Scholarly article on topic 'The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of Clostridium butyricum DSP1'

The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of Clostridium butyricum DSP1 Academic research paper on "Industrial Biotechnology"

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{"Batch culture" / " Clostridium butyricum " / "Environmental stress" / "1 / 3-Propanediol" / "Substrate resistant"}

Abstract of research paper on Industrial Biotechnology, author of scientific article — Daria Szymanowska-Powałowska

Abstract Background The production of biofuels from renewable energy sources is one of the most important issues in biotechnology today. The process is known to generate various by-products, for example glycerol that is obtained in the making of biodiesel from rapeseed oil. Crude glycerol may be utilized in many ways, including microbial conversion to 1,3-propanediol. The main drawback of that technology is the use of high concentrations of glycerol, which inhibits the growth of bacterial cells. Results This study investigated the impact of crude glycerol on Clostridium butyricum DSP1 and its ability to adapt to an environment of high osmotic pressure. It was found that a crude glycerol concentration of up to 70g/L did not have an inhibitory effect on C. butyricum DSP1. Adaptation procedures involving the passage of metabolically active biomass from a fermentation medium with a lower concentration of crude glycerol to one with a greater substrate concentration allowed breaking the barrier of high osmotic pressure (150g/L crude glycerol) and receiving a 1,3-PD concentration of 74g/L in a batch culture operation. The work looked into intracellular modifications shown by proteomic profiling in order to explain the mechanisms underlying the response and adaptation of bacterial cells exposed to unfavorable environmental conditions. Conclusions This study of the effect of glycerol on the growth and metabolism of C. butyricum DSP1 demonstrated that the maximum substrate concentrations that do not inhibit the metabolic activity of bacterial cells are 90g/L and 70g/L for pure and crude glycerol, respectively.

Academic research paper on topic "The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of Clostridium butyricum DSP1"

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Electronic Journal of Biotechnology

The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of Clostridium butyricum DSP1

Daria Szymanowska-Powalowska

Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Wojska Polskiego 48, 60-527 Poznan, Poland

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ABSTRACT

Article history: Received 8 August 2014 Accepted 18 November 2014 Available online xxxx

Keywords: Batch culture Clostridium butyricum Environmental stress 1,3-Propanediol Substrate resistant

Background: The production of biofuels from renewable energy sources is one of the most important issues in 16 biotechnology today. The process is known to generate various by-products, for example glycerol that is 17 obtained in the making of biodiesel from rapeseed oil. Crude glycerol may be utilized in many ways, including 18 microbial conversion to 1,3-propanediol. The main drawback of that technology is the use of high 19 concentrations of glycerol, which inhibits the growth of bacterial cells. 20

Results: This study investigated the impact of crude glycerol on Clostridium butyricum DSP1 and its ability to adapt 21 to an environment of high osmotic pressure. It was found that a crude glycerol concentration of up to 70 g/L did 22 not have an inhibitory effect on C. butyricum DSP1. Adaptation procedures involving the passage of metabolically 23 active biomass from a fermentation medium with a lower concentration of crude glycerol to one with a greater 24 substrate concentration allowed breaking the barrier of high osmotic pressure (150 g/L crude glycerol) and 25 receiving a 1,3-PD concentration of 74 g/L in a batch culture operation. The work looked into intracellular 26 modifications shown by proteomic profiling in order to explain the mechanisms underlying the response and 27 adaptation of bacterial cells exposed to unfavorable environmental conditions. 28

Conclusions: This study of the effect of glycerol on the growth and metabolism of C. butyricum DSP1 demonstrated 29 that the maximum substrate concentrations that do not inhibit the metabolic activity of bacterial cells are 90 g/L 30 and 70 g/L for pure and crude glycerol, respectively. 31

© 2015 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction

One of the most pressing problems of industrial biotechnology today is a need to select microorganisms robustly resistant to adverse environmental conditions. The metabolic and molecular mechanisms of cellular response to various stress factors have been dealt with in many publications concerned predominantly with laboratory research rather than studies of industry-scale fermentation. It is vital, however, to understand the dependence of microbial behavior on conditions that occur during industrial processes in order to ensure optimal selection of strains and efficient management of mass-scale fermentation [1]. An important biotechnological issue is the inhibition of bioconversion that may arise from considerable concentrations of substrates or products (high osmotic pressure in the fermentation medium) and/or the presence of toxic substances (metabolites and/or substrate impurities) [2,3]. Bacterial growth inhibition also affects microorganisms of the genus Clostridium. The non-pathogenic clostridia have a great potential for industrial applications [4]. These

