub>
            <article-id pub-id-type="doi">10.5256/f1000research.13468.r25289</article-id>
            <title-group>
                <article-title>Reviewer response for version 1</article-title>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author">
                    <name>
                        <surname>Nugroho</surname>
                        <given-names>Rudy Agung</given-names>
                    </name>
                    <xref ref-type="aff" rid="r25289a1">1</xref>
                    <role>Referee</role>
                    <uri content-type="orcid">https://orcid.org/0000-0001-9006-7329</uri>
                </contrib>
                <aff id="r25289a1">
                    <label>1</label>Animal Physiology, Development and Molecular Laboratory, Department of Biology, Faculty of Mathematic and Natural Science, Mulawarman University, Samarinda, Indonesia</aff>
            </contrib-group>
            <author-notes>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>5</day>
                <month>9</month>
                <year>2017</year>
            </pub-date>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2017 Nugroho RA</copyright-statement>
                <copyright-year>2017</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <related-article ext-link-type="doi" id="relatedArticleReport25289" related-article-type="peer-reviewed-article" xlink:href="10.12688/f1000research.12438.1"/>
            <custom-meta-group>
                <custom-meta>
                    <meta-name>recommendation</meta-name>
                    <meta-value>approve</meta-value>
                </custom-meta>
            </custom-meta-group>
        </front-stub>
        <body>
            <p>1. Title is suitable and clearly defined the research that has been done.</p>
            <p> </p>
            <p> 2. Abstract is well written and attract the reader. Please add information regarding the CFU of the bacillus (ex: 10 mL inoculants bacteria with density of 
                <italic>Bacillus</italic> sp. about 5&#x00d7;10
                <sup>10</sup> colony forming units (CFU) ....).</p>
            <p> </p>
            <p> 3. Introduction: the introduction is well constructed and supported with current references.</p>
            <p> </p>
            <p> 4. Methods: a) please be specific on the ethical guidelines that author's performed in this research. Which international/national ethical guidelines. b) Please explain why the author use 5 days interval in this research. Is there any previous/preliminary research?</p>
            <p> </p>
            <p> 5. Results: Survival rate of B2 groups was only 65% (Raw data), any explanation?</p>
            <p> </p>
            <p> 6. Discussion: Good Discussion.</p>
            <p> </p>
            <p> 7. Conclusion: Well constructed summary.</p>
            <p> </p>
            <p> 8. Reference: please revise reference #1 :&#x00a0; (Sahwan MF: The feed fish and shrimp. 
                <italic>Penebar Swadaya.</italic> Jakarta. 1999. ), with original title and give translation. otherwise it cannot be traced.</p>
            <p>Is the work clearly and accurately presented and does it cite the current literature?</p>
            <p>Yes</p>
            <p>If applicable, is the statistical analysis and its interpretation appropriate?</p>
            <p>Yes</p>
            <p>Are all the source data underlying the results available to ensure full reproducibility?</p>
            <p>Partly</p>
            <p>Is the study design appropriate and is the work technically sound?</p>
            <p>Yes</p>
            <p>Are the conclusions drawn adequately supported by the results?</p>
            <p>Yes</p>
            <p>Are sufficient details of methods and analysis provided to allow replication by others?</p>
            <p>Yes</p>
            <p>Reviewer Expertise:</p>
            <p>Animal Physiology, Fish nutrition, Fish Immunology</p>
            <p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.</p>
        </body>
    </sub-article>
    <sub-article article-type="reviewer-report" id="report25287">
        <front-stub>
            <article-id pub-id-type="doi">10.5256/f1000research.13468.r25287</article-id>
            <title-group>
                <article-title>Reviewer response for version 1</article-title>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author">
                    <name>
                        <surname>Syandri</surname>
                        <given-names>Hafrijal</given-names>
                    </name>
                    <xref ref-type="aff" rid="r25287a1">1</xref>
                    <role>Referee</role>
                    <uri content-type="orcid">https://orcid.org/0000-0002-3684-8842</uri>
                </contrib>
                <aff id="r25287a1">
                    <label>1</label>Department of Aquaculture, Faculty of Fisheries and Marine Science, Bung Hatta University&#x00a0;, Padang, Indonesia</aff>
            </contrib-group>
            <author-notes>
                <fn fn-type="conflict">
                    <p>
                        <bold>Competing interests: </bold>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>25</day>
                <month>8</month>
                <year>2017</year>
            </pub-date>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2017 Syandri H</copyright-statement>
                <copyright-year>2017</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <related-article ext-link-type="doi" id="relatedArticleReport25287" related-article-type="peer-reviewed-article" xlink:href="10.12688/f1000research.12438.1"/>
            <custom-meta-group>
                <custom-meta>
                    <meta-name>recommendation</meta-name>
                    <meta-value>approve</meta-value>
                </custom-meta>
            </custom-meta-group>
        </front-stub>
        <body>
            <p>A comment for abstract: the fish was fed a commercial diet that contain 38% crude protein, twice a day at satiation, could you please show the time?</p>
            <p> </p>
            <p> A comment for the methodology:</p>
            <p> 1. Please state the type of commercial feed used? floating or drowned feed?</p>
            <p> 2. The weight gain of fish measured every 12 day for 60 days, please show the data weight gain every 12-days in bar diagram or line graph?</p>
            <p> 3. Please explain the version (including city) of software used in statistical analysis?</p>
            <p>Is the work clearly and accurately presented and does it cite the current literature?</p>
            <p>Yes</p>
            <p>If applicable, is the statistical analysis and its interpretation appropriate?</p>
            <p>Partly</p>
            <p>Are all the source data underlying the results available to ensure full reproducibility?</p>
            <p>Partly</p>
            <p>Is the study design appropriate and is the work technically sound?</p>
            <p>Yes</p>
            <p>Are the conclusions drawn adequately supported by the results?</p>
            <p>Yes</p>
            <p>Are sufficient details of methods and analysis provided to allow replication by others?</p>
            <p>Yes</p>
            <p>Reviewer Expertise:</p>
            <p>NA</p>
            <p>I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.</p>
        </body>
    </sub-article>
</article>
