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For Review OnlyViable But Nonculturable State of Escherichia coli, Pseudomonas putida and Lactococcus lactis during

Exposure to Toxic Chemicals, as Revealed by Headspace Gas Chromatography and Indirect Conductivity Techniques

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2018-0175.R2

Manuscript Type: Original Article

Date Submitted by the Author: 21-Mar-2019

Complete List of Authors: Payongsri, Panwajee; Mahidol University, BiotechnologyCharoenrat , Nattapat; Mahidol University, BiotechnologyPongtharangkul, Thunyarat; Mahidol University, BiotecnologyWongkongkatep, Jirarut; Biotechnology

Keyword: gas chromatography, indirect conductivity, metabolic activity, viable but nonculturable (VBNC), emulsion

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Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep

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1 Tittle page

2 Viable But Nonculturable State of Escherichia coli, Pseudomonas putida and

3 Lactococcus lactis during Exposure to Toxic Chemicals, as Revealed by Headspace

4 Gas Chromatography and Indirect Conductivity Techniques

5

6 Panwajee Payongsri, Nattapat Charoenrat, Thunyarat Pongtharangkul and Jirarut

7 Wongkongkatep*

8 Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama 6

9 Road, Bangkok 10400 Thailand

10

11 Corresponding author

12 Jirarut Wongkongkatep email: [email protected]

13 Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama 6

14 Road, Bangkok 10400 Thailand

15 TEL: +662-201-5302

16 FAX: +662-354-7160

17 Panwajee Payongsri email: [email protected]

18 Nattapat Charoenrat [email protected]

19 Thunyarat Pongtharangkul [email protected]

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1 SJST MANUSCRIPT TEMPLATE FOR A TEXT FILE

2

3 Original Article

4

5 Viable But Nonculturable State of Escherichia coli, Pseudomonas putida and

6 Lactococcus lactis during Exposure to Toxic Chemicals, as Revealed by Headspace

7 Gas Chromatography and Indirect Conductivity Techniques

8

9 Abstract

10 The bioconversion of water-immiscible chemicals by microbes may occur

11 through viable but nonculturable cells (VBNC) and the evaluation of such cellular

12 activity is important for a deeper understanding the metabolic process. In this study, the

13 metabolic CO2 production in a bacteria interface emulsion consisting of bacteria,

14 chitosan and solvent has been monitored using headspace gas chromatography (GC) and

15 indirect conductivity (IC). The results from GC in comparison to the standard culture

16 technique revealed the presence of VBNC state when E. coli DH5α and P. putida F1

17 were in contact with n-hexane. L. lactis IO-1 was the most sensitive strain, but the VBNC

18 cells were obvious in the case of soybean and n-decane. Although GC showed better

19 detection sensitivity, the IC technique was more practical and cost-effective. Therefore,

20 GC and IC could be extremely simple and useful methods for monitoring of VBNC

21 state of bacteria which generally are under environmental stresses.

22

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23 Keywords: gas chromatography, indirect conductivity, metabolic activity, viable but

24 nonculturable (VBNC), emulsion

25

26 1. Introduction

27 Are the bacterial cells already dead or still alive? This question is definitely

28 important on the basis of decisions when people are concerning about the function of

29 the metabolically active bacteria especially in the bioconversion of toxic water-

30 immiscible chemicals when the cells are utilized as a micro-scaled factory for the

31 production of value added products and in close contact with organic media (Aono,

32 Tsukagoshi and Miyamoto, 2001; de Carvalho and da Fonseca, 2004; Lee, Yun, Lee

33 and Park, 2015; León, Fernandes, Pinheiro and Cabral, 1998; Siriphongphaew et al.,

34 2012; Wangrangsimagul et al., 2012). The generally used plate count is a traditional

35 method used for enumeration of the culturable bacteria based on an ability to grow and

36 produce colonies on the agar media. However, many recent studies revealed that both

37 Gram-negative and Gram-positive bacteria enter the viable but nonculturable (VBNC)

38 state, meaning that the bacterial cells are alive and show metabolic activity and

39 respiration, but cannot be seen as colony forming units on the agar media (Capozzi et al.,

40 2016; Kell, Kaprelyants, Weichart, Harwood and Barer, 1998; Rizzotti, Levav,

41 Fracchetti, Felis and Torriani, 2015; Wu, Liang and Kan, 2016; Zhao et al., 2017).

42 Therefore, knowing the real status of the bacteria is crucial in the aspect of biosafety

43 concern as well as the utilization of the bacterial cells as biocatalyst for the

44 bioconversion purpose.