E-mail address: darszy@up.poznan.pl. Peer review under responsibility of Pontificia Universidad Católica de Valparaíso.

bacteria ferment organic compounds such as carbohydrates and 55 release large amounts of CO2 and H2 as well as organic acids (e.g. 56 butyric, lactic, acetic, and succinic acids) and solvents such as butanol, 57 acetone, and isopropanol (solventogenic clostridia) [4,5]. Among these 58 substances, 1,3-propanediol (1,3-PD) is of industrial interest as a 59 monomer for light-insensitive plastics. 1,3-PD is an industrially 60 valuable chemical intermediate with potential uses in the production 61 of polymers (e.g. polyesters, polyethers, polyurethanes), cosmetics, 62 foods, lubricants, medicines, and as an intermediate for the synthesis 63 of heterocyclic compounds such as indole and quinolines [4,5,6,7,8]. 64 1,3-PD may be produced chemically or microbiologically [9,10,11,12]. 65 At present chemical methods are being replaced by microbiological 66 technologies. 1,3-PD is effectively synthesized microbiologically from 67 crude glycerol generated during biodiesel production [3,8]. In the 68 microbiological conversion of glycerol to 1,3-PD the most common are 69 Clostridium spp., Klebsiella spp., Citrobacter spp., Lactobacillus spp. and 70 Hafnia spp. [8,13,14,15]. The main factors responsible for inhibiting 71 that process include a high substrate concentration (pure or crude 72 glycerol), the accumulation of toxic products such as 1,3-PD, and the 73 presence of organic acids. This study focused on defining the 74 maximum concentration of glycerol that allows bacterial growth and 75 efficient synthesis of 1,3-PD. Additionally, an attempt was made to 76

http://dx.doi.org/m1016/j.ejbt2015.01.006

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adapt Clostridium butyricum DSP1 to high concentrations of crude glycerol.

2. Materials and methods

2.1. Microorganism

The strain used in the process of converting crude glycerol to 1,3-PD was C. butyricum DSP1 [16]. It was previously isolated from ruminal fluid and put in the collection of the Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, Poland, as well as deposited at the Polish Collection of Microorganisms (PCM).

2.2. Culture medium

The strain was maintained in Reinforced Clostridial Medium (RCM, Oxoid, UK) in serum bottles at 4°C. Pre-cultures of pure culture inoculum were cultivated in Hungate test tubes in an appropriate cultivation medium (37°C, 18 h). Clostridium bacteria were cultured in a chamber for cultivation of anaerobic microorganisms (Whitley MG500, Don Whitley Scientific, Shipley, United Kingdom), without pH regulation or stirring.

23. Fermentation medium

The first fermentation medium consisted of (per 1 L of deionized water) 0.26 g K2HPO4, 0.02 g KH2PO4, 1.23 g (NH4)2SO4, 0.1 g MgSO4 x 7H2O, 0.01 g CaCl2 x 2H2O, 0.01 g FeCl2 x 7H2O, and yeast extract. The concentration of the yeast extract in the fermentation medium was increased as the glycerol concentration rose (Table 1). The fermentation medium was supplemented with pure (Avantor Performance Materials Poland S.A, Gliwice) or crude glycerol (Wratislavia-Bio, Wroclaw, Poland) at a concentration of 50.0170.0 ±1.0 g/L. The crude glycerol contained (v/v) 72% glycerol, 12% NaCl, and 13.6% water, and had a pH of 6.5. The media were autoclaved at 121°C for 20 min.

2.4. Fermentation experiments

All experiments were performed in a 6.6 L bioreactor (Sartorius Stedim, Germany) with a working volume of 2.0 L. The bioreactor was equipped with controls for the temperature, pH, agitation speed and aeration rate. The pH was controlled at 7.0 by automatic addition of 1 M NaOH and all fermentation experiments were carried out at 37°C. Anaerobic conditions were sustained by continuous nitrogen sparging at a flow rate of 0.1 vvm. During the experiments aimed at examining the influence of high glycerol concentrations on C. butyricum DSP1 the biomass was passaged from the culture medium to production media where pure or crude glycerol concentrations were 50, 70, 90, 110, 130, 150, and 170 g/L. An evaluation of the adaptation ability of C. butyricum DSP1 was performed using only crude glycerol and comprised several stages of which the first one involved a batch culture with a substrate concentration of 70 g/L. The moment that biomass accumulation reached a plateau, an amount of the

Table 1

The concentration of yeast extract depending on the applied concentration of glycerol.

Concentration of glycerol (g/L) Concentration of yeast extract (g/L)

130 10

150 12

170 14

bioreactor's contents equal to 10% ofits working capacity was pumped 123

into another one filled with a production medium containing 90 g/L 124

crude glycerol. The next passages were carried out to production 125

media with glycerol concentrations of 110,130,150, and 170 g/L in a 126

similar manner. 127

2.5. Analytical methods 128

The 1,3-PD, glycerol and organic acids were assayed by 129

high-performance liquid chromatography. Samples for chemical 130

analysis were first centrifuged at 10,000 x g for 10 min at 4°C Q4

(Multifuge 3SR, Germany), filtered through a 0.22 |am membrane 132

filter (Millex-GS, Millipore, USA), and then analyzed on an HPLC 133

system (Agilent Technologies 1200 series). 134

An Agilent Technologies 1200 series system equipped with a 135

refractive index detector was used. Analyses were performed 136

isocratically at a flow rate of 0.6 mL/min on an Aminex HPX-87H 137

300 x 7.8 columns (Bio-Rad, CA, USA) at a constant temperature of 138

65°C. H2SO4 (0.5 mN) was the mobile phase. External standards were 139

applied for identification and quantification of peak areas. Retention 140

times (Rt) determined for the target compounds were: 1,3-PD — 141

17.17 min; glycerol — 13.03 min; butyric acid — 20.57 min; acetic acid 142

— 14.4 min and lactic acid — 11.19 min. 143

The cell concentration (g/L) was determined using a linear equation 144

derived from the relationship of cell dry weight (90°C until constant 145

weight) and optical density (OD) at 600 nm (AnalytikJena Specord 50). 146

2.6. Protein analyses 147

Proteins were reduced (10 mM DTT, 30 min, 56°C) and alkylated 148

with iodoacetamide in darkness (45 min, 20°C) and digested 149

overnight with 10 ng/^L trypsin. The resulting peptide mixtures were 150

applied to the RP-18 pre-column of a UPLC system (Waters) using 151

water containing 0.1% FA as a mobile phase and then transferred to a 152

nano-HPLC RP-18 column (internal diameter 75 |am, Waters) using 153

ACN gradient (0-35% ACN in 160 min) in the presence of 0.1% FA at a 154

flow rate of 250 mL/min. The column outlet was coupled directly to 155

the ion source of an Orbitrap Velos mass spectrometer (Thermo). Each 156

sample was measured in duplicate — once for protein sequencing 157

(data-dependent MS to MS/MS switch) and once for quantitative 158

information (MS only, sequencing disabled). 159

The acquired MS/MS data were pre-processed with Mascot Distiller 160

software (v. 2.3, MatrixScience) and a search was performed with the 161

Mascot Search Engine MatrixScience, Mascot Server 2.4 against the set 162

of Clostridium protein sequences derived from Uniprot, merged with 163

its randomized version (16294 sequences; 5095802 residues). 164

Those proteins that exactly matched the same set of peptides were 165

combined into a single cluster. Mass calibration and data filtering 166

were carried out with MScan software. 167

The lists of peptides that matched the acceptance criteria from the 168

LC-MS/MS runs were merged into one common list. This was overlaid 169

onto 2-D heat maps generated from the LC-MS profile datasets by 170

tagging the peptide-related isotopic envelopes with corresponding 171

peptide sequence tags on the basis of the measured/theoretical mass 172

difference, the deviation from the predicted elution time, and the 173

match between the theoretical and observed isotopic envelopes. The 174

abundance of each peptide was determined as the height of a 2-D fit 175

to the monoisotopic peak of the tagged isotopic envelope. Quantitative 176

values were normalized with LOWESS, proteins with more than 80% 177

common peptides were clustered and the peptides unique for the 178

cluster were used for statistical analysis. Only proteins with q-value 179

below 0.05 or those present in only one of two compared analytical 180

groups were taken into consideration during further analysis. The 181

protein concentration was measured by Bradford's method [17]. 182

