A Novel Approach To Improve the Efficiency of Block Freeze

Feb 27, 2017 - methods still face the challenge of achieving high concentration .... cycles ended when the salt concentration in the melted ice fracti...
0 downloads 0 Views 2MB Size
Subscriber access provided by The University of Liverpool

Article

A Novel Approach to Improve Efficiency of Block Freeze Concentration Using Ice Nucleation Proteins with Altered Ice Morphology Jue Jin, Edward J. Yurkow, Derek Adler, and Tung-Ching Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03710 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

Journal of Agricultural and Food Chemistry

1

A Novel Approach to Improve Efficiency of Block Freeze Concentration Using

2

Ice Nucleation Proteins with Altered Ice Morphology

3

Jue Jin a, Edward J. Yurkow b, Derek Adler b and Tung-Ching Lee a,*

4 5

a

6

Road, New Brunswick, New Jersey 08901, USA

7

b

8

University of New Jersey, 41 Gordon Road, Suite D, Piscataway, New Jersey 08854,

9

USA

Department of Food Science, Rutgers, the State University of New Jersey, 65 Dudley

Molecular Imaging Center, Rutgers Translational Sciences, Rutgers, the State

10 11 12

*

13

[email protected] (Tung-Ching Lee).

Correspondent author. Tel: +1 848 9325536, Fax: +1 732 9326776, E–mail:

14 15 16 17 18 19 20 21 22 1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

23

ABSTRACT

24

Freeze concentration is a separation process with high success in product quality. The

25

remaining challenge is to achieve high efficiency with low cost. This study aims to

26

evaluate the potential of using ice nucleation proteins (INPs) as an effective method to

27

improve the efficiency of block freeze concentration while also exploring the related

28

mechanism of ice morphology. Our results show that INPs are able to significantly

29

improve the efficiency of block freeze concentration in a desalination model. Using

30

this experimental system, we estimate that approximately 50% of the energy cost can

31

be saved by the inclusion of INPs in desalination cycles while still meeting the EPA

32

standard of drinking water (99.9%), sodium

125

chloride,

126

magnesium sulfate (MgSO4), and calcium chloride (CaCl2) were obtained from Fisher

127

Scientific (Fair Lawn, NJ, USA). L-serine, L-alanine, potassium iodide (KI) and

128

magnesium chloride (MgCl2) were purchased from Sigma-Aldrich (St. Louis, MO,

129

USA). All reagents were of analytical grade, and deionized water from Milli-Q was

130

used throughout the experiments. Seawater was prepared artificially in the lab by

131

dissolving 26.73g NaCl, 2.26g MgCl2, 3.25g MgSO4 and 1.15g CaCl2 in 1L of

132

deionized water.26

Tris

(Hydroxymethyl)

aminomethane,

potassium

6

ACS Paragon Plus Environment

sulfate

(K2SO4),

Page 7 of 42

Journal of Agricultural and Food Chemistry

133

Preparation and purification of ice nucleation proteins. Erwinia herbicola was

134

stored frozen at -60 °C and grown in yeast extract (YE) media (20g/L) containing

135

sucrose (10g/L), L-serine (2g/L), L-alanine (2g/L), K2SO4

136

(4g/L). Following culture expansion to a density of 108/L, the cells were collected by

137

high-speed centrifugation (10,000×g, 20 mins @ 4 oC), and the resulting pellet was

138

re-suspended in 20mM Tris buffer containing 20mM MgCl2. The suspension was then

139

sonicated on ice, using three brief (10 sec) sonication bursts generated by a Brandson

140

sonicator (Danbury, CT) set at 4.5 power output. Following sonication, the suspension

141

was centrifuged again as described above and the supernatant was isolated and

142

ultra-centrifuged at 4 oC and 160,000×g for 2h. Finally, the resultant pellet was

143

re-suspended in 20mM Tris buffer with 20mM MgCl2 and freeze-dried to obtain the

144

INP powder. Lyophilized INPs isolated in this manner were stored at -18 oC prior to

145

use.27

146

Determination of the supercooling point using differential scanning calorimetry.

147

The supercooling point of seawater containing different concentrations of INPs was

148

measured by differential scanning calorimetry using a DSC 823E thermal analyzer

149

(Mettler-Toledo Inc., Columbus, OH) charged with liquid nitrogen and compressed

150

nitrogen gas as described in the manufacturer’s instructions. Briefly, 30µL of the

151

seawater samples were transferred into 40-µL aluminum crucibles with lids and

152

positioned in the unit. The temperature ramp of the DSC unit was set at a freezing rate

153

of 1 oC/min, from 4 oC to -25 oC. An empty crucible was used as the reference. The

154

temperature point exhibiting the maximum observed heat flow was recorded as the 7

ACS Paragon Plus Environment

(8.6g/L) and MgSO4

Journal of Agricultural and Food Chemistry

155

supercooling point.

156

Centrifugal freeze concentration procedures. The artificial seawater (40mL) used

157

in this study was frozen in plastic centrifuge tubes (internal diameter of 29mm) by

158

radial freezing using a static cooling bath containing a mixture of water and ethylene

159

glycol.28 The samples were then removed from the cooling bath and rapidly subjected

160

to refrigerated centrifugation to separate the brine from the ice fractions. After

161

centrