Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments.

In the context of research for new cytotoxic compounds, obtaining bioactive molecules from renewable sources remain a big challenge. Microorganisms and more specifically Actinobacteria from original sources are well known for their biotechnological potential and are hotspots for the discovery of new bioactive compounds. The strain DP94 studied here had shown an interesting cytotoxic activity of its culture broth (HaCaT: IC50 = 8.0 ± 1.5 μg/mL; B16: IC50 = 4.6 ± 1.8 μg/mL), which could not been explained by the compounds isolated in a previous work. The increase of the cytotoxic activity of extracts was investigated, based on a Taguchi L9 orthogonal array design, after DP94 culture in TY medium using two different vessels (bioreactor or Erlenmeyer flasks). Various culture parameters such as temperature, pH and inoculum ratio (%) were studied. For experiments conducted in a bioreactor, stirring speed was included as an additional parameter. Significant differences in the cytotoxic activities of different extracts on B16 melanoma cancer cell lines, highlighted the influence of culture temperature on the production of cytotoxic compound(s) using a bioreactor. A culture in Erlenmeyer flasks was also performed and afforded an increase of the production of the active compounds. The best conditions for the highest cytotoxicity (IC50 on B16: 6 ± 0.5 μg/mL) and the highest yield (202.0 mg/L) were identified as: pH 6, temperature 37°C and 5% inoculum.

Introduction

Some antibiotics such as actinomycin, streptothricin and streptomycin have been discovered since the forties [1]. Actinobacteria also produce a wide variety of secondary metabolites with interesting biological activities such as insecticidal (e.g. avermectins from S. avermitilis [2]), antifungal (e.g. amphotericin B from S. nodosus [3]), antiviral (e.g. hygromycin from S. hygroscopicus [4]), and cytotoxic activities (e.g. bleomycin from Streptomyces verticillus [5] or asterobactin from Nocardia asteroides [6]). These bacteria are also well described for their interesting biotechnological applications [7]. Actinobacteria are widespread in environment making them accessible sources of novel bioactive metabolites as we can see with Streptomyces and Nocardia strains isolated from underground lake and moonmilk [8].

Microbial population associated with lichens have been recently characterized in the lichen symbiosis classically defined like a dual symbiosis between a green alga (or a cyanobacterium) and a fungus [9]. Indeed, a plethora of bacteria have been identified from lichens using culture-independent (reviewed by Suzuki et al [10]) or culture-dependent approaches [11,12] and their bacterial community seems to be specific to the lichen species [13]. This long-lasting ecological niche is a novel reservoir for interesting bacterial strains. The three main bacterial phyla associated with lichens are Proteobacteria, Firmicutes and Actinobacteria [12-14]. The chemical production of several strains associated to lichens has already been described [10,15] highlighting their ability to produce active compounds.

In this study, an Actinobacterium isolated from a terrestrial lichen species Lathagrium auriforme [12] (ex Collema auriforme), close to Nocardia sp. (100% 16S rRNA sequence similarity with Nocardia soli, N. cummidelens and N. salmonicida and 98.59% with N. ignorata), was selected. To our knowledge only our previous study has described the chemical production of this Nocardia strain [16]. In our attempts to focus on the discovery of novel agents to treat melanoma, one of the most fastest growing forms of cancer, we have selected the culture broth extract of this bacterium which showed an interesting cytotoxic activity against murine B16 melanoma cell lines (IC50 = 23 ± 3 μg/mL). In the previous work, the cytotoxic compound(s) involved in the biological activity of this strain were not identified. In this context, the optimization of culture conditions leading to the increase of the cytotoxicity of the bacterial broth was necessary to highlight bioactive compound(s). The aim of this study was to estimate the impact of different culture parameters on this cytotoxicity. We have chosen herein to evaluate various parameters: pH, temperature, stirring speed and the inoculum ratio (%) (v/v). The evaluation of all the possible combinations of these parameters requires 81 experiments, which is very time consuming. A Taguchi L9 orthogonal array design, which is a robust statistical DOE (Design of Experiments) method, allowing to study a set of variables with a limited number of trials, was employed to perform 9 experiments instead of 81 [17]. At the end of the stationary growth phase the cultures were stopped and we determined the yield of extraction and the cytotoxic activity of the extracts against a cancer cell line (B16) and a non-cancer cell line (HaCaT) which will eventually led us to determine a selectivity of action. ANOVA (Analysis Of Variance) test was used to highlight which parameters exhibited a significant influence on the cytotoxicity of the bacterial extracts. Finally, the chemical analyses of the most active extracts were also carried out using HPLC (High Performance Liquid Chromatography) in order to highlight the common metabolites.

Materials and methods

Microorganism

The strain used for this study was isolated from a terrestrial lichen Lathagrium auriforme (ex Collema auriforme) collected in Kesselfallklamm in Austria (47°12’21.26” N, 15°23’57.27” E) in November 2012 by Parrot et al [12]. Its 16S rRNA gene was then sequenced using Sanger sequencing (874 pb) and the close phylogenetic neighbors of our strain were identified as Nocardia ignorata DQ659907 at 98.59% sequence identity by comparison of these data with sequences in the Eztaxon server type strain database [18]. A second sequencing of 993 pb following the same protocol as described by Parrot et al [12] allowed the identification of three closer phylogenetic neighbors Nocardia soli, N. cummidelens and N. salmonicida at 100% sequence identity using the Eztaxon server. The strain was stored after growth in ISP2 medium [19] with 50% v/v glycerol or 5% v/v DMSO (Dimethylsulfoxyde) at −80°C and referenced as DP94 [12]. This strain was deposited at the Institute of Plant Sciences, University of Graz, Austria.

Preliminary assays

Precultures

The precultures were obtained by inoculation of 30 mL of medium with an isolated colony collected on an agar plate. The culture was incubated until the optical density reached 0.6 for further uses. A purity control was performed on agar plate to check the absence of contamination.

Small-scale cultures

Four media cited below were used for preliminary assays:

TY (Tryptone Yeast medium): 10 g/L yeast extract (Sigma-Aldrich, St Louis, MO, USA), 16 g/L tryptone (Sigma-Aldrich, St Louis, MO, USA), and 5 g/L NaCl (Sigma-Aldrich, St Louis, MO, USA),

Modified LB (Lysogeny Broth) (LBm): 5 g/L peptone (Sigma-Aldrich, St Louis, MO, USA), 5 g/L NaCl (Sigma-Aldrich, St Louis, MO, USA) and 3 g/L yeast extract (Sigma-Aldrich, St Louis, MO, USA)

ISP2 (International Streptomyces Project medium 2): 4 g/L yeast extract (Sigma-Aldrich, St Louis, MO, USA), 10 g/L malt extract (Sigma-Aldrich, St Louis, MO, USA) and 4 g/L glucose

MB (Marine Broth): 37.4 g/L of commercial marine broth (Difco ® 2216, ThermoFisher, Waltham, MA, USA)

30 mL of each medium were inoculated in duplicate with the bacterial strain and incubated at 25°C and at 110 rpm in an incubator New Brunswick® Innova 42 (New Brunswick®, Edison, NJ, USA). After 8, 11 or 14 days of incubation the culture broth was centrifuged at 3000 rpm (Thermo scientific Sorvall ST40R, ThermoFisher scientific, Waltham, MA, USA), at 4°C for 15 min. The supernatant was extracted with 2 x 30 mL of ethyl acetate and the organic layer was dried on anhydrous sodium sulfate and the solvent was evaporated in vacuo leading to a raw extract.

Scale-up process

5.4 L of TY medium and 7.2 L of LBm medium were divided in 500 mL Erlenmeyer flasks and inoculated with 1% of preculture of the bacterial strain. The cultures were incubated at 25°C, 110 rpm in an incubator New Brunswick® Innova 42 (New Brunswick®, Edison, NJ, USA). After 11 days of incubation the culture broth was extracted as described below. The monitoring of the bacterial growth of these scale-up cultures is reported in S1 Fig.

Culture extraction

After fermentation, the broth was collected and centrifuged at 3000 rpm (Thermo scientific Sorvall ST40R, ThermoFisher scientific, Waltham, MA, USA), at 4°C for 15 min. The supernatant was collected and 40 g of a resin Amberlite® XAD 7HP (Sigma-Aldrich, St Louis, MO, USA) were added per liter. After 4h of stirring at 150 rpm the resin was filtered and desorbed three times successively with 400 mL of a mixture of MeOH/acetone (1/1, v/v) per 40 g of resin. The solvents were evaporated in vacuo and the residue was dissolved in 100 mL of water and extracted with 2 x 100 mL of EtOAc. The organic layer was then dried with anhydrous sodium sulfate, filtered and evaporated in vacuo leading to the resin extract (RE). The supernatant previously treated with the resin was extracted two times with EtOAc (1/2, v/v) and the organic layer was dehydrated with anhydrous sodium sulfate, filtered and dried in vacuo leading to the supernatant extract (SE). An extraction yield was defined as follows:

Culture in bioreactor

Culture was performed in 2.5 L of TY medium in a bioreactor BioFlo/Celligen® 115 (New Brunswick®, Edison, NJ, USA). The oxygenation rate of the medium was regulated at 0.4 vvm (volume of gas per volume of liquid per minute) and the percentage of dissolved oxygen was maintained up to 40% by using the cascade mode with the stirring. Temperature, pH, stirring and inoculum ratio (%) (v/v) were selected according to the design of experiments (Tables 1 and 2). The cultures were stopped after 5 days of fermentation. An example of monitoring of culture parameters and of bacterial growth has been reported in S2 and S3 Figs.

Table 1

Assignment of factors and level setting of the orthogonal array design L9 (33) for cultures in bioreactor.

Factors	Level 1	Level 2	Level 3
[A] Temperature (°C)	25	30	37
[B] pH	6	7	8
[C] inoculum ratio %	1	2	5
[D] stirring speed (rpm)	150	200	250

Table 2

Assignment of the experimental conditions in the orthogonal design L9 (34) for cultures in bioreactor.

Exp n°	Factora
	[A]	[B]	[C]	[D]
B1	1	1	1	1
B2	2	1	2	2
B3	3	1	3	3
B4	1	2	2	3
B5	2	2	3	1
B6	3	2	1	2
B7	1	3	3	2
B8	2	3	1	3
B9	3	3	2	1

[A]: Temperature, [B]: pH, [C]: inoculum ratio (%), [D]: stirring

Culture in Erlenmeyer flasks

Culture of the strain was performed in 300 mL of TY medium in 500 mL Erlenmeyer flask. The pH of the medium was adjusted with a solution of NaOH 5 M or H2SO4 0.5 N. Flasks were then incubated in an incubator New Brunswick® Innova 42 (New Brunswick®, Edison, NJ, USA) with a stirring fixed at 150 rpm and a temperature fixed at a value according to the design of experiments (See Tables 3 and 4). The cultures were stopped after 11 days of fermentation. The monitoring of bacterial growth was performed by measurement of OD (Optical Density) as shown in S4 Fig.