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183 3. Results

184 3.1. The effect of high concentrations of pure and crude glycerol on the

185 growth and metabolism ofC. butyricum DSP1

186 This part of the study involved tests designed to evaluate the

187 resistance of C. butyricum DSP1 to various concentrations of pure and

188 crude glycerol within a range of 50-170 g/L. It was found that

189 although the growth of C. butyricum DSP1 bacteria and the synthesis

190 of 1,3-PD were still possible at a pure glycerol concentration of 110 g/L,

191 complete substrate utilization occurred in the range of 50-90 g/L. The

192 concentration of pure glycerol at which the 1,3-PD yield reached a

193 maximum of 0.56 g/g and the productivity stood at 1.13 g/L/h was

194 70 g/L.

195 The highest concentration of 1,3-PD, 48.3 g/L, was obtained for the

196 initial substrate concentration of 90 g/L, though the productivity

197 declined markedly to 0.78 g/L/h. The highest value of the initial

198 substrate concentration allowing for a maximum of biomass

199 aggregation and a satisfying level of 1,3-PD yield was further reduced

200 after using crude glycerol as a carbon source and dropped to 50 g/L.

201 Under those conditions a slight accumulation of the biomass

202 still progressed at a crude glycerol concentration not exceeding

203 110 g/L whereas complete substrate utilization was observed for

204 concentrations of 50 and 70 g/L. A significant fall in the productivity

205 was registered as increasing initial substrate concentrations were

206 applied. At the initial concentration of crude glycerol of 110 g/L the

207 productivity was 0.22 g/L/h, 1.5-fold lower than that for pure glycerol

208 of the same concentration (Table 2). The 1,3-PD yield also decreased

209 compared to the synthesis from the pure substrate. The profile of

210 metabolites other than 1,3-PD changed as well. Due to the fact that

211 the use of rising concentrations of the crude glycerol caused a greater

212 production of lactic acid, the selectivity of synthesizing particular

213 metabolites, especially 1,3-PD, was considered to be an important

214 kinetic parameter. The selectivity of 1,3-PD synthesis expressed

215 as percentage was obtained by dividing the 1,3-PD concentration

216 by the sum of the concentrations of all liquid metabolites, and

217 multiplying by 100%. Thus, the selectivities of 1,3-PD synthesis from

218 50 g/L and 110 g/L crude glycerol were 74% and 63%, respectively.

219 Biomass accumulation was stopped in the experiments with

220 high concentrations of pure as well as crude glycerol. A 130 g/L

221 concentration of crude glycerol prevented bacterial growth and even

t2.1 Table 2

t2.2 Effect of initial glycerol concentration on growth and kinetic parameters during batch t Q1 fermentation of C. butyricum DSP1.

t2.4 Ccly (g/L) C13-PD (g/L) Y13-PD (g/g) X (g/L) Q (g/L/h) S1,3-PD (%)

t2.5 Pure

t2.6 50 27.5 ± 0.9 0.55 1.2 ±0.1 1.18 82

t2.7 70 38.5 ± 1.0 0.56 1.3 ± 0.2 1.13 84

t2.8 90 48.3 ± 1.1 0.54 0.9 ± 0.1 0.78 73

t2.9 110 36.1 ± 1.1 0.51 0.9 ± 0.1 0.31 68

t2.10 130 25.3 ± 0.8 0.48 0.7 ± 0.1 0.21 65

t2.11 150 20.3 ± 0.6 0.42 0.5 ± 0.0 0.23 62

t2.12 170 0.3 ± 0.0 r- 0.2 ± 0.0 - -

t2.13 t2.14 Crude

t2.15 50 26.2 ± 0.9 0.52 1.3 ± 0.3 0.94 74

t2.16 70 35.8 ± 0.9 0.51 1.2 ±0.1 0.86 77

t2.17 90 28.3 ± 0.8 0.47 0.8 ± 0.1 0.62 64

t2.18 110 30.8 ± 0.8 0.48 0.5 ± 0.0 0.22 63

t2.19 130 0.5 ± 0.0 - 0.2 ± 0.0 - -

t2.20 150 0.0 ± 0.0 - 0.2 ± 0.0 - -

t2.21 170 0.0 ± 0.0 - 0.1 ± 0.0 - -

t2.22 CGiy: concentration of glycerol in fermentation medium (gram per liter); C13-PD0:

t2.23 produced 1,3-propanediol (gram per liter); Y13-PD: yield of 1,3-propanediol produced

t2.24 per glycerol consumed (gram per gram); X: biomass (gram per liter); Q:

t2.25 1,3-propanediol productivity (gram per liter per h); S13-PD: selectivity of 1,3-PD

t2.26 synthesis (percentage); dividing 1,3-PD concentration by sum of concentrations of all

t2.27 liquid metabolites, and multiplying by 100%.

resulted in autolysis of the existing cells supplied with the culture 222 medium, which was reflected by a sharp increase in the pH. 223

3.2. The adaptation ofC. butyricum DSP1 to large concentrations of crude 224 glycerol 225

In this part of the study an attempt was made to adapt C. butyricum 226

DSP1 bacteria to both osmotic (large substrate concentrations) and 227

toxic (crude glycerol) stress conditions. Five batches of metabolically 228

active biomass were passaged from the first fermentation medium 229

(70 g/L crude glycerol) to the next ones containing successively 230

greater glycerol concentrations (90,110,130,150,170 g/L) (Table 3). 231

It was observed that the kinetic parameters of 1,3-PD fermentation 232

improved significantly as compared to the experiment results shown 233

in Table 2. Complete substrate utilization occurred when the 234

concentrations of glycerol in the media were 90,110,130, and 150 g/L, 235

corresponding to noteworthy productivity values of 1.01, 1.03, 1.09, 236

and 0.74 g/L/h, respectively. The metabolite profile typified a proper 237

process of microbiological glycerol conversion by C. butyricum 238

DSP1 and the 1,3-PD synthesis selectivity ranged from 64 to 75% 239

(Table 3). Microscopic imaging did not indicate any concern-raising 240

morphological cell alterations (Fig. 1a). The crude glycerol 241

concentration at which the 1,3-PD concentration reached a maximum 242

of 74.2 g/L was 150 g/L. The process was inhibited following the 243

passage of the biomass to the medium containing 170 g/L glycerol. 244

Despite the availability of nutrients the productivity fell substantially 245

to 0.31 g/L/h. Substrate utilization did not exceed 60%. Biomass 246

accumulation reached 50% of the highest level obtained in the stage 247

with 150 g/L crude glycerol. A marked increase in the concentration 248

of lactic acid caused a drop in the yield of the main metabolite to 249

0.44 g/g. In addition, elongated forms were noticed under a microscope 250

that appeared to be conglomerates of bacterial cells (Fig. 1b). 251

In order to investigate changes in the metabolism of cells exposed to 252

various environmental stressors (high osmotic pressure, increasing 253

concentration of toxic agents, stirring), the level of intracellular 254

proteins in C. butyricum DSP1 bacteria was analyzed. The results of 255

proteomic analysis collected in Fig. 2 represent the physiologic status 256

of the cells during 1,3-PD synthesis at two stages using diametrically 257

different substrate concentrations. Fig. 2a shows the analytical results 258

for the stage with the first passage of the biomass to the fermentation 259

medium containing 90 g/L glycerol. Changes in the levels of cell stress 260

response markers selected for analysis observed in the stage where 261

the fermentation was inhibited and the substrate concentration was 262

170 g/L are presented in Fig. 2b. The markers of interest were the 263

proteins HSP20, HSP60, HSP70, the sporulation-related transcription 264

factor Spo0A as well as two enzymatic proteins connected with the 265

pathway of 1,3-PD synthesis followed by C. butyricum: glycerol 266

dehydratase and 1,3-PD dehydrogenase. The analysis demonstrated 267

considerable alterations to the proteomic profile of C. butyricum DSP1. 268

High levels of the HSP proteins (particularly the HSP60) and the 269

Table 3 t3.1

Kinetic parameters of fermentation during adaptation to large concentrations of crude t3.2 glycerol of C. butyricum DSP1. t3.3

Number of CQy C13-PD Y13-PD X Q S13-PD t3.4

passage (g/L) (g/L) (g/g) (g/L) (g/L/h) (%) t3.5

0 70 34.9 ± 1.16 0.50 1.2 ± 0.1 0.86 73 t3.6

1 90 45.6 ± 1.53 0.51 1.1 ±0.1 1.01 75 t3.7

2 110 55.6 ± 1.68 0.50 1.1 ± 0.1 1.03 73 t3.8

3 130 64.8 ± 1.58 0.49 0.9 ± 0.1 1.09 72 t3.9

4 150 74.2 ± 1.32 0.49 0.8 ± 0.1 0.74 72 t3.10

5 170 43.1 ± 1.12 0.44 0.4 ± 0.1 0.31 64 t3.11

C1,3-PD: produced 1,3-propanediol (gram per liter); Y1,3-PD: yield of 1,3-propanediol t3.12

produced per glycerol consumed (gram per gram); X: biomass (gram per liter); t3.13

Q: 1,3-propanediol productivity (gram per liter per hour) S13-PD: selectivity of 1,3-PD t3.14

synthesis (percentage); dividing 1,3-PD concentration by sum of concentrations of all t3.15

liquid metabolites, and multiplying by 100%. t3.16

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Fig. 1. Environmental impact on morphology of C. butyricum DSP1 (a) neutral conditions and (b) stress conditions. Neutral conditions: initial glycerol concentration 70 ± 1.0 g/L, T = 37°C, pH 7.0, growth in 2 L bioreactor. Stress conditions: initial glycerol concentration 170 ± 1.0 g/L, T = 37°C, pH 7.0, growth in 2 L bioreactor.

Spo0A factor registered in the stage with 170 g/L glycerol (Fig. 2b) indicated activation of cell defense mechanisms, which was not observed when bioconversion was not disturbed (Fig. 2a). Moreover, the process of autolysis (typically marked by a surge in the pH of the fermentation medium) was not triggered in either of the stages. The level of enzymatic proteins strictly associated with the pathway of metabolic conversion of glycerol was greater in the first stage (Fig. 2a) than in the second stage (Fig. 2b) suggesting an inhibiting impact of stress conditions on their synthesis.

4. Discussion

The majority of technological processes using microorganisms proceed in conditions generating various environmental stressors that negatively affect their growth and metabolism [2,5]. One of them is a high substrate concentration, which is beneficial only in economic terms as it affords high product concentrations, thus reducing the cost of product separation and the number of fermentation cycles required. Therefore, stress factors are often applied in selection tests used in the search for new microorganisms with biotechnological potential allowing elimination of stress-prone strains at the isolation stage [18,19]. In this study an attempt was made to define the maximum concentrations of a pure and a crude glycerol allowing the growth and

effective metabolism of C. butyricum DSP1. Since glycerol is an 291 osmotically active substance with a significant influence on 292 the osmotic potential of a fermentation medium, it may limit the 293 production capacity of microorganisms [2]. It was observed that the 294 growth and metabolism of C. butyricum DSP1 were not inhibited 295 until the concentration of pure glycerol in the fermentation 296 medium exceeded 90 g/L. However, industrial biotechnology has a 297 particularly important task in selecting microbial strains able to 298 metabolize waste materials, including crude glycerol [20,21,22]. 299 The use of substrates originating as waste generates stress factors 300 resulting from the accumulation of toxic agents in the fermentation 301 medium, not to mention the problem of varying substrate quality 302 even within the same operation. Many authors studying the 303 conversion of crude glycerol to 1,3-PD have reported deterioration 304 of kinetic parameters, especially final product concentration, as 305 compared to pure glycerol [14,23,24]. Similar findings were made in 306 this work (Table 2). Interestingly, a decrease was observed in the 307 productivity and selectivity of 1,3-PD synthesis for high concentrations 308 of both pure and crude glycerol, probably due to an increased synthesis 309 of lactic acid by the bacteria under stress conditions. An increased 310 content of lactic acid indicates that the process is blocked probably due 311 to substrate excess, a high concentration of toxic carbon monoxide or 312 stoppage at the stage of pyruvate generation. However, it should not be 313 assumed that this tendency is always observed for the synthesis of 314 1,3-PD and other metabolites [7,25,26]. The maximum concentrations 315 of a pure and a crude glycerol determined in the present study that did 316 not inhibit C. butyricum DSP1 were 90 and 70 g/L, respectively, which is 317 below the levels reported by Ringel et al. [19]. The differences between 318 results of experiments with C. butyricum AKR102a and C. butyricum 319 DSP1 were probably mainly due to their varied origin, including 320 different selection factors used in the screening, and differences in the 321 composition of the carbon-providing substrate [16,19]. Considering the 322 unsatisfying parameters received with C. butyricum DSP1, in order to 323 adapt the bacteria to conditions caused by using crude glycerol at a 324 high concentration, batches of metabolically active biomass were 325 passaged to the fermentation media containing successively greater 326 substrate concentrations. This enabled a complete utilization of 150 g/L 327 glycerol and provided a 1,3-PD concentration of 74.2 g/L, which was 328 only 5.9 g/L less than the highest value of 80.1 g/L reported previously 329 by Hirschmann et al. [27]. In addition, proteomic analysis conducted in 330 this study gave an insight into modifications within C. butyricum DSP1 331 bacteria cells at the molecular level. Since changes in the levels of some 332 intracellular proteins reflect the physiologic status of cells, it may be 333 possible to obtain a detailed profile of a strain with potential for 334 industrial application with emphasis on its metabolic capacity [28]. 335 Heat shock proteins, similar to the transcription factor Spo0A, are 336 markers for the response of C. butyricum to adverse environmental 337 conditions [28,29]. An increase in their levels is a signal that the cell 338 defense mechanism is active and that the metabolic processes are 339 redirected toward protecting the cells from the harmful action of the 340 environment. Consequently, an intensified synthesis of proteins that do 341 not normally participate in the main metabolic activity slows or stops 342 the entire process. A rise in the HSP60, a protein also known as GroEL, 343 at the stage when the crude glycerol concentration was 170 g/L 344 indicated the efforts of the cells to defend against the high osmotic 345 potential in the early hours of the process. The literature points to 346 HSP60 as a protein associated with the response of the genus 347 Clostridium to osmotic, toxic and temperature stresses [29,30]. During 348 that period biomass accumulation was not observed, which pointed to 349 the interruption of some life-sustaining biochemical reactions, probably 350 to save metabolic energy. The level of enzymatic proteins associated 351 with the metabolism of glycerol continued to grow, though not as 352 markedly as when the crude glycerol concentration was 90 g/L. The 353 fact that it still exceeded the initial value proved the ability of the 354 microorganisms to metabolize the substrate at such an elevated 355 concentration despite their defensive activity. That progressed until 356

