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Biotechnology and Industrial Microbiology
Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus
Kaio César da Silva Rodriguesa, Arianne Tairyne de Souzab, Alberto Colli Badinoa, Danielle Biscaro Pedrollic, Marcel Otavio Cerric,
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marcel@fcfar.unesp.br

Corresponding author.
a Universidade Federal de São Carlos, Departamento de Engenharia Química, São Carlos, SP, Brazil
b Universidade Federal de São João del-Rei, Campus Alto Paraopeba, Departamento de Química, Biotecnologia e Bioprocessos, Ouro Branco, MG, Brazil
c Universidade Estadual Paulista (UNESP), Departamento de Bioprocessos e Biotecnologia, Araraquara, SP, Brazil
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0015">Introduction</span><p id="par0005" class="elsevierStylePara elsevierViewall">Clavulanic acid &#40;CA&#41; is a &#946;-lactam compound with potent inhibitory activity against &#946;-lactamases&#46;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">1</span></a> These enzymes catalyze the hydrolysis of &#946;-lactam ring in antibiotics&#44; hence inactivating them&#46;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">2</span></a> Although CA is considered an antibiotic&#44; it has weak antibacterial activity and is ineffective if administrated alone&#46; Therefore&#44; CA in the form of the potassium salt is used in conjunction with &#946;-lactamase-sensitive antibiotics such as penicillin and cephalosporin&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">3</span></a> Such combinations increase the effectiveness of infection treatments&#44; which makes CA clinically and economically important&#46;</p><p id="par0010" class="elsevierStylePara elsevierViewall">Traditionally&#44; CA is produced from cultures of <span class="elsevierStyleItalic">Streptomyces clavuligerus</span>&#44; a filamentous aerobic bacterium belonging to the actinomycetes group&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">4</span></a> Although numerous studies concerning CA production have been published in recent decades&#44;<a class="elsevierStyleCrossRef" href="#bib0180"><span class="elsevierStyleSup">5</span></a> the process can still be improved&#46; Strategies to enhance CA production include manipulation of bioreactor operation mode<a class="elsevierStyleCrossRef" href="#bib0185"><span class="elsevierStyleSup">6</span></a> and conditions of agitation and aeration&#44;<a class="elsevierStyleCrossRef" href="#bib0190"><span class="elsevierStyleSup">7</span></a> as well as optimization of medium composition&#46;<a class="elsevierStyleCrossRef" href="#bib0195"><span class="elsevierStyleSup">8</span></a> Many different culture media have been reported for CA production&#46; Recent studies have used glycerol or lipid as carbon and energy sources&#46;<a class="elsevierStyleCrossRef" href="#bib0200"><span class="elsevierStyleSup">9</span></a> Soybean derivatives &#40;flour&#44; protein isolate&#44; and meal&#41; have been used as sources of nitrogen&#46;<a class="elsevierStyleCrossRefs" href="#bib0205"><span class="elsevierStyleSup">10&#44;11</span></a> Compounds containing phosphorus and magnesium are also included in culture media used for CA production&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> Ortiz et al&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">13</span></a> investigated the effects of soybean flour and soybean protein isolate as primary sources of nitrogen on CA production by <span class="elsevierStyleItalic">S&#46; clavuligerus</span>&#46; Soybean flour provided the highest CA production&#46; Teodoro et al&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a> evaluated the effect of the initial concentration of the nitrogen source in the medium&#46; A high initial nitrogen concentration &#40;about 4&#46;5<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> total N&#41; resulted in greater release of catabolic products in the broth&#44; inhibiting CA production&#46; Use of an initial total N concentration of about 3&#46;0<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> provided good cell growth and the highest CA production among the cultivations tested&#46;</p><p id="par0015" class="elsevierStylePara elsevierViewall">Some compounds are directly used to build the CA molecule and are essential for CA biosynthesis&#46; This phenomenon has been extensively studied and is well understood&#46; The first reaction is the condensation of precursors of C5 and C3 units&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">15</span></a> The C5 precursor is arginine&#44;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">16</span></a> which can be synthesized from ornithine and glutamate&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">17</span></a> Khaleeli et al&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">15</span></a> showed that the C3 precursor originates from glycerol&#46; Chen et al&#46;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">18</span></a> studied the effects of feeding glycerol&#44; ornithine and arginine on CA production in batch cultures&#46; The combination glycerol and ornithine &#40;molar ratio of 22&#58;1&#41; significantly increased CA production and provided greater stimulation of CA biosynthesis&#44; compared to glycerol and arginine &#40;molar ratio of 22&#58;1&#41;&#46; Wang et al&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> examined the effect of the addition of ornithine at different concentrations&#46; The highest CA yield was obtained using 1&#46;2<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> of ornithine&#46; Saudagar and Singhal<a class="elsevierStyleCrossRef" href="#bib0250"><span class="elsevierStyleSup">19</span></a> evaluated the influence of feeding with glycerol&#44; arginine&#44; and threonine in fed-batch cultures&#44; finding that the two amino acids acted to stimulate CA production&#46; Domingues et al&#46;<a class="elsevierStyleCrossRef" href="#bib0255"><span class="elsevierStyleSup">20</span></a> optimized the glycerol&#58;ornithine molar ratio in the feed medium in continuous cultures using a 5<span class="elsevierStyleHsp" style=""></span>L working volume bioreactor&#46; The best molar ratio was approximately 40&#58;1&#46;</p><p id="par0020" class="elsevierStylePara elsevierViewall">Progress has been made in elucidating the roles of amino acids such as ornithine&#44; arginine&#44; threonine&#44; and glutamate in CA biosynthesis&#46; However&#44; few studies have quantitatively determined the effects of these amino acids in CA production&#46; The aim of this study was therefore to screen several compounds involved in CA biosynthesis&#44; using experimental design&#46; This is a very useful tool that enables analysis not only of the effects of individual factors&#44; but also the interactions between factors&#44; in order to find the best conditions for the process&#46; Firstly&#44; cultivations were carried out in order to evaluate the influence of the primary nitrogen source and salts composition on CA production&#46; Subsequently&#44; the effect on CA production of supplementation of the primary nitrogen source with the amino acids arginine&#44; threonine&#44; ornithine&#44; and glutamate was evaluated using experimental design and response surface analysis&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0020">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0025">Microorganism</span><p id="par0025" class="elsevierStylePara elsevierViewall">The strain of <span class="elsevierStyleItalic">S&#46; clavuligerus</span> used in this study was purchased from the American Type Culture Collection &#40;ATCC-27064&#41;&#46; Vegetative cells were stored at &#8722;70<span class="elsevierStyleHsp" style=""></span>&#176;C in cryotubes with 10&#37; &#40;v<span class="elsevierStyleHsp" style=""></span>v<span class="elsevierStyleSup">&#8722;1</span>&#41; glycerol&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Culture media</span><p id="par0030" class="elsevierStylePara elsevierViewall">The seed medium had the following composition &#40;g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> in distilled water&#41;&#44; as proposed by Rosa et al&#46;<a class="elsevierStyleCrossRef" href="#bib0260"><span class="elsevierStyleSup">21</span></a>&#58; glycerol&#44; 10&#46;0&#59; yeast extract&#44; 1&#46;0&#59; bacto peptone&#44; 10&#46;0&#59; malt extract&#44; 10&#46;0&#59; K<span class="elsevierStyleInf">2</span>HPO<span class="elsevierStyleInf">4</span>&#44; 2&#46;5&#59; MgSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&#44; 0&#46;75&#59; MnCl<span class="elsevierStyleInf">2</span>&#183;4H<span class="elsevierStyleInf">2</span>O&#44; 0&#46;001&#59; FeSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&#44; 0&#46;001&#59; ZnSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&#44; 0&#46;001&#59; 3-&#40;N-morpholino&#41; propanesulfonic acid &#40;MOPS&#41; buffer&#44; 21&#46;0 &#40;100<span class="elsevierStyleHsp" style=""></span>mM&#41;&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">The inoculum and production media had the same composition&#46; First&#44; the influence of the primary nitrogen source &#40;soybean flour &#8211; SF and soybean protein isolate &#8211; SPI&#41; and the salts composition &#40;FeSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&#44; K<span class="elsevierStyleInf">2</span>HPO<span class="elsevierStyleInf">4</span>&#44; KH<span class="elsevierStyleInf">2</span>PO<span class="elsevierStyleInf">4</span>&#44; and MgSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&#41; was investigated&#46; Five different production media &#40;M1&#8211;M5&#41; were tested&#44; based on those reported previously by Teodoro et al&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a> and Wang et al&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a><a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a> presents the media compositions &#40;g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> in distilled water&#41;&#46; Subsequently&#44; the influence on CA production of supplementation of the primary nitrogen source with the amino acids arginine&#44; threonine&#44; ornithine&#44; and glutamate was investigated using an experimental design&#46; The M2 medium containing SPI was used as the basis for supplementation and the amounts of amino acids were altered according to the experimental design&#46; In all cases&#44; the pH of the medium was adjusted to 6&#46;8&#44; followed by autoclaving at 121<span class="elsevierStyleHsp" style=""></span>&#176;C for 15<span class="elsevierStyleHsp" style=""></span>min&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Experimental design</span><p id="par0040" class="elsevierStylePara elsevierViewall">A two-level central composite rotatable design &#40;CCRD&#41; and response surface methodology<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">22</span></a> were employed in this study&#46; Previous reports<a class="elsevierStyleCrossRefs" href="#bib0215"><span class="elsevierStyleSup">12&#44;16&#44;18&#44;19</span></a> indicated that arginine&#44; threonine&#44; ornithine&#44; and glutamate significantly affect CA biosynthesis&#46; In this study&#44; these amino acids were chosen as the independent variables&#44; denoted <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">1</span>&#44; <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">2</span>&#44; <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">3</span>&#44; and <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">4</span>&#46; The response variable was the CA concentration&#46; A second order polynomial was used to calculate the predicted response &#40;Eq&#46; <a class="elsevierStyleCrossRef" href="#eq0005">&#40;1&#41;</a>&#41;&#46;<elsevierMultimedia ident="eq0005"></elsevierMultimedia></p><p id="par0045" class="elsevierStylePara elsevierViewall">In Eq&#46; <a class="elsevierStyleCrossRef" href="#eq0005">&#40;1&#41;</a>&#44; y&#710; represents the predicted response&#59; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">0</span> is the intercept&#59; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">1</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">2</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">3</span>&#44; and <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">4</span> are the linear terms&#59; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">11</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">22</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">33</span>&#44; and <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">44</span> are the quadratic terms&#59; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">12</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">13</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">14</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">23</span>&#44; <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">24</span>&#44; and <span class="elsevierStyleItalic">b</span><span class="elsevierStyleInf">34</span> are the interaction terms&#59; and <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">1</span>&#44; <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">2</span>&#44; <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">3</span>&#44; and <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">4</span> are the independent variables studied&#46;</p><p id="par0050" class="elsevierStylePara elsevierViewall">A total of 26 experiments were carried out&#44; with 16 factorial points&#44; 8 axial points &#40;<span class="elsevierStyleItalic">&#945;</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>2&#41;&#44; and 2 central points&#46; Statistica &#40;version 7&#46;0&#41; software was used for the regression and for constructing the response surface for analysis of the experimental data&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Cultivation procedure</span><p id="par0055" class="elsevierStylePara elsevierViewall">Vegetative cell suspensions kept in cryotubes &#40;3&#46;5<span class="elsevierStyleHsp" style=""></span>mL&#41; were inoculated into 50<span class="elsevierStyleHsp" style=""></span>mL of seed medium in 500<span class="elsevierStyleHsp" style=""></span>mL Erlenmeyer flasks&#44; followed by incubation in a rotary shaker &#40;New Brunswick Scientific&#41; at 27<span class="elsevierStyleHsp" style=""></span>&#176;C and 250<span class="elsevierStyleHsp" style=""></span>rpm for 24<span class="elsevierStyleHsp" style=""></span>h&#46; Erlenmeyer flasks &#40;250<span class="elsevierStyleHsp" style=""></span>mL&#41; containing 22&#46;5<span class="elsevierStyleHsp" style=""></span>mL of inoculum medium were inoculated with 2&#46;5<span class="elsevierStyleHsp" style=""></span>mL of the cultivated seed broth and incubated at 27<span class="elsevierStyleHsp" style=""></span>&#176;C and 250<span class="elsevierStyleHsp" style=""></span>rpm for 24<span class="elsevierStyleHsp" style=""></span>h&#46; In the production stage&#44; the inoculum suspensions were transferred &#40;using a proportion of 10&#37; v<span class="elsevierStyleHsp" style=""></span>v<span class="elsevierStyleSup">&#8722;1</span>&#41; to 250<span class="elsevierStyleHsp" style=""></span>mL Erlenmeyer flasks containing 22&#46;5<span class="elsevierStyleHsp" style=""></span>mL of production medium&#44; followed by incubation at 27<span class="elsevierStyleHsp" style=""></span>&#176;C and 250<span class="elsevierStyleHsp" style=""></span>rpm for 72&#8211;84<span class="elsevierStyleHsp" style=""></span>h&#46; The M1&#8211;M5 cultivations were performed in duplicate&#44; with samples &#40;1<span class="elsevierStyleHsp" style=""></span>mL&#41; being withdrawn at 48&#44; 60&#44; 72&#44; and 84<span class="elsevierStyleHsp" style=""></span>h&#46; In the factorial design runs&#44; samples were only removed at 72<span class="elsevierStyleHsp" style=""></span>h&#46; The samples were centrifuged at 3720<span class="elsevierStyleHsp" style=""></span>&#215;<span class="elsevierStyleHsp" style=""></span><span class="elsevierStyleItalic">g</span> for 15<span class="elsevierStyleHsp" style=""></span>min to obtain a clear supernatant for CA analyses&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Determination of clavulanic acid concentration</span><p id="par0060" class="elsevierStylePara elsevierViewall">The CA concentration in the fermentation broth was determined spectrophotometrically&#44; as described by Bird et al&#46;<a class="elsevierStyleCrossRef" href="#bib0270"><span class="elsevierStyleSup">23</span></a> A volume of 0&#46;4<span class="elsevierStyleHsp" style=""></span>mL of sample was added to 2&#46;0<span class="elsevierStyleHsp" style=""></span>mL of imidazole solution &#40;60<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> and pH 6&#46;8&#41;&#46; After a 15<span class="elsevierStyleHsp" style=""></span>min period at 30<span class="elsevierStyleHsp" style=""></span>&#176;C&#44; the derivative produced by the reaction between CA and imidazole was detected at 311<span class="elsevierStyleHsp" style=""></span>nm&#46; The standard used was CA contained in the pharmaceutical product Clavulin<span class="elsevierStyleSup">&#174;</span> &#40;Glaxo-SmithKline Farmac&#234;utica&#44; Rio de Janeiro&#44; Brazil&#41;&#46;</p></span></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Results</span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Influence of primary nitrogen source and salts composition</span><p id="par0065" class="elsevierStylePara elsevierViewall">Firstly&#44; in order to evaluate the effects of the primary nitrogen source and the salts composition&#44; cultivations were carried out using the five production media &#40;M1&#8211;M5&#41;&#46; The time courses of CA production &#40;C<span class="elsevierStyleInf">CA</span>&#41; are shown in <a class="elsevierStyleCrossRef" href="#fig0005">Fig&#46; 1</a>&#46; The M1 medium was based on that proposed by Teodoro et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">14</span></a> in which SPI was used as the primary nitrogen source&#46; The M2 and M3 media were similar to M1&#44; but with modification of the salts composition&#46; The M4 medium was the same as the one used by Wang et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> containing SF as the primary nitrogen source&#44; while the M5 medium had an altered salt composition&#44; compared to M4&#46; The total nitrogen concentration was the same in all the media&#46; The highest and lowest maximum CA production &#40;<span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span>&#41; in the broth were obtained after 60<span class="elsevierStyleHsp" style=""></span>h in M2 &#40;437<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41; and 48<span class="elsevierStyleHsp" style=""></span>h in M1 &#40;41<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; respectively&#46; The main difference between the compositions of the media used in these assays was the presence of ferrous sulfate in M2&#44; which indicated the importance of this salt for CA biosynthesis&#46; <span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span> in M2 was 1&#46;34-fold higher than in M3 &#40;325<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; which contained ornithine&#46; With respect to primary nitrogen sources tested&#44; SPI supported higher CA production than SF present in M4 &#40;<span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>317<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41; and M5 media &#40;<span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>196<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#46; In contrast&#44; the CA production remained steady for longer in M4 and M5&#46;</p><elsevierMultimedia ident="fig0005"></elsevierMultimedia></span><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Quantitative effects of amino acids supplementation</span><p id="par0070" class="elsevierStylePara elsevierViewall">Since the highest <span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span> was achieved in the M2 medium&#44; which contained SPI as primary nitrogen source&#44; M2 was chosen as the basis for supplementation with amino acids&#44; according to the experimental design&#46; <a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a> summarizes the two-level four-factor CCRD&#44; with the ranges and levels of the independent variables &#40;arginine&#44; threonine&#44; ornithine&#44; and glutamate&#41; and the corresponding CA concentrations after 72<span class="elsevierStyleHsp" style=""></span>h of cultivation &#40;response variable&#41;&#46; The runs were conducted randomly in order to minimize errors in the experimental design&#46; The results shown in <a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a> were used to evaluate the effects of the variables on the CA concentration and obtain the regression coefficients for the second order polynomial model&#46; Statistica software &#40;version 7&#46;0&#41; was employed for these analyses&#44; adopting a 95&#37; confidence level&#46;</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><p id="par0075" class="elsevierStylePara elsevierViewall">The Pareto diagram shown in <a class="elsevierStyleCrossRef" href="#fig0010">Fig&#46; 2</a> illustrates the influence of the independent variables on the CA concentration&#44; considering the effects of linear &#40;l&#41; and quadratic &#40;q&#41; terms&#46; The horizontal bars show the magnitudes of the effects and the vertical line &#40;<span class="elsevierStyleItalic">p</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;05&#41; indicates the limit above which an effect was considered statistically significant&#46; It can be seen that CA production was significantly influenced by glutamate and ornithine&#46; The linear effect of glutamate was most important in reducing the CA concentration&#46; Although the quadratic term for glutamate showed a positive effect&#44; the negative effect of the linear term was considerably higher&#46; Ornithine also had a negative effect on CA production&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><p id="par0080" class="elsevierStylePara elsevierViewall">Statistical evaluation of the significance of the fitted model was performed using analysis of variance &#40;ANOVA&#41;&#44; with the results shown in <a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>&#46; This analysis was carried out using the <span class="elsevierStyleItalic">F</span>-test to determine the quality of the fit and the associated probability <span class="elsevierStyleItalic">p</span>-value&#46; Calculation of the determination coefficient &#40;<span class="elsevierStyleItalic">R</span><span class="elsevierStyleSup">2</span>&#41; provided a measure of the extent of variation &#40;using the regression equation&#41;&#46; The ANOVA results revealed that the model was statistically significant &#40;at the 95&#37; confidence level&#41; and was able to satisfactorily describe the data&#44; since the calculated <span class="elsevierStyleItalic">F</span> value &#40;54&#46;70&#41; was higher than the tabulated value &#40;2&#46;84&#41;&#46; The coefficient of determination &#40;<span class="elsevierStyleItalic">R</span><span class="elsevierStyleSup">2</span><span class="elsevierStyleHsp" style=""></span>&#61;<span class="elsevierStyleHsp" style=""></span>0&#46;9124&#41; was also satisfactory&#44; indicating that 91&#46;24&#37; of the variation in the CA concentration could be explained&#46; The observed and predicted CA concentrations are shown in <a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#46;</p><elsevierMultimedia ident="tbl0015"></elsevierMultimedia><elsevierMultimedia ident="fig0015"></elsevierMultimedia><p id="par0085" class="elsevierStylePara elsevierViewall">Eq&#46; <a class="elsevierStyleCrossRef" href="#eq0010">&#40;2&#41;</a> shows the second order regression equation with the estimated coefficients&#46; The model was simplified by the elimination of statistically insignificant terms&#46; The independent variables <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">3</span> &#40;ornithine&#41; and <span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">4</span> &#40;glutamate&#41; are expressed as their coded values and the CA concentration values at 72<span class="elsevierStyleHsp" style=""></span>h of cultivation are expressed in mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#46;<elsevierMultimedia ident="eq0010"></elsevierMultimedia></p><p id="par0090" class="elsevierStylePara elsevierViewall">The response surface method consists of the association of the independent and dependent variables of a model in the form of a surface graph&#46; This method was used to identify the best conditions for amino acid supplementation of the primary nitrogen source&#44; maximizing CA production&#46; <a class="elsevierStyleCrossRef" href="#fig0020">Fig&#46; 4</a> shows the response surface for CA concentration as a function of the concentrations of the amino acids ornithine and glutamate&#46; The most evident feature was the decrease in CA yield at higher glutamate concentrations&#46; The model predicted a maximum CA concentration when the glutamate concentration was minimal&#46; The distorted contours associated with the axes of ornithine and glutamate suggested interaction between these two parameters&#46;</p><elsevierMultimedia ident="fig0020"></elsevierMultimedia></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Discussion</span><p id="par0095" class="elsevierStylePara elsevierViewall">The substantial difference between the <span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span> values in M1 and M2 media indicated the importance of the ferrous sulfate for CA biosynthesis&#44; as mentioned by Wang et al&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> The CA biosynthesis employs <span class="elsevierStyleSmallCaps">l</span>-arginine &#40;C5-precursor&#41; and <span class="elsevierStyleSmallCaps">d</span>-glyceraldehyde 3-phosphate &#40;C3-precursor&#41; in nine catalyzed reactions&#44; as shown in <a class="elsevierStyleCrossRef" href="#fig0025">Fig&#46; 5</a>&#46; The conversion of N-glycylclavaminic acid to clavaldehyde &#40;step 8&#41; is the only reaction for which the enzyme has not yet been characterized&#46; Six enzymes are known to catalyze the other eight steps&#46; Five monofunctional enzymes catalyze steps 1&#44; 2&#44; 4&#44; 7&#44; and 9&#44; while one multifunctional enzyme&#44; clavaminate synthase &#40;cas&#44; EC 1&#46;14&#46;11&#46;21&#41;&#44; catalyzes steps 3&#44; 5&#44; and 6&#46; Clavaminate synthase is a nonheme Fe<span class="elsevierStyleSup">2&#43;</span>-dependent enzyme present as two isoforms in <span class="elsevierStyleItalic">S&#46; clavuligerus</span>&#46;<a class="elsevierStyleCrossRefs" href="#bib0275"><span class="elsevierStyleSup">24&#44;25</span></a> The iron dependency of this essential enzyme in the CA biosynthesis explains the improvement in CA production observed when the medium was enriched with ferrous sulfate&#46;</p><elsevierMultimedia ident="fig0025"></elsevierMultimedia><p id="par0100" class="elsevierStylePara elsevierViewall">The lower <span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span> value achieved in M3 &#40;containing ornithine&#41; compared to M2 contrasts with the findings of Chen et al&#46;&#44;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">18</span></a> who reported that CA production could be enhanced by adding ornithine to a batch culture&#46; In M4&#44; <span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CAmax</span> &#40;317<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41; was lower than those reported by Wang et al&#46;<a class="elsevierStyleCrossRef" href="#bib0215"><span class="elsevierStyleSup">12</span></a> &#40;670<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41; using the same medium&#46; An explanation for this difference lies in the cultivation procedures used and the media employed for strain preservation and propagation&#46;<a class="elsevierStyleCrossRefs" href="#bib0290"><span class="elsevierStyleSup">27&#44;28</span></a> According to Mayer and Deckwer&#44;<a class="elsevierStyleCrossRef" href="#bib0300"><span class="elsevierStyleSup">29</span></a> the presence of soybean flour particles in the medium might induce the production of extracellular proteases by <span class="elsevierStyleItalic">S&#46; clavuligerus</span>&#46; These proteases maintained the degradation of soybean flour during the growth phase&#44; providing a steady supply of essential nutrients to the microorganism&#46; This explains the maintenance of the CA production steady for longer in M4 and M5 relative to M2&#46;</p><p id="par0105" class="elsevierStylePara elsevierViewall">With respect to experimental design results&#44; in the biosynthetic pathway in <span class="elsevierStyleItalic">S&#46; clavuligerus</span> &#40;<a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>&#41;&#44; glutamate can be converted to 2-oxoglutarate&#44; which enters the citric acid cycle&#46; The conversion is catalyzed by a glutamate dehydrogenase and releases ammonium &#40;see <a class="elsevierStyleCrossRef" href="#fig0030">Fig&#46; 6</a>&#41;&#44; which inhibits CA biosynthesis<a class="elsevierStyleCrossRef" href="#bib0305"><span class="elsevierStyleSup">30</span></a> and induces CA degradation&#44;<a class="elsevierStyleCrossRef" href="#bib0310"><span class="elsevierStyleSup">31</span></a> hence explaining the negative influence of glutamate on CA production&#46; Although the glycerol supply was high&#44; supplementation with C5 precursors &#40;arginine&#44; ornithine&#44; and glutamate&#41; did not result in any increase in CA production&#46; This could be attributed to possible overload of the capacity of carboxyethylarginine synthase &#40;CeaS2&#41; to process the substrates&#46; In such a scenario&#44; the soybean protein isolate would provide sufficient arginine &#40;as a C5 precursor&#41; for the enzyme dosage in the cell&#44; making supplementation unnecessary under the selected production conditions&#46; The commercial soybean protein isolate used in this study &#40;Nutrisoy Alimentos&#41; contained arginine &#40;8&#46;0&#37; w&#47;w&#41;&#44; threonine &#40;3&#46;6&#37; w&#47;w&#41;&#44; and glutamate &#40;20&#46;4&#37; w&#47;w&#41;&#44; which reinforces this assumption&#46;</p><elsevierMultimedia ident="fig0030"></elsevierMultimedia></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Conclusion</span><p id="par0110" class="elsevierStylePara elsevierViewall">The findings demonstrated that ferrous sulfate should be included in the composition of fermentation media used for CA production&#44; because the enzymes involved in CA biosynthesis are Fe<span class="elsevierStyleSup">2&#43;</span>-dependent&#46; In contrast&#44; according to the results of the experimental design&#44; supplementation of media containing soybean protein isolate with amino acids &#40;arginine&#44; threonine&#44; ornithine&#44; and glutamate&#41; is unnecessary&#44; since the isolate provides enough C5 precursor &#40;arginine&#41; for CA biosynthesis&#44; under the conditions tested&#46; This study simultaneously investigated the influence of several medium constituents in order to determine the best formulation for CA production by <span class="elsevierStyleItalic">S&#46; clavuligerus</span>&#46; This approach is especially important for maximizing CA yields&#44; as well as for reducing the cost of fermentative production of CA&#46;</p></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Conflicts of interest</span><p id="par0115" class="elsevierStylePara elsevierViewall">The authors declare no conflicts of interest&#46;</p></span></span>"
    "textoCompletoSecciones" => array:1 [
      "secciones" => array:10 [
        0 => array:3 [
          "identificador" => "xres1091272"
          "titulo" => "Abstract"
          "secciones" => array:1 [
            0 => array:1 [
              "identificador" => "abst0005"
            ]
          ]
        ]
        1 => array:2 [
          "identificador" => "xpalclavsec1034394"
          "titulo" => "Keywords"
        ]
        2 => array:2 [
          "identificador" => "sec0005"
          "titulo" => "Introduction"
        ]
        3 => array:3 [
          "identificador" => "sec0010"
          "titulo" => "Materials and methods"
          "secciones" => array:5 [
            0 => array:2 [
              "identificador" => "sec0015"
              "titulo" => "Microorganism"
            ]
            1 => array:2 [
              "identificador" => "sec0020"
              "titulo" => "Culture media"
            ]
            2 => array:2 [
              "identificador" => "sec0025"
              "titulo" => "Experimental design"
            ]
            3 => array:2 [
              "identificador" => "sec0030"
              "titulo" => "Cultivation procedure"
            ]
            4 => array:2 [
              "identificador" => "sec0035"
              "titulo" => "Determination of clavulanic acid concentration"
            ]
          ]
        ]
        4 => array:3 [
          "identificador" => "sec0040"
          "titulo" => "Results"
          "secciones" => array:2 [
            0 => array:2 [
              "identificador" => "sec0045"
              "titulo" => "Influence of primary nitrogen source and salts composition"
            ]
            1 => array:2 [
              "identificador" => "sec0050"
              "titulo" => "Quantitative effects of amino acids supplementation"
            ]
          ]
        ]
        5 => array:2 [
          "identificador" => "sec0055"
          "titulo" => "Discussion"
        ]
        6 => array:2 [
          "identificador" => "sec0060"
          "titulo" => "Conclusion"
        ]