Table 3

Assignment of factors and level setting of the orthogonal array design L9 (33) for cultures in Erlenmeyer flasks.

Factors	Level 1	Level 2	Level 3
[A] Temperature (°C)	25	30	37
[B] pH	6	7	8
[C] inoculum ratio %	1	2	5

Table 4

Experimental conditions in the orthogonal design L9 (33) for cultures in Erlenmeyer flasks.

Exp n°	Factora
	[A]	[B]	[C]
E1	1	1	1
E2	1	2	2
E3	1	3	3
E4	2	1	2
E5	2	2	3
E6	2	3	1
E7	3	1	3
E8	3	2	1
E9	3	3	2

a[A]: Temperature, [B]: pH, [C]: inoculum ratio (%)

Taguchi experimental design

The Taguchi method was selected to establish the experimental design and led to 9 experiments either in bioreactor (Tables 1 and 2) nor in Erlenmeyer flask (Tables 3 and 4). For each experiment, the responses evaluated were the yield and the cytotoxicity of extracts against B16 and HaCaT cell lines. For the fermentation in bioreactor we selected 4 independent parameters and attributed 3 levels for each. The four parameters selected were temperature (25°C, 30°C and 37°C), pH (6, 7 and 8), percentage of inoculum (v/v) (1%, 2% and 5%) and stirring speed (150, 200 and 250 rpm) (Table 1). The experimental design and ANOVA analysis were performed using Excel 2016.

For the fermentation in Erlenmeyer flasks the stirring was fixed at 150 rpm and only 3 parameters were selected with 3 levels for each (Tables 3 and 4).

Cytotoxic assays

The cytotoxic activity of the resin extracts and the supernatant extracts was evaluated for each condition of the experimental design with a standard tetrazolium assay (MTT) [20]. Two cell lines (B16: murine melanoma, HaCaT: human keratinocytes) were cultivated in RPMI (Roswell Park Memorial Institute) 1640 medium (Thermo Fisher Scientific, USA) supplemented with 5% fetal calf serum (FCS) and 1% antibiotics (penicillin 10 000 UI/ml and streptomycin 10 000 μg/ml, Eurobio®, Les Ulis, France). Cells were then seeds in 96-wells plate (B16: 6000 cells/well and HaCaT 10,000 cells/well) at day 0 and incubated for 24h at 37°C and 5% CO2. Extracts were then added at different concentrations (1, 10, 50, 100, and 200 μg/mL) for all the experiments. For experimental design in bioreactor and Erlenmeyer flasks the extracts were tested a second time at 5, 50, 100, 250 and 500 μg/mL to expand the range of IC50 values. After 24h of incubation (37°C, 5% CO2) the cell viability was measured at 540 nm using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay [20]. Each experiment was repeated three times.

Chemical analyses

Chemical analyses of the most active extracts were carried out on a HPLC system—Diode Array Detector (LC-DAD) (Shimadzu, Marne La Vallée, France). A Prevail C18 column (5 μm, 250 × 4.6 mm, GRACE, Columbia, MD, USA) was used and a gradient system was applied: A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The following gradient was applied at a flow rate of 0.8 mL/min in the HPLC system: initial: 100% (A); from 0 to 5 min: 100% (A); from 5 to 35 min: 100% (A)/0% (B) to 0% (A)/100% (B); from 35 to 45 min: 100% B; from 45 to 50 min: 0% (A)/100% (B) to 100% (A)/0% (B); from 50 to 55 min: 100% (A). Samples were prepared by dissolving extracts in MeOH at 1 mg/mL and 20 μL were injected after filtration (45 μm). The LabSolutions software (Shimadzu, Marne La Vallée, France) was used for data analyses.

Results

The Nocardia sp. strain used in this study achieved the stationary growth phase after 7 days of growth. We have cultivated this strain using 30 mL of four different media in 50 mL tubes, with a 7 days of exponential growth phase followed by 1, 4 or 7 days of stationary phase (respectively 8, 11 and 14 days in total). The best cytotoxic activity against B16 cells was achieved with the culture in LBm medium for 11 days (IC50 = 8 ± 2 μg/mL) and with the ISP2 medium after 14 days of culture (IC50 = 7.5 ± 0.5 μg/mL). Similar activities have been shown on HaCaT cells mentioning no selectivity of action. The highest amount of extract was obtained when the strain was grown in TY medium (Table 5). A scale-up assay with 300 mL of media in 500 mL Erlenmeyer flasks was then performed using LBm and TY for 11 days of culture (4 days of stationary phase). In order to have sufficient amount of LBm extracts and based on the results obtained with the 30 mL process, we have inoculated 24 Erlenmeyer flasks with LBm medium (total volume 7.2 L) and 18 Erlenmeyer flasks with TY medium (total volume 5.4 L). The culture extract from TY medium gave the best activity against B16 cell line (IC50 = 4.6 ± 1.8 μg/mL) even though the amount obtained was low (yield = 16.1 mg/L) but higher than for the culture in LBm (Table 6). These culture conditions (TY medium and 4 days of stationary phase) were applied to the Taguchi design experiments.

Table 5

Weight and cytotoxicity of extracts in preliminary assays.

