for review onlyrdo.psu.ac.th/sjstweb/ar-press/2019sep/22.pdf · 2019. 9. 10. · for review only 4...
Post on 19-Aug-2020
0 Views
Preview:
TRANSCRIPT
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
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
For Review Only
1
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: jirarut.chu@mahidol.ac.th
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: panwajee.pay@mahidol.ac.th
18 Nattapat Charoenrat natty.auzy@gmail.com
19 Thunyarat Pongtharangkul thunyarat.pon@mahidol.ac.th
Page 5 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
1
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
Page 6 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
2
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.
Page 7 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
3
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.,
Page 8 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
4
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
Page 9 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
5
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
Page 10 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
6
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
Page 11 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
7
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
Page 12 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
8
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
Page 13 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
9
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).
Page 14 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
10
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
Page 15 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
11
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.
Page 16 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
12
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.
Page 17 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
13
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
Page 18 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
14
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
Page 19 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
15
296 Aono, R., Tsukagoshi, N., & Miyamoto, T. (2001). Evaluation of the growth inhibition
297 strength of hydrocarbon solvents against Escherichia coli and Pseudomonas putida
298 grown in a two-liquid phase culture system consisting of a medium and organic solvent.
299 Extremophiles, 5, 11-15. doi:10.1007/s007920000167
300 Archakunakorn, S., Charoenrat, N., Khamsakhon, S., Pongtharangkul, T.,
301 Wongkongkatep, P., Suphantharika, M., & Wongkongkatep, J. (2015). Emulsification
302 efficiency of adsorbed chitosan for bacterial cells accumulation at the oil-water
303 interface. Bioprocess and Biosystems Engineering, 38, 701-709. doi:10.1007/s00449-014-
304 1310-6
305 Bolton, F. J. (1990). An investigation of indirect conductimetry for detection of some
306 food - borne bacteria. Journal of Applied Bacteriology, 69, 655-661.
307 Capozzi, V., Di Toro, M. R., Grieco, F., Michelotti, V., Salma, M., Lamontanara, A.,
308 Russo, P., Orrù, L., Alexandre, H., & Spano, G. (2016). Viable But Not Culturable
309 (VBNC) state of Brettanomyces bruxellensis in wine: New insights on molecular basis of
310 VBNC behaviour using a transcriptomic approach. Food Microbiology, 59, 196-204.
311 doi:10.1016/j.fm.2016.06.007
312 Chai, X. S., Dong, C., & Deng, Y. (2008). In situ determination of bacterial growth by
313 multiple headspace extraction gas chromatography. Analytical Chemistry, 80(20), 7820–
314 7825. doi:10.1021/ac801537x
Page 20 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
16
315 Chaikham, P. (2015). Stability of probiotics encapsulated with Thai herbal extracts in
316 fruit juices and yoghurt during refrigerated storage. Food Bioscience,12, 61–66.
317 doi:10.1016/j.fbio.2015.07.006
318 Davis, C. (2014). Enumeration of probiotic strains: Review of culture-dependent and
319 alternative techniques to quantify viable bacteria. Journal of Microbiological Methods,
320 103, 9–17. doi:10.1016/j.mimet.2014.04.012
321 de Carvalho, C. C. C. R., & da Fonseca, M. M. R. (2004). Solvent toxicity in organic-
322 aqueous systems analyzed by multivariate analysis. Bioprocess and Biosystems
323 Engineering, 26, 361-375. doi:10.1007/s00449-004-0381-1
324 Gardini, F., Lanciotti, R., Sinigaglia, M., & Guerzoni, M. E. (1997). A head space gas
325 chromatographic approach for the monitoring of the microbial cell activity and the cell
326 viability evaluation. Journal of Microbiological Methods, 29, 103–114.
327 Gong, H., Li, Y., Bao, M., Lv, D., & Wang, Z. (2015). Petroleum hydrocarbon degrading
328 bacteria associated with chitosan as effective particle-stabilizers for oil emulsification.