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45 In combination with conventional plate count, several cell viability assessment

46 techniques have been employed to estimate the VBNC cells and these include SYBR

47 green real-time PCR (Tirapattanun, Chomvarin, Wongboot and Kanoktippornchai,

48 2015), propidium monoazide combined with qPCR (Rizzotti et al., 2015; Wu et al, 2016)

49 and fluorescein diacetate staining/flow cytometry (Capozzi et al., 2016). CO2 can be

50 appointed as a decent candidate for quantification of viable cells. Headspace gas

51 chromatography (GC) has been used to monitor metabolic CO2 for estimating bacterial

52 cell number in food systems (Gardini, Lanciotti, Sinigagli and Guerzoni, 1997;

53 Guerzoni, Gardini, Cavazza and Piva, 1987) as well as in culture medium (Chai, Dong

54 and Deng, 2008). Alternatively, the level of CO2 can be indirectly measured by trapping

55 CO2 in alkaline solution placed in the same vessel of the bacterial cell and monitoring

56 the changes in the conductivity of the alkaline solution which referred as Indirect

57 Conductivity (IC) (Bolton, 1990; Taok, Cochet, Pauss and Schoefs, 2007; Timms,

58 Colquhoun and Fricker, 1996). Such system is feasible because the conductivity of

59 hydroxide ion (OH-) is completely different from that of carbonate ion (CO32-). Despite of

60 their simplicity and availability, both GC and IC techniques have not yet been reported

61 for an evaluation of the VBNC state of bacteria. In this study, a commercial portable

62 conductivity probe has been successfully applied for the IC measurement of the

63 metabolic CO2 in the 3D emulsion system containing bacterial cells and hydrophobic

64 substrate for the first time. Bioconversion of a hydrophobic substrate using bacterial

65 cells have attracted much interest during the past decades because the technologies are

66 considered cleaner when compared with conventional chemical processes (Schmid et al.,

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67 2001). However, most bacterial cell surfaces are hydrophilic and may limit their use as a

68 whole-cell biocatalyst for the oil-like substrates. Enabling industrially important and

69 generally used bacteria such as E. coli, P. putida and L. lactis to access water-insoluble

70 substrates can extend their applications for both bioconversion and bioremediation. Our

71 original research has demonstrated that a bacteria-chitosan network has an excellent

72 ability in stabilization of oil droplets in an aqueous solution and allows the formation of

73 a 3D scaffold of bacteria interface emulsion (Archakunakorn et al., 2015;

74 Wongkongkatep et al., 2012). The bacteria interface emulsion has also been successfully

75 developed into biocatalyst for effective degradation of petroleum hydrocarbon (Gong,

76 Li, Bao, Lv and Wang, 2015) and a bacterial imprinting at the interface (Shen et al.,

77 2014). To achieve efficient bioconversion, the process control is inevitable and the

78 biological status of the bacterial cells associated with the bacteria interface emulsion is

79 important. In this study, GC and IC techniques coupled with the standard culture

80 technique were used to differentiate the real biological status of the bacterial cells when

81 they are in contact with toluene, n-hexane, n-decane and soybean oil. Toluene is an

82 industrially important aromatic hydrocarbon (C7H8) that is widely used as a cleaning

83 solvent in the manufacturing of paints and coatings, adhesives, resins, and leather

84 industry. The n-hexane (C6H14) has been used as an organic solvent in extraction of

85 cooking oils, production of furniture and painting, rubber, pharmaceutical, perfume,

86 footwear, leather and textiles. The n-decane (C10H22) was employed because of its

87 intermediate length of carbon chain while soybean oil is one of the most widely

88 consumed cooking oils. Together with the standard culture technique, this common

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89 instrumental analysis allowed us to distinguish the VBNC cells out of culturable and

90 metabolically active (live) cells, dead cells and dormant cells. Therefore, GC and IC

91 techniques provided a simple and useful tool which allows a precise bioprocess control.