Days of culture	Mediuma	Raw extract amount (mg)	IC50 (μg/mL)
			B16	HaCaT
8	ISP2	2.3 ± 0.0	45 ± 5	59 ± 5
	TY	5.2 ± 0.1	41 ± 5	72 ± 7
	MB	2.2 ± 0.3	105 ± 20	115 ± 15
	LBm	2.5 ± 0.4	25 ± 4	95 ± 30
11	ISP2	4.0 ± 0.7	31 ± 10	64 ± 28
	TY	5.4 ± 0.9	25 ± 4	47 ± 13
	MB	2.1 ± 0.7	57 ± 18	120 ± 15
	LBm	2.5 ± 0.1	8 ± 2	31 ± 12
14	ISP2	3.6 ± 0.2	7.5 ± 0.5	41 ± 5
	TY	6.4 ± 0.3	35 ± 8	86 ± 42
	MB	3.8 ± 0.2	85 ± 10	79 ± 9
	LBm	4.2 ± 0.2	14 ± 6	70 ± 4

aISP2 = International Streptomyces Project 2, TY = Tryptone Yeast, MB = Marine Broth, LBm = modified Lysogeny Broth

Table 6

Extraction yield and cytotoxicity of extracts from scale up in Erlenmeyer flasks.

Medium	Extract	Yield (mg/L)	IC50 (μg/mL)
			B16	HaCaT
TY	Resin extract	101.4	>200	>200
	Supernatant extract	16.1	4.6 ± 1.8	8 ± 1.5
LBm	Resin extract	60.3	>200	>200
	Supernatant extract	3.0	52 ± 20	73 ± 20

Experimental design in bioreactor

An experimental design was applied in order to find the best culture conditions to improve the yield and the cytotoxic activity of extracts. Varying temperature [A] (25, 30 and 37°C), pH [B] (6, 7 and 8), percentage of inoculum ratio (% v/v) [C] (1, 2 and 5%) and stirring speed [D] (150, 200 and 250 rpm), 9 experiments were carried out. In all experiments, the yield of the resin extracts (RE) and the supernatant extracts (SE) were determined and their cytotoxicity was measured against B16 and HaCaT cell lines (Table 7). When similar activities have been exhibited against the two cell lines, we have focused on activities against B16.

Table 7

Extraction yield and cytotoxicity of the extracts from the experimental design using bioreactor.

Exp n°	RE			SE
	Yield (mg/L)	IC50 (μg/mL)		Yield (mg/L)		IC50 (μg/mL)
		B16	HaCaT			B16	HaCaT
B1	146.1	157 ± 77	92 ± 29	46.3	>500		460 ± 140
B2	160.5	205 ± 100	185 ± 65	1859.3	>500		>500
B3	216.6	53 ± 16	152 ± 28	478.6	41 ± 8		20 ± 8
B4	158.0	92 ± 19	70 ± 8	152.3	250		320
B5	177.8	345 ± 95	120 ± 40	506.9	100 ± 30		150
B6	223.5	45 ± 15	80 ± 20	36.1	270 ± 35		225 ± 45
B7	126.8	270 ± 30	185 ± 15	66.5	430 ± 90		180 ± 20
B8	703.0	340 ± 25	397 ± 27	18.4	67 ± 4		38 ± 19
B9	318.0	90 ± 50	110 ± 25	43.9	350		270

The mean IC50 values of RE on B16 cells, were classified into three groups: a first group with IC50 > 300 μg/mL (exp. B5 and B8); a second group with 100 < IC50 < 300 μg/mL (exp. B1, B2 and B7) and the last group with IC50 < 100 μg/mL (exp. B3, B4, B6 and B9) (Table 7). In a similar manner, the results were classified into three groups for SE extracts based on their activity on B16: the first group with IC50 > 400 μg/mL (exp. B1, B2 and B7); a second group with 100 < IC50 < 400 μg/mL (exp. B4, B6 and B9) and the last group with IC50 < 100 μg/mL (exp. B3, B5 and B8) (Table 7).

Most of RE extracts exhibited cytotoxic activities whereas only a few of SE extracts were active. Main-effect plots (Fig 1) indicated that the IC50 values decreased with the maximum level of factor B at 37°C. It was also noticed that the IC50 values increased in culture extracts at 30°C, highlighting the importance of culture temperature on the cytotoxicity of the extracts.

Fig 1

Main-effect plots for optimization of culture conditions in bioreactor.

Finally, the experiment with the highest cytotoxic activity was the experiment B3 (RE, IC50 = 53 ± 16 μg/mL and SE IC50 = 41 ± 8 μg/mL on B16) with the following culture conditions: pH 6, temperature 37°C, an inoculum of 5% and a stirring of 250 rpm.

Surprisingly, the experiment B2 (culture conditions: pH 6, temperature of 30°C, inoculum of 2% and stirring speed of 200 rpm) gave the highest supernatant extraction (SE) yield.

Based on the cytotoxic activity against B16 and the extraction yield, ANOVA test was used to highlight which parameter(s) could significantly influence the production of active compound(s).

Statistical analysis

The F-ratio and the p-value determined from the ANOVA analysis on the cytotoxicity of the resin extracts showed that the temperature significantly influenced the activity (p < 0.05) (Table 8 and Fig 1). Conversely, none of the parameters selected for this analysis showed a significant effect on the extraction yield as well as for the cytotoxicity of the supernatant extracts (Table 9).

Table 8

Summary of analysis of variance in the ANOVA test for cytotoxicity on B16 of RE from bioreactor.

Factorsa	Sum of squares	f	Average of square	F-ratio	F0.95(2,2)	p
A	14804.2	2	7402.1	7.46	19	0.118
B	82222.9	2	41111.4	41.44	19	0.024
C	1984.2	2	992.1	1.00	19	0.500
D	13206.9	2	6603.4	6.66	19	0.131
Error	1984.2	2	992.1
Total	112218.2

[A]: Temperature, [B]: pH, [C]: inoculum ratio (%), [D]: stirring

Table 9

Summary of p-value from the ANOVA test on all extracts from bioreactor.

Factorsa	p
	RE		SE
	Effect on IC50 on B16	Effect on extraction yield	Effect on IC50 on B16	Effect on extraction yield
A	0.118	0.408	0.500	0.303
B	0.024	0.475	0.333	0.312
C	0.500	0.490	0.192	0.500
D	0.131	0.500	0.388	0.385

[A]: Temperature, [B]: pH, [C]: inoculum ratio (%), [D]: stirring

Validation of the optimal conditions

The experiment B3 with the following parameters, pH 6, temperature 37°C, inoculum 5% and stirring 250 rpm, was repeated once using bioreactor to confirm the previous results on HaCaT and B16. The repetition of this experiment showed close values of IC50 for SE on B16 and HaCaT cell lines (IC50 = 33 ± 8 μg/mL on B16 and IC50 = 23 ± 5 μg/mL on HaCaT) and a decrease of the activity of RE against B16 (IC50 > 200 μg/mL). This experiment confirms nevertheless that the culture conditions used are optimal for the cytotoxicity of the extracts from bioreactor.

Experimental design in Erlenmeyer flasks

The data obtained in Erlenmeyer flasks were analyzed in a same manner using a Taguchi array and the yields and cytotoxic activities of the both extracts (RE and SE) were measured on B16 and HaCaT cell lines (Table 10).

Table 10

Extraction yield and cytotoxicity of the extracts on B16 and HaCaT cell lines for the experimental design in Erlenmeyer flasks.

Exp n°	RE			SE
	Yield (mg/L)	IC50 (μg/mL)		Yield (mg/L)	IC50 (μg/mL)
		B16	HaCaT		B16	HaCaT
E1	306.7	280 ± 160	110 ± 60	199.3	375 ± 110	40 ± 15
E2	279.7	96 ± 9	122 ± 4	77.9	73 ± 20	77 ± 16
E3	218.7	70 ± 8	71 ± 19	66.0	74 ± 34	56 ± 24
E4	96.3	145 ± 15	122 ± 22	74.0	380 ± 105	75 ± 50
E5	184.0	130 ± 4	127 ± 13	57.3	390 ± 100	240 ± 150
E6	305.7	64 ± 8	72 ± 1	58.3	170 ± 10	152 ± 18
E7	202.0	6 ± 0.5	30 ± 7	583.3	480 ± 230	140 ± 70
E8	176.7	81 ± 10	50 ± 7	840.3	>500	260 ± 110
E9	489.0	63 ± 7	79 ± 15	39.7	137 ± 7	32 ± 10

The most important cytotoxic activity was demonstrated with experiment E7 (RE, IC50 = 6 ± 0.5 μg/mL on B16) with the following culture conditions: pH 6, temperature 37°C and 5% inoculum and led to a sufficient amount of extract (202.0 mg/L) (Table 10). Interestingly, these conditions were close to those obtained for the culture in bioreactor (excepted for the value of stirring fixed at 150 rpm for Erlenmeyer process), confirming that these parameters are the best to improve the cytotoxicity of the extracts. Conversely, the highest extraction yield (840.3 mg/mL) was obtained with E8 and the conditions (37°C, pH 7 and inoculum 1%, Table 10) were completely different than those obtained for culture in bioreactor.

Nevertheless, the ANOVA analysis of the results obtained from these Erlenmeyer cultures showed that none of the selected parameters had a significant effect on the cytotoxicity of the extracts on B16 cell lines or on the extraction yield.

Impact of the culture vessel on the activity of extracts

In order to determine the effect of the culture conditions on the production of cytotoxic metabolites, we compared the results obtained for three processes performed using either bioreactor or Erlenmeyer flasks and possessing similar culture conditions with a stirring speed fixed at 150 rpm (Table 11). Among them, the most active extracts were obtained at 37°C, pH 6 and a 2% inoculum ratio (experiments B9 and E9). The yields were also higher in these experiments in comparison to the other ones.

Table 11

Comparison of the results obtained for cultures in bioreactor and in Erlenmeyer flasks with the same culture conditions.

Experiences	Factors			RE			SE
	Temperature (°C)	pH	Inoculum ratio (%)	Yield (mg/L)	IC50 (μg/mL)		Yield (mg/L)	IC50 (μg/mL)
					B16	HaCaT		B16	HaCaT
B1	25	6	1	146.1	157 ± 77	92 ± 29	46.3	>500	460 ± 140
E1	25	6	1	306.7	280 ± 160	110 ± 60	199.3	375 ± 110	40 ± 15
B5	30	7	5	177.8	345 ± 95	120 ± 40	506.9	100 ± 30	39 ± 10
E5	30	7	5	184.0	130 ± 4	127 ± 13	57.3	390 ± 100	240 ± 150
B9	37	8	2	318.0	90 ± 50	110 ± 25	43.9	390 ± 90	45 ± 2
E9	37	8	2	489.0	63 ± 7	79 ± 15	39.7	137 ± 7	32 ± 10

Comparison of HPLC profiles of the most active extracts

The HPLC chromatograms of these two experiments B9 and E9 were compared in Fig 2. Those of the most active extracts obtained during this study (SE of the large scale culture in TY shown in preliminary assays, SE from B3 and RE from E7) were reported in Fig 3. The compounds previously isolated from this Nocardia strain [16] and known compounds such as cyclo (L-Ala-L-Phe) [21], adenine, adenosine and 1-(5-deoxy-β-D-erythro-pent-4-enofuranosyl) [22] were identified by comparison with a standard and data from literature (Figs 2A, 2B and 3).