329 RSC Advances, 5, 37640-37647. doi:10.1039/c5ra01360g
330 Guerzoni, M. E., Gardini, F., Cavazza, A., & Piva, M. (1987). Gas-liquid chromatographic
331 method for the estimation of coliforms in milk. International Journal of Food
332 Microbiology, 4, 73-78.
Page 21 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
17
333 Hanpanich, O., Wongkongkatep, P., Pongtharangkul, T., & Wongkongkatep, J. (2017).
334 Turning hydrophilic bacteria into biorenewable hydrophobic material with potential
335 antimicrobial activity via interaction with chitosan. Bioresource Technology, 230, 97–
336 102. doi:10.1016/j.biortech.2017.01.047
337 Heipieper, H., & de Bont, J. A. M. (1994). Adaptation of Pseudomonas putida S12 to
338 ethanol and toluene at the level of fatty acid composition of membranes. Applied
339 Environmental Microbiology, 60, 4440-4444.
340 Huertas, M-J., Duque, E., Marques, S., & Ramos, J. L. (1998). Survival in soil of different
341 toluene-degrading Pseudomonas strains after solvent shock. Applied and Environmental
342 Microbiology, 64(1), 38-42.
343 Kantachote, D., Ratanaburee, A., Hayisama-ae, W., Sukhoom, A., & Nunkaew, T. (2017).
344 The use of potential probiotic Lactobacillus plantarum DW12 for producing a novel
345 functional beverage from mature coconut water. Journal of Functional Foods, 32, 401–
346 408. doi:10.1016/j.jff.2017.03.018
347 Kell, D. B., Kaprelyants, A. S., Weichart, D. H., Harwood, C. R., & Barer, M. R. (1998).
348 Viability and activity in readily culturable bacteria: a review and discussion of the
349 practical issues. Antonie van Leeuwenhoek, 73, 169–87.
350 Laane, C., Boeren, S., Vos, K., & Veeger, C. (1987). Rules for optimization of
351 biocatalysis in organic solvents. Biotechnology and Bioengineering, 30, 81–87.
Page 22 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
18
352 Lee, N. R., Yun, J. Y., Lee, S. M., & Park, J. B. (2015). Cyclohexanone-induced stress
353 metabolism of Escherichia coli and Corynebacterium glutamicum. Biotechnology and
354 Bioprocess Engineering, 20, 1088-1098. doi:10.1007/s12257-015-0607-x
355 Lee, W. S., Park, C. S., Kim, J. E., Yoon, B. D., & Oh, H. M. (2002). Characterization of
356 TCE-degrading bacteria and their application to wastewater treatment. Journal of
357 Microbiology and Biotechnology, 12(4), 569–575.
358 León, R., Fernandes, P., Pinheiro, H. M., & Cabral, J. M. S. (1998). Whole-cell biocatalysis
359 in organic media. Enzyme and Microbial Technology, 23, 483–500.
360 Li, S-S., Hu, X., Zhao, H., Li, Y-X., Zhang, L., Gong, L-J., Guo, J., & Zhao, H-B. (2015).
361 Quantitative analysis of cellular proteome alterations of Pseudomonas putida to
362 naphthalene-induced stress. Biotechnology Letters, 37, 1645-1654. doi:10.1007/s10529-
363 015-1828-y
364 Millet, V., & Lonvaud-Funel, A. (2000). The viable but non-culturable state of wine
365 micro-organisms during storage. Letters in Applied Microbiology, 30, 136–141.
366 Panikov, N. S., Mandalakis, M., Dai, S., Mulcahy, L. R., Fowle, W., Garrett, W. S., &
367 Karger, B. L. (2015). Near-zero growth kinetics of Pseudomonas putida deduced from
368 proteomic analysis. Environmental Microbiology, 17(1), 215–228. doi: 10.1111/1462-
369 2920.12584
Page 23 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
19
370 Phuapaiboon, P., Leenanon, B., & Levin, R. E. (2013). Effect of Lactococcus lactis
371 immobilized within pineapple and yam bean segments, and Jerusalem artichoke powder
372 on its viability and quality of yogurt. Food and Bioprocess Technology 6, 2751–2762.