        7 => array:2 [
          "identificador" => "sec0065"
          "titulo" => "Conflicts of interest"
        ]
        8 => array:2 [
          "identificador" => "xack370740"
          "titulo" => "Acknowledgments"
        ]
        9 => array:1 [
          "titulo" => "References"
        ]
      ]
    ]
    "pdfFichero" => "main.pdf"
    "tienePdf" => true
    "fechaRecibido" => "2017-08-15"
    "fechaAceptado" => "2018-01-24"
    "PalabrasClave" => array:1 [
      "en" => array:1 [
        0 => array:4 [
          "clase" => "keyword"
          "titulo" => "Keywords"
          "identificador" => "xpalclavsec1034394"
          "palabras" => array:4 [
            0 => "Clavulanic acid"
            1 => "<span class="elsevierStyleItalic">Streptomyces clavuligerus</span>"
            2 => "Ferrous sulfate"
            3 => "Amino acids supplementation"
          ]
        ]
      ]
    ]
    "tieneResumen" => true
    "resumen" => array:1 [
      "en" => array:2 [
        "titulo" => "Abstract"
        "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall">Clavulanic acid is a &#946;-lactam compound with potent inhibitory activity against &#946;-lactamases&#46; Studies have shown that certain amino acids play essential roles in CA biosynthesis&#46; However&#44; quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production&#46; Here&#44; we report a study of the nutritional requirements of <span class="elsevierStyleItalic">Streptomyces clavuligerus</span> for CA production&#46; Firstly&#44; the influence of the primary nitrogen source and the salts composition was investigated&#46; Subsequently&#44; soybean protein isolate was supplemented with arginine &#40;0&#46;0&#8211;3&#46;20<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; threonine &#40;0&#46;0&#8211;1&#46;44<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; ornithine &#40;0&#46;0&#8211;4&#46;08<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; and glutamate &#40;0&#46;0&#8211;8&#46;16<span class="elsevierStyleHsp" style=""></span>g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#44; according to a two-level central composite rotatable design&#46; A medium containing ferrous sulfate yielded CA production of 437<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#44; while a formulation without this salt produced only 41<span class="elsevierStyleHsp" style=""></span>mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span> of CA&#46; This substantial difference suggested that Fe<span class="elsevierStyleSup">2&#43;</span> is important for CA biosynthesis&#46; The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence&#46; The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis&#44; so that supplementation was unnecessary&#46; Screening of medium components&#44; together with experimental design tools&#44; could be a valuable way of enhancing CA titers and reducing the process costs&#46;</p></span>"
      ]
    ]
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        "identificador" => "fig0005"
        "etiqueta" => "Fig&#46; 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr1.jpeg"
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        "descripcion" => array:1 [
          "en" => "<p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Time courses of CA concentration &#40;<span class="elsevierStyleItalic">C</span><span class="elsevierStyleInf">CA</span>&#41; for cultivations with the M1&#44; M2&#44; M3&#44; M4&#44; and M5 media&#46;</p>"
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        "etiqueta" => "Fig&#46; 2"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
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        "figura" => array:1 [
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        "descripcion" => array:1 [
          "en" => "<p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">Pareto diagram for CA concentration&#44; at a 95&#37; confidence level&#46;</p>"
        ]
      ]
      2 => array:7 [
        "identificador" => "fig0015"
        "etiqueta" => "Fig&#46; 3"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
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          "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">Observed versus predicted CA concentrations &#40;mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&#46;</p>"
        ]
      ]
      3 => array:7 [
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        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
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            "Tamanyo" => 139522
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        "descripcion" => array:1 [
          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">Response surface for CA concentration as a function of the independent variables ornithine and glutamate&#46;</p>"
        ]
      ]
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        "etiqueta" => "Fig&#46; 5"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "fuente" => "Adapted from Ozcengiz and Demain&#46;<a class="elsevierStyleCrossRef" href="#bib0285"><span class="elsevierStyleSup">26</span></a>"
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        "descripcion" => array:1 [
          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">CA biosynthetic pathway starting from C3 and C5 precursors&#46; Solid lines represent steps catalyzed by known enzymes&#59; the broken line indicates the step for which no enzyme has yet been characterized&#46; ceaS&#44; carboxyethylarginine synthase&#59; gcaS&#44; glycylclavaminate synthase&#59; bls&#44; &#946;-lactam synthetase&#59; cas&#44; clavaminate synthase&#59; pah&#44; proclavaminate amidinohydrolase&#59; cad&#44; clavaldehyde dehydrogenase&#46;</p>"
        ]
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        "identificador" => "fig0030"
        "etiqueta" => "Fig&#46; 6"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "fuente" => "Adapted from Bushell et al&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">17</span></a>"
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr6.jpeg"
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          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">CA biosynthetic pathway in <span class="elsevierStyleItalic">S&#46; clavuligerus</span> showing the conversion of glutamate to 2-oxoglutarate&#44; which causes the release of ammonium&#46;</p>"
        ]
      ]
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                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="table-head  " align="left" valign="top" scope="col" style="border-bottom: 2px solid black">Composition &#40;g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">M1&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">M2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">M3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">M4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">M5&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Glycerol&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">15&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">15&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">15&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">18&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">18&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Yeast extract&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">1&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">1&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">1&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Soybean protein isolate&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">20&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">20&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">20&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Soybean flour&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">38&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">38&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">Ornithine&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">1&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">1&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">1&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MOPS&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">21&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">21&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">21&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">21&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">21&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">K<span class="elsevierStyleInf">2</span>HPO<span class="elsevierStyleInf">4</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;8&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;5&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;5&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">KH<span class="elsevierStyleInf">2</span>PO<span class="elsevierStyleInf">4</span>&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;125&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MgSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;75&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;2&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;5&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">FeSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">MnCl<span class="elsevierStyleInf">2</span>&#183;4H<span class="elsevierStyleInf">2</span>O&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">0&#46;001&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td-with-role" title="table-entry ; entry_with_role_rowhead " align="left" valign="top">ZnSO<span class="elsevierStyleInf">4</span>&#183;7H<span class="elsevierStyleInf">2</span>O&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</td><td class="td" title="table-entry  " align="left" valign="top">&#8211;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td-with-role" title="table-head ; entry_with_role_rowhead " align="left" valign="top" scope="col">Run&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " colspan="4" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Independent variables &#40;g<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;</th><th class="td" title="table-head  " align="center" valign="top" scope="col">CA concentration &#40;mg<span class="elsevierStyleHsp" style=""></span>L<span class="elsevierStyleSup">&#8722;1</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th></tr><tr title="table-row"><th class="td" title="table-head  " align="" valign="top" scope="col" style="border-bottom: 2px solid black">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Arginine &#40;<span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">1</span>&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Threonine &#40;<span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">2</span>&#41;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t</th><th class="td" title="table-head  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Glutamate &#40;<span class="elsevierStyleItalic">x</span><span class="elsevierStyleInf">4</span>&#41;&nbsp;\t\t\t\t\t\t\n
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ISSN: 15178382
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