Fig 2

Comparison of chemical profiles of B9 (red) and E9 (blue). A. Resin extracts, B. Supernatant extracts. All samples were analysed at 220 nm on Prevail® reversed phase C18 column with a gradient of H2O (A)/acetonitrile (B) (10 min 100% of A, 30 min from 0% of B to 100% of B, 10 min 100% of B).

Fig 3

Comparison of the chemical profiles of the most active extracts.

SE of the large scale culture in TY shown in preliminary assays (red), SE of B3 (blue) and RE of E7 (black). All samples were analysed at 220 nm on Prevail® reversed phase C18 column with a gradient of H2O (A)/acetonitrile (B) (10 min 100% of A, 30 min from 0% of B to 100% of B, 10 min 100% of B).

The main differences between the production of metabolites by the strain in the RE of B9 and E9 are due to the presence of an unidentified compound with a r.t. of 19 min and of a diketopiperazine cyclo-(L-Ala-L-Phe) which were produced in large amount in bioreactor B9 but not in Erlenmeyer flask E9 (Fig 2A). Variability was also observed for the production of compounds eluted between 26 and 32 min (Fig 2A). In the SE of E9 a compound eluted at the beginning of the gradient appeared. Moreover, a difference of the production of compounds eluted between 22 and 28 min was also highlighted between the two culture vessels (Fig 2B). The production of adenine was most important in bioreactor than in Erlenmeyer reaction set-up (Fig 2). Despite these differences in the chemical profiles, the activity of the extracts were similar between bioreactor and Erlenmeyer flasks (except for the SE on B16 cells) meaning that the compounds highlighted above are not implicated in the cytotoxicity of the extracts.

Finally, the comparison of the most active extracts led to the highlighting of unidentified compounds common in all the three active extracts which were indicated with black squares in Fig 3.

Discussion

Lichens are a novel ecological niche of considerable interest for the discovery of bacterial strains with biotechnological potential [12]. We selected and studied a lichen-associated Actinobacterium, Nocardia sp that demonstrated interesting cytotoxic activities. The results detailed herein showed that the production of cytotoxic compounds by this Nocardia sp. could be modulated by culture conditions according to the OSMAC (One Strain/Many Compounds) approach previously described [23]. The data obtained in preliminary assays are in accordance with our expectations. The marine broth currently used in our experiments performed on strains isolated from marine lichens [12,24] is not suitable to optimize the cytotoxicity of our Nocardia strain isolated from a terrestrial lichen. Moreover, the amount of production of metabolites increases with rich medium such as TY. This observation is close to those of Abdelmohsen et al [25] who indicated that the production of actinosporin by a marine sponge associated-Actinokineospora was widely affected depending on the culture medium used. Our results suggest in the same manner that the production of the compound(s) involved in the cytotoxicity of the extracts is influenced by the difference in the medium growth used.

A significant effect of temperature on cytotoxicity of the resin extracts (p < 0.05) with an improvement of the activity at 37°C was exhibited by ANOVA analysis of the data from experimental design in bioreactor. The importance of culture parameters such as temperature on increasing of the biomass and of the activity has been already demonstrated for Actinobacteria strains. Thus, the effect of the temperature has been described on the biomass and geosmin production by strains of N. cummidelens and N. fluminea. The highest biomass production was thus obtained at the warmest temperature tested, 30°C and 25°C, for the both strains and the production of geosmin was optimal at 25°C [26]. Similar effects has been reported on the production of rapamycin by a Streptomyces strain, where the optimal temperature was established at 23°C, the lowest tested value [27].

In spite of employing similar culture conditions (temperature 37°C, pH 6 and inoculum 5%,) in both the bioreactor (B3) and Erlenmeyer flasks (E7), the best activity (IC50 = 6 ± 0.5 μg/mL on B16) was displayed by extracts obtained from Erlenmeyer flasks (E7) highlighting the importance of the culture process used.

Various compounds have been previously isolated from the culture broth of this bacterial strain [16]. Compounds identified were mainly diketopiperazines. An auxin derivative and purine derivatives (e.g. adenine) have also been isolated (data not published) but none of these compounds explained the cytotoxic activity of the bacterial extracts.

The comparison of the chemical profiles of extracts obtained by culture in bioreactor and in Erlenmeyer flasks highlighted several differences which also revealed the impact of the system used. These variations of production could be explained by the different aeration and the stirring system used [28] leading to different stress conditions for bacteria.

The comparison of the chemical profiles of the most active extracts (Fig 3) showed a very similar production and the major compounds already isolated possess no cytotoxic properties on B16 cell line. Nevertheless, some zones of the chromatogram corresponding to unidentified compounds have been highlighted (Fig 3).

Conclusion

In conclusion, the Taguchi method for design of experiments combined with an ANOVA test were shown to be powerful tools for the optimization of culture parameters of lichen-associated Nocardia sp. for the production of cytotoxic compounds. Further separative experiments will be carried out with a focus on the zones highlighted on HPLC profiles during this study in order to identify compound(s) involved in the cytotoxic activity of these bacterial extracts.

Monitoring of bacterial growth of scale-up in TY and LBm.

(TIF)

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Monitoring of culture parameters of B1.

(TIF)

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Monitoring of bacterial growth of B1.

(TIF)

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Monitoring of bacterial growth of E1.

(TIF)

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16 Sep 2019

PONE-D-19-18890

Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments

PLOS ONE

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript presented by Tomasi et al entitled "Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments" addresses the impact of culture media, parameters (bioreactor/shake flask) and process parameters (Temperature, pH, inoculation and stirring) on the yield and cytotoxic effects of the culture fluid on two mammalian cell lines. However, the study has been done in details but there are some concerns in the manuscript. The rational of this study and each experiment should be written clearly.