373 doi:10.1007/s11947-012-0940-4
374 Rizzotti, L., Levav, N., Fracchetti, F., Felis, G. E., & Torriani, S. (2015). Effect of UV-C
375 treatment on the microbial population of white and red wines, as revealed by
376 conventional plating and PMA-qPCR methods. Food Control, 47, 407-412.
377 doi:10.1016/j.foodcont.2014.07.052
378 Schmid, A., Dordick, J. S., Hauer, B., Kiener, A., Wubbolts, M., & Witholt, B. (2001).
379 Industrial biocatalysis today and tomorrow. Nature, 409, 258-268.
380 Shen, X., Bonde, J. S., Kamra, T., Bulow, L., Leo, J. C., Linke, D., & Ye, L. (2014).
381 Bacterial Imprinting at Pickering Emulsion Interfaces. Angewante Chemie International
382 Edition, 53, 10687-10690. doi:10.1002/anie.201406049
383 Siriphongphaew, A., Pisnupong, P., Wongkongkatep, J., Inprakhon, P., Vangnai, A. S.,
384 Honda, K., Ohtake, H., Kato, J., Ogawa, J., Shimizu, S., Urlacher, V. B., Schmid, R. D., &
385 Pongtharangkul, T. (2012). Development of a whole-cell biocatalyst co-expressing P450
386 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane. Applied
387 Microbiology and Biotechnology, 95, 357–367. doi:10.1007/s00253-012-4039-7
Page 24 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
20
388 Taok, M., Cochet, N., Pauss, A., & Schoefs, O. (2007). Monitoring of microbial activity
389 in soil using biological oxygen demand measurement and indirect impedancemetry.
390 European Journal of Soil Biology, 43, 335-340. doi:10.1016/j.ejsobi.2007.03.007
391 Timms, S., Colquhoun, K. O., & Fricker, C. R. (1996). Detection of Escherichia coli in
392 potable water using indirect impedance technology. Journal of Microbiological
393 Methods, 26, 125-132.
394 Tirapattanun, A., Chomvarin, C., Wongboot, W., & Kanoktippornchai, B. (2015).
395 Application of SYBR green real-time PCR for detection of toxigenic Vibrio cholera O1
396 in the aquatic environment. Chiang Mai Journal of Science, 42(3), 588-598.
397 Wangrangsimagul, N., Klinsakul, K., Vangnai, A. S., Wongkongkatep, J., Inprakhon, P.,
398 Honda, K., Ohtake, H., Kato, J., & Pongtharangkul, T. (2012). Bioproduction of vanillin
399 using an organic solvent-tolerant Brevibacillus agri 13. Applied Microbiology and
400 Biotechnology, 93, 555–563. doi:10.1007/s00253-011-3510-1
401 Wongkongkatep, P., Manopwisedjaroen, K., Tiposoth, P., Archakunakorn, S.,
402 Pongtharangkul, T., Suphantharika, M., Honda, K., Hamachi, I., & Wongkongkatep, J.
403 (2012). Bacteria interface Pickering emulsions stabilized by self-assembled bacteria-
404 chitosan network. Langmuir, 28, 5729-5736. doi:10.1021/la300660x
Page 25 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
21
405 Wu, B., Liang, W., & Kan, B. (2016). Growth phase, oxygen, temperature, and starvation
406 affect the development of viable but non-culturable state of Vibrio cholerae. Frontiers in
407 Microbiology, 7, 404. doi:10.3389/fmicb.2016.00404
408 Zhao, X., Zhong, J., Wei, C., Lin, C-W., & Ding, T. (2017) Current perspectives on viable
409 but non-culturable state in foodborne pathogens. Frontiers in Microbiology, 8, 580.
410 doi:10.3389/fmicb.2017.00580
411 End of Text file………………………………………………………………………….
Page 26 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
1
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
Page 27 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
2
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
Page 28 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
3
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.
Page 29 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
4
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).
Page 30 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
For Review Only
1
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…………………………………………………………………………
Page 31 of 31
For Proof Read only
Songklanakarin Journal of Science and Technology SJST-2018-0175.R2 Wongkongkatep
123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960
top related