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Time of fermentation (h)

HSP20 HSP60 HSP70 SpoOA Glycerol dehydratase 1,3-PD dehydrogenase

10 20 30

Time of fermentation (h)

HSP20 ■ HSP60 ■ HSP70 ■ Spo0A Glycerol dehydratase 1,3-PD dehydrogenase

Fig. 2. Proteomic analysis in C. butyricum DSP1 (a) neutral conditions and (b) stress conditions. **Fold change in selected proteins in samples from batch fermentation with 90 g/L crude glycerol; *fold change in selected proteins in samples from batch fermentation with 170 g/L crude glycerol.

357 an increase in the SpoOA was observed, as the synthesis of 1,3-PD

358 stopped, leading to sporulation and the arrest of the cells'

359 life-sustaining functions [31]. It should be emphasized that by the time

360 1,3-PD synthesis came to a halt there was a high concentration

361 of the diol as well as organic acids inside and outside the cells. A

362 simultaneous presence of various stressors, which in this context may

363 be referred to as multifunctional environmental stress, very likely

364 added to the impact of unfavorable factors on the bacterial cells as was

365 further confirmed by the observation of cell conglomerates (Fig. 1b),

366 whose formation is believed to be a protective measure [32].

367 5. Concluding remarks

368 This study of the effect of glycerol on the growth and metabolism

369 of C. butyricum DSP1 demonstrated that the maximum substrate

370 concentrations that do not inhibit the metabolic activity of bacterial

371 cells are 90 g/L and 70 g/L for a pure and a crude glycerol, respectively.

372 It was also found that an adaptation procedure involving the passage of

metabolically active biomass from a fermentation medium with a 373 lower concentration of crude glycerol to media with successively 374 greater concentrations allows breaking the barrier of high osmotic 375 pressure and affords a 1,3-PD concentration of 74 g/L in a batch culture 376 operation. Due to multifunctional environmental stress affecting the 377 fermentation medium there is a need to determine maximum 378 non-inhibiting concentrations for 1,3-PD and other metabolites in the 379 next stage of research in order to define the production capacity of 380 C. butyricum DSP1 and to advance an understanding of its resistance to 381 stress factors. 382

Financial support 383

The work was prepared within the framework of the project PO IG 384 01.01.02-00-074/09, co-funded by the European Union from The 385 European Regional Development fund within the framework of the Q5 Innovative Economy Operational Program 2007-2013. Q6

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