1. Order of authors is different in the beginning table of submitted file compared to first page of manuscript.

2. Abstract:

- The abstract should be revised and included method section. Moreover, it should be well reflected whole significant information of the manuscript. In the abstract, the advantages of this work should be reflected shortly.

3. Introduction:

- In the introduction section the authors should be briefly clarify about the application of recent work and drawbacks.

- (Lines 65 to 68): What is the reason for choosing B16 cell line to check the cytotoxicity? Authors should be explain more about the application such cytotoxic effect of broth extract on clarified targets such as therapeutic purposes.

- In addition, there is no explanation about the HaCaT cell line in introduction section of the manuscript.

- (Lines 69 to 70): Four parameters were targeted to evaluation in this study such as pH, Temperature, stirring speed and inoculum ratio, so please explain that based on which experimental design basically you reached to 81 experiments then in continue you reduced the number to 9 experiment?

4. Materials and method:

- (Line 110): How you ensure that the number of inoculated bacteria (precultured) were same in all experimental conditions? Please provide method for inoculum preparation and the number of bacteria (CFU/ml) added in both small scale and scaled up experimental condition.

- (Line 147): To keep the oxygen on 40% in the bioreactor, the cascade mode with the stirring was selected. Otherwise, based on experimental design the rpm is one of the variables which was adjusted on 150, 200 and 250 in each experimental condition. So the question arises is that when the amount of oxygen is reduced, the rpm may increase to compensate the aeration/oxygenation. Please explain how you justify such rpm variations during the experimental design.

- Please add the software name and version used for experimental design and statistical analysis.

5. Results:

- The quality of figure no. 2 is not good. I suggest having alternate presentation of this figure.

- The number of repetition in validation of the optimal condition and the results of them should be reported clearly in results section of the manuscript.

- The value of “yield” as a parameter should be followed by standard deviation.

6. Discussion:

- According below statement of the manuscript, the differences between bioreactor and shake flask are well known and they are not much comparable to each other. I strongly recommend to revise the discussion part of manuscript.

“In fact, the stirring in bioreactor is performed by agitation blade submerged in the culture broth which implicates hyphal breaks, while in shaken flasks the agitation is performed with an orbital shaker allowing the hyphal formation. In a similar way, the aeration system in bioreactor consisted of a tube submerged in the culture broth that conducted oxygen into the medium causing the formation of bubbles, while in the shaken flasks there is no specific system for aeration. These main differences could explain the variation of bacterial production profile due to a different oxygenation and stirring methods inducing different stress conditions for bacteria.”

7. Minor comments:

- Some minor comment and suggestions are included in the pdf file of manuscript.

Reviewer #2: The work is interesting since explore the culture conditions to have the highest cytotoxic activity from Nocardia sp in bioreactor and flask. Cultures in flask result better since possibly cells are not as affected by hydrodynamic stresses as in bioreactor. The main different peaks separated by HPLC are not the responsible for the cytotoxic activity, so, there is pending to identify the molecules responsible. There have been identified some molecules with cytotoxic activity from Nocardia, so authors can trying to identify if activity could correspond to this kind of molecules.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE-D-19-18890_reviewer#1.pdf

Click here for additional data file.

5 Nov 2019

RESPONSES TO REVIEWER’S COMMENTS

Rewiever’s 1 comments :

The manuscript presented by Tomasi et al entitled "Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments" addresses the impact of culture media, parameters (bioreactor/shake flask) and process parameters (Temperature, pH, inoculation and stirring) on the yield and cytotoxic effects of the culture fluid on two mammalian cell lines. However, the study has been done in details but there are some concerns in the manuscript. The rational of this study and each experiment should be written clearly.

1. Order of authors is different in the beginning table of submitted file compared to first page of manuscript.

Due to a problem appeared during the submission process an error occured in the order of authors. The good order is: Alba Noël, Gwendoline Van Soen, Isabelle Rouaud, Eric Hitti, Sophie Tomasi.

2.Abstract:

- The abstract should be revised and included method section. Moreover, it should be well reflected whole significant information of the manuscript. In the abstract, the advantages of this work should be reflected shortly.

The abstract has been rewritten p 2 line 18- 42 following the reviewer’s comments.

3. Introduction:

- In the introduction section the authors should be briefly clarify about the application of recent work and drawbacks.

According to the reviewer’s comments it has been clarified p.4 l.89-92 as: “In the previous work, the cytotoxic compound(s) involved in the biological activity of this strain were not identified. In this context, the optimization of culture conditions leading to the increase of the cytotoxicity of the bacterial broth was necessary to highlight bioactive compound(s).”

- (Lines 65 to 68): What is the reason for choosing B16 cell line to check the cytotoxicity? Authors should be explain more about the application such cytotoxic effect of broth extract on clarified targets such as therapeutic purposes.

The B16 cell line corresponds to murine melanoma cells. We used routinely these cells in our laboratory for screening cytotoxic activity of various extracts or compouds as B16 cells are a mouse model for human melanoma.

To clarify the application of our work in therapeutic purpose the paragraph was modified as follows (p 4-5 lines 85-92): “In our attempts to focus on the discovery of novel agents to treat melanoma, one of the most fastest growing forms of cancer, we have selected the culture broth extract of this bacterium which showed an interesting cytotoxic activity against murine B16 melanoma cell lines (IC50 = 23 ± 3 µg/mL). In the previous work, the cytotoxic compounds involved in the biological activity of this strain were not identified. In this context, the optimization of culture conditions leading to the increase of the cytotoxicity of the bacterial broth was necessary to highlight bioactive compound(s).