92 These simple and rapid methods using GC and IC techniques for the detection of VBNC

93 state of the bacterial cells can also be used to assure the biological safety of the

94 manufacturing process in food and pharmaceutical industries.

95

96 2. Materials and Methods

97 2.1 Microorganisms and Culture Conditions

98 The bacterial strains used in this study included E. coli DH5α, P. putida F1 and L.

99 lactis IO-1. E. coli DH5α and P. putida F1 were cultivated in LB (Lab M, Lancashire,

100 UK) at 37°C and 30°C, respectively. L. lactis IO-1 was cultivated in MRS (Lab M,

101 Lancashire, UK) at 30°C. An overnight culture was used to inoculate 200 ml of either

102 LB or MRS (in 1 l-flask) to an initial optical density at 660 nm (OD660) of 0.1. E. coli

103 DH5α, P. putida F1 and L. lactis IO-1 were harvested at an early stationary phase with an

104 OD660 of 2.4, 2.6 and 1.0, respectively by centrifugation at 2,760×g, 4°C for 15 min. Cell

105 pellets were washed twice using 200 ml of sterile 0.1 M sodium phosphate buffer (pH

106 6.8) and re-suspended in the same buffer to the final OD660 of 35, 12 and 35 for E. coli

107 DH5α, P. putida F1 and L. lactis IO-1 (corresponding to a cell concentration of 1109

108 cfu/ml, respectively).

109 2.2 Preparation of the Bacteria Interface Emulsions

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110 The white powder of chitosan was available from Sigma-Aldrich with Product

111 No. C3646 and Lot No. 068K00851 with the deacetylation degree of 96% and MW of 1.6

112 106, and then dissolved in 1% (v/v) acetic acid solution to prepare 1% (w/v) chitosan

113 stock solution. The mixture was stirred continuously at room temperature for 24 h until a

114 clear solution was obtained. The stock solution was then sterilized at 121oC for 15 min

115 and diluted as needed by sterile distilled water. Then, three volumes of bacterial cells

116 suspended in 0.1 M sodium phosphate buffer solution (pH 6.8) were mixed with one

117 volume of the 0.3% (w/v) chitosan solution. Then, the aqueous phase was mixed with an

118 oil phase (soybean oil, n-decane, n-hexane or toluene) at a volume ratio of 1:1. To obtain

119 the bacteria interface emulsion, the mixture was homogenized by using a homogenizer

120 (IKA® T18 basic, ULTRA-TURRAX®) at 11,200 rpm for 3 cycles (30 sec/cycle with 2

121 min rest). The stability of the emulsion was evaluated as %Emulsification Index (%EI) or

122 the percentage of an emulsion phase volume compared with the total volume of the

123 mixture. Cell viability was determined by staining the bacteria interface emulsion with

124 fluorescein diacetate (FDA) as reported by Capozzi et al. (2016). The emulsions were

125 kept at room temperature and sampling at 0.5, 1, 2, 3, 4, 5 day intervals then stained

126 with FDA for 15 min, at a final concentration of 15 mM, in 500 mM sodium phosphate

127 buffer (pH 7.0). The suspension of FDA-stained cells was imaged by epifluorescent

128 microscope (Olympus BX 50, Japan).

129 2.3 GC Technique for Monitoring of Metabolic CO2

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130 The emulsion, the positive control containing the same amount of bacterial cells

131 as in the emulsion and the negative control consisting of the sterile 0.1 M sodium

132 phosphate buffer (pH 7.4) (2 ml each) were transferred into 4 ml gas-tight vial. At a

133 specific time, one vial was sacrificed and the headspace (1 ml) was withdrawn and

134 analyzed by GC (GC-2014, Shimadzu, Japan) equipped with ShinCarbon ST 80/100 (2 m

135 in length, 2 mm inner diameter; 1/8 inch outer diameter) and a thermal conductivity

136 detector. Helium was used as a carrier gas at a flow rate of 10 ml/min. The column

137 temperature was raised from 120°C to 250°C at a rate of 20°C/min. The temperature for

138 injector and TCD were set at 100°C and 280°C, respectively. The culturable cells were

139 evaluated using a conventional plate count.

140 2.4 IC Technique for Monitoring of Metabolic CO2

141 The prepared emulsion (2 ml) was then transferred into a 4 ml-vial which was

142 placed inside a 25 ml-vial containing 10 ml of 0.007 M (equivalent to 0.4 g/l)

143 standardized KOH. An accumulation of metabolic CO2 was measured as the change in

144 conductivity of the KOH solution using a commercial conductivity probe (CON-BTA,

145 Vernier, USA; detection range 0-2000 µS/cm). The signal was recorded every 15 s with a

146 resolution of 1 µS/cm at time intervals. Untreated bacterial cell suspension was used as a

147 positive control while the sterile buffer was used as a negative control. IC change was

148 calculated as follow.

149 IC change = i - t

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150 where i is the initial conductance of the KOH solution and t is the time-

151 dependent conductance of the KOH solution after reacted with CO2.

152 2.5 Statistical Analysis

153 All experiments are performed in at least 2 replicates and a statistical analysis

154 was performed using Minitab software (Release 15, State College, PA).

155 3. Results and Discussion

156 3.1 Enumeration of Culturable Bacterial Cells in 3D Scaffold of Emulsion

157 The growth profiles of E. coli DH5, P. putida F1 and L. lactis IO-1 were

158 constructed and the cells were harvested at the early stationary phase. Oil-in-water

159 emulsions from these 3 strains were obtained after associated with chitosan and

160 emulsified with soybean oil, decane, hexane and toluene as shown in Figure 1. Data of

161 %EI has been analyzed as Factorial Experiment. The obtained results confirmed the

162 significant effects of ‘Type of microorganism’ (p-value = 0.044) and ‘Type of solvent’

163 (p-value = 0.000) but not of ‘Day’ (p-value = 0.920) on the value of %EI. As there were

164 significant interactions between ‘Type of microorganism’ versus ‘Day’ (p-value =

165 0.023) and between ‘Type of solvent’ versus ‘Day’ (p-value = 0.034), the data were

166 then analyzed further using General Linear Model (GLM) option of a Minitab software.

167 Interestingly, while the significant effect of ‘Type of microorganism’ on %EI could be

168 observed on Day 1 (p-value = 0.027), the effect was insignificant on Day 5 (p-value =

169 0.307). On the other hand, the significant effect of ‘Type of solvent’ could be observed

170 throughout an entire experiment period. Overall, the emulsion prepared with soybean oil

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171 provided the highest %EI (85-94%) which was in good agreement with our recent report

172 (Hanpanich, Wongkongkatep, Pongtharangkul and Wongkongkatep, 2017), whereas

173 hexane showed the lowest %EI (69-78%), possibly due to its low boiling point compared

174 with other oil phases used in this study (hexane: 69oC, decane: 174oC, toluene: 111oC,

175 smoke point of soybean oil: 238oC). Consequently, high level of hexane vaporization

176 during homogenization could be expected and may lead to the lowest %EI observed.

177 The partition coefficient (P) is defined as a particular ratio of the concentrations

178 of a target solvent between the two immiscible solvents, generally water and n-octanol

179 which is abbreviated as Po/w. The Po/w value represents the degree of hydrophobicity of

180 the target solvent. Solvents with log Po/w values higher than 4 are generally

181 biocompatible, while those with lower log Po/w are not (Laane, Boeren, Vos and Veeger,

182 1987). The solvents used for emulsion preparation in this study covered a broad range of

183 log Po/w values so as to provide systems with different levels of metabolic activity and

184 culturability, with decreasing levels of toxicity predicted from log Po/w values: toluene >

185 hexane > decane > soybean oil. The results from the standard culture technique

186 confirmed such ranking in terms of toxicity. The culturable cells of E. coli DH5α were

187 retained at approximately 109 cfu/ml in the positive control where the cells were

188 suspended in the buffer without any treatment over the 5 days of study (Figure 3A),

189 while a slight decrease in the value of culturable cells was observed in the case of P.