- In addition, there is no explanation about the HaCaT cell line in introduction section of the manuscript.

The HaCaT cell line are the non-cancer cell line that we use routinely in our lab to evaluate the cytotoxic activity and to determine an eventual selectivity of the compounds tested.

It has been explained in the sentence p 5 lines 101-104: “At the end of the stationary growth phase the cultures were stopped and we determined the yield of extraction and the cytotoxic activity of the extracts against a cancer cell line (B16) and a non- cancer cell line (HaCaT) of the extracts which will eventually led us to determine a selectivity of action”

We have completed a sentence p 12 lines 262-263 as : Similar activities have been shown on HaCaT cells mentioning no selectivity of action.

- (Lines 69 to 70): Four parameters were targeted to evaluation in this study such as pH, Temperature, stirring speed and inoculum ratio, so please explain that based on which experimental design basically you reached to 81 experiments then in continue you reduced the number to 9 experiment?

The number of 81 experiments is necessary if you not use any experimental design. It corresponds to the number of trials necessary to test all the possible combinations with 3 levels of 4 parameters. The application of the Taguchi orthogonal design allow us to realise only 9 experiments to evaluate the impact of each parameter on the cytotoxic activity of the culture broth. It has been clarified by modification of the sentence p 5 lines 97-101: “The evaluation of all the possible combinations of these parameters requires 81 experiments, which is very time consuming. A Taguchi L9 orthogonal array design, which is a robust statistical DOE (Design of Experiments) method, allowing to study a set of variables with a limited number of trials, was employed to perform 9 experiments instead of 81 [17]”

4. Materials and method:

- (Line 110): How you ensure that the number of inoculated bacteria (precultured) were same in all experimental conditions? Please provide method for inoculum preparation and the number of bacteria (CFU/ml) added in both small scale and scaled up experimental condition.

All the precultures were obtained in a same way, the number of inoculated bacteria was controlled by monitoring the OD. It has been added in the manuscript p 6 lines 127-131 as:

“Precultures

The precultures were obtained by inoculation of 30 mL of medium with an isolated colony collected on an agar plate. The culture was incubated until the optical density reached 0.6 for further uses. A purity control was performed on agar plate to check the absence of contamination.

- (Line 147): To keep the oxygen on 40% in the bioreactor, the cascade mode with the stirring was selected. Otherwise, based on experimental design the rpm is one of the variables which was adjusted on 150, 200 and 250 in each experimental condition. So the question arises is that when the amount of oxygen is reduced, the rpm may increase to compensate the aeration/oxygenation. Please explain how you justify such rpm variations during the experimental design.

The oxygenation reduced below 40% only during the exponential growth phase of the bacteria. As the production of compounds involved in the cytotoxic activity mainly happens during the stationary phase it means that at this time the stirring conditions are not affected by the cascade mode. The experimental design is based on variations occurred during stationary phase, to study the impact on the production of secondary metabolites.

- Please add the software name and version used for experimental design and statistical analysis.

A sentence was added p 9 lines 206-607 : The experimental design and ANOVA analysis were performed using Excel 2016.

5. Results:

- The quality of figure no. 2 is not good. I suggest having alternate presentation of this figure.

The figure 2 has been retreated to increase the quality as suggested by the reviewer.

- The number of repetition in validation of the optimal condition and the results of them should be reported clearly in results section of the manuscript.

It has been modified p 17 lines 347-350 as: “The experiment B3 with the following parameters, pH 6, temperature 37°C, inoculum 5% and stirring 250 rpm, was repeated once using bioreactor to confirm the previous results on HaCaT and B16 cells.”

- The value of “yield” as a parameter should be followed by standard deviation.

The experimental design allowed to achieve the different culture conditions only once which explain why we do not have standard deviation for these results. It is the interest to do a experimental design. Only the optimal conditions were repeated.

6. Discussion:

- According below statement of the manuscript, the differences between bioreactor and shake flask are well known and they are not much comparable to each other. I strongly recommend to revise the discussion part of manuscript.

“In fact, the stirring in bioreactor is performed by agitation blade submerged in the culture broth which implicates hyphal breaks, while in shaken flasks the agitation is performed with an orbital shaker allowing the hyphal formation. In a similar way, the aeration system in bioreactor consisted of a tube submerged in the culture broth that conducted oxygen into the medium causing the formation of bubbles, while in the shaken flasks there is no specific system for aeration. These main differences could explain the variation of bacterial production profile due to a different oxygenation and stirring methods inducing different stress conditions for bacteria.”

Following the reviewer’s comment, this part has been modified p 22 lines 461-462 as: “These variations of production could be explained by the different aeration and the stirring system used [28] leading to different stress conditions for bacteria.”

7. Minor comments:

- Some minor comment and suggestions are included in the pdf file of manuscript.

We have taken account all the comments of the reviewer 1.

Reviewer #2: The work is interesting since explore the culture conditions to have the highest cytotoxic activity from Nocardia sp in bioreactor and flask. Cultures in flask result better since possibly cells are not as affected by hydrodynamic stresses as in bioreactor. The main different peaks separated by HPLC are not the responsible for the cytotoxic activity, so, there is pending to identify the molecules responsible. There have been identified some molecules with cytotoxic activity from Nocardia, so authors can trying to identify if activity could correspond to this kind of molecules.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

31 Dec 2019

Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments

PONE-D-19-18890R1

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Yoshihiro Uesawa

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

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Reviewer #1: Yes: Shayan Maleknia

6 Jan 2020

PONE-D-19-18890R1

Optimization of cytotoxic activity of Nocardia sp culture broths using a design of experiments

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