190 putida F1 (Figure 3B).

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191 L. lactis IO-1, which is lactic acid bacteria generally used in fermented food and

192 claimed to have several health benefit (Chaikham, 2015; Kantachote, Ratanaburee,

193 Hayisama-ae, Sukhoom and Nunkaew, 2017; Phuapaiboon, Leenanon and Levin, 2013),

194 seemed to be the most sensitive strain used in this study because more than 3-log

195 reduction of the culturable cells was observed in the positive control within 5 days

196 (Figure 3C). After treated with chitosan and homogenized with the oil phase, the

197 numbers of culturable cells of E. coli DH5α and P. putida F1 were in the following

198 order; soybean oil > decane > hexane > toluene, which was inversely proportional to the

199 toxicity of the solvent predicted by the value of log Po/w (Laane et al., 1987). A similar

200 trend was also observed in the case of L. lactis IO-1.

201 The cell viability of the bacteria located at the oil droplet interface was accessed

202 by staining with fluorescein diacetate, which will be non-florescent when the cells were

203 metabolically inactive. If the cells are viable, indicating by the activation of membrane

204 esterase during active metabolism, non-fluorescent fluorescein diacetate will be cleaved

205 and liberate the fluorescent fluorescein, therefore the bright fluorescence will be

206 observed through the fluorescent channel of the epifluorescent microscope. In this study,

207 the bright fluorescence of fluorescein was observed from the surface of the emulsion,

208 indicating the viable cells locating at the interface even after the culturable cells count

209 was below the detection limit of 103 cfu/mL (Figure 3 D-F).

210 3.2 Detection of VBNC State of Bacteria in 3D Emulsion as Evaluated by GC and

211 IC

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212 GC has been considered as a rapid determination method of the number of

213 viable bacterial cells since 1987 (Guerzoni et al., 1987), and the technique was

214 developed for the in situ determination of bacterial growth as reported in 2008 (Chai et

215 al, 2008). GC measured the CO2 peak area corresponding to the concentration, while IC

216 is the measurement of conductivity change of the KOH solution upon CO2 absorption.

217 IC provided several advantages over GC as it offers an opportunity for real-time

218 monitoring with an inexpensive equipment. A key design parameter for the sensitivity of

219 the IC method is the concentration of the KOH solution. In this study, an average

220 conductivity of the KOH solution as a function of concentration was measured and

221 presented in Table 1.

222 When bacterial cells are in contact with toxic chemicals, their metabolic activity

223 can be suppressed, leading to low CO2 production. Too high concentration of KOH will

224 reduce the sensitivity of the quantitative analysis of CO2, especially when the small

225 amount of metabolic CO2 was expected. On the other hand, the low concentration of

226 KOH solution may not be suitable as the chemical absorption of ambient air CO2 by

227 KOH solution, as expressed by the chemical reactions below (Taok et al., 2007), would

228 reduce the value of conductivity below the sensitivity of the equipment. In this study, the

229 optimum concentration of 0.4 g/l KOH solution with an average conductivity value of

230 1,338 S/cm was used corresponding to the medium sensitivity of the commercial

231 portable conductivity probe with the detection range of 0-2,000 S/cm and the resolution

232 of 1 S/cm.

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233 CO2(g) + H2O(l) H2CO3 (aq)

234 H2CO3(aq) + 2OH-(aq) CO23- (aq) + 2H2O(l)

235 In the positive control prepared without chitosan and organic phase, %metabolic

236 CO2 (GC) increased sharply during the first 12 h and then slowly increased until it

237 reached 100% for all species of bacteria tested (Figure 4), where the 100% of metabolic

238 CO2 refers to the highest CO2 production detected in the positive control over 5 days. In

239 general, higher number of culturable cells would be accounted for higher %metabolic

240 CO2 observed by GC and IC. However, exceptions were very clear especially in

241 emulsion systems of P. putida F1 prepared with soybean oil and decane because their

242 metabolic CO2 was the highest among the tested strains and even higher than that of the

243 positive control (Figure 4B, E). It should be noted that the number of culturable cells in

244 solvent emulsion systems did not exceed those of the positive controls (Figure 3). The

245 results clearly indicated the presence of VBNC P. putida F1 cells in the 3D scaffold of

246 emulsion. Furthermore, this result indicated that P. putida F1 exhibited higher tolerance

247 towards a wide range of organic solvents, as reported elsewhere (Lee, Park, Kim, Yoon

248 and Oh, 2002; Li et al., 2015), compared with other bacteria including E. coli DH5 and

249 L. lactis IO-1 tested in this study. Moreover, P. putida F1 seemed to be able to recover

250 from hexane exposure as indicated by a continuous increase of metabolic CO2 obtained

251 from both GC and IC (Figure 4B, E) and the recovery of the culturable cells number

252 from day 1 to day 5 (Figure 3B). This result agreed well with a previous report that P.

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253 putida F1 cells could recover from hexane shock due to an unknown innate ability and

254 environmental factors (Heipieper and de Bont, 1994; Huertas, Duque, Marques and

255 Ramos, 1998). Panikov et al. (2015) reported that proteomic analysis showed the

256 upregulated (transporters, stress response, self-degrading enzymes and extracellular

257 polymers) and downregulated (ribosomal, chemotactic and primary biosynthetic

258 enzymes) proteins found during VBNC state of P. putida.

259 L. lactis IO-1 was apparently the most sensitive of the strains tested, because the

260 culturable cell count was gradually decreased to a level below the detection limit at day

261 3 even in the least toxic soybean oil-emulsion (Figure 3C). The degree of solvent

262 toxicity ranging from the most toxic toluene > hexane > decane > soybean oil was most

263 obvious in the case of L. lactis IO-1 as suggested by the highest to lowest metabolic CO2

264 values recorded by GC when the cells were in contact with soybean oil, followed by

265 decane, hexane and toluene (Figure 4C). Therefore, instrumental analysis of metabolic

266 CO2 by GC and IC coupled with conventional plate count, suggested the presence of

267 VBNC L. lactis IO-1 cells in the emulsion system, which was in good agreement with

268 the fluorescein diacetate staining method as shown in Figure 3F. In fact, the VNBC state

269 of lactic acid bacteria has been previously reported during the storage of wine (Millet

270 and Lonvaud-Funel, 2000; Rizzotti et al., 2015).

271 4. Conclusions

272 The metabolic activity of E. coli DH5α, P. putida F1 and L. lactis IO-1 after an

273 exposure to toluene, hexane, decane and soybean oil in the 3D scaffold of bacteria

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274 interface emulsion was evaluated using a conventional plate counting in combination

275 with an instrumental analysis of metabolic CO2. These GC and IC techniques enabled

276 the detection of VBNC state of all three strains tested when contact with hexane and

277 decane. The ability of P. putida F1 to recover from stress shock during the emulsion

278 formation could be clearly observed from a conventional plate count and metabolic

279 activity detected by both GC and IC. Other methods for enumeration of VBNC state of

280 bacteria as summarized by Davis (2014), such as fluorescent in situ hybridization (FISH),

281 real-time quantitative PCR (RT-qPCR or qPCR), reverse transcriptase (RT-PCR),

282 propidium monoazide-PCR, and flow cytometry (FC)/fluorescent activated cell sorting

283 (FACS), all depend on expensive fluorescent dye/labelling techniques and the use of

284 sophisticated instruments. The common GC and simple IC techniques for the detection

285 of VBNC state of bacterial cells demonstrated for the first time in this study can serve

286 as the standard and basic instrumental analysis for detection of VBNC state of bacteria

287 in various applications. The application of metabolically active P. putida and E. coli in

288 bioconversion using the bacteria interface emulsion system is now being investigated.

289

290 Acknowledgments

291 This work was supported by the Faculty of Science, Mahidol University and the

292 Thailand Research Fund (IRG5980001). Academic scholarship has been awarded to NC

293 by the Young Scientist Strengthening Program, Faculty of Science, Mahidol University.

294

295 References

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411 End of Text file………………………………………………………………………….

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1 All figures caption list should precede all the figures.

2 Figure 1. Bacteria interface emulsion preparation scheme. SEM images of E. coli

3 DH5 before (A) and after network formation with chitosan (B). Scale bars (white)

4 represent 3 m for SEM images. Appearance and micro-image of 3D scaffold bacteria

5 interface emulsion (C). Scale bar (black) represent 100 m.

6 Figure 2. %Emulsification index (EI) of the emulsion prepared using E. coli DH5

7 (white), P. putida F1 (gray), and L. lactis IO-1 (black) stored at room temperature for 1

8 and 5 days.

9 Figure 3. Culturable cells count of E. coli DH5α (A), P. putida F1 (B) and L. lactis IO-

10 1 (C) in emulsion prepared from soybean oil (black square), decane (white circle),

11 hexane (white triangle) and toluene (white square). The same amount of cells in the

12 suspension without any oil phase was referred as a positive control (black triangle) and

13 the abiotic 0.1 M sodium phosphate buffer (pH 7.4) was used as a negative control

14 (cross). Fluorescent images of the emulsions provided by E. coli DH5α (D), P. putida

15 F1 (E) and L. lactis IO-1 (F) using fluorescein diacetate staining method. The

16 fluorescence observed from the surface of emulsion indicated the viable cells with

17 active metabolism.

18 Figure 4. %Metabolic CO2 of of E. coli DH5α (A, D), P. putida F1 (B, E) and L. lactis

19 IO-1 (C, F) evaluated by GC (upper panels) and IC change (lower panels) in emulsion

20 prepared from soybean oil (black square), decane (white circle), hexane (white triangle)

21 and toluene (white square). The same amount of cells in the suspension without any oil

22 phase was referred as a positive control (black triangle) and the abiotic 0.1 M sodium

23 phosphate buffer (pH 7.4) was used as a negative control (cross).

24

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25

26 Figure 1. Bacteria interface emulsion preparation scheme. SEM images of E. coli

27 DH5 before (A) and after network formation with chitosan (B). Scale bars (white)

28 represent 3 m for SEM images. Appearance and micro-image of 3D scaffold bacteria

29 interface emulsion (C). Scale bar (black) represent 100 m.

30

31 Figure 2. %Emulsification index (EI) of the emulsion prepared using E. coli DH5

32 (white), P. putida F1 (gray), and L. lactis IO-1 (black) stored at room temperature for 1

33 and 5 days.

34

35

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36

37 Figure 3. Culturable cells count of E. coli DH5α (A), P. putida F1 (B) and L. lactis IO-

38 1 (C) in emulsion prepared from soybean oil (black square), decane (white circle),

39 hexane (white triangle) and toluene (white square). The same amount of cells in the

40 suspension without any oil phase was referred as a positive control (black triangle) and

41 the abiotic 0.1 M sodium phosphate buffer (pH 7.4) was used as a negative control

42 (cross). Fluorescent images of the emulsions obtained from E. coli DH5α (D), P. putida

43 F1 (E) and L. lactis IO-1 (F) in emulsion prepared from hexane at 3, 0.5 and 2 days of

44 storage respectively at room temperature using fluorescein diacetate staining method.

45 The arrows indicate the live cells with active metabolism.

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46

47 Figure 4. %Metabolic CO2 of of E. coli DH5α (A, D), P. putida F1 (B, E) and L. lactis

48 IO-1 (C, F) evaluated by GC (upper panels) and IC change (lower panels) in emulsion

49 prepared from soybean oil (black square), decane (white circle), hexane (white triangle)

50 and toluene (white square). The same amount of cells in the suspension without any oil

51 phase was referred as a positive control (black triangle) and the abiotic 0.1 M sodium

52 phosphate buffer (pH 7.4) was used as a negative control (cross).

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1 Table 1. Average conductivity values of the different KOH concentrations

KOH concentration (g/l) Average conductivity value (S/cm)

0.1

0.4

0.5

1

5

353

1,338

2,165

4,311

18,693

2

3 End of Table file…………………………………………………………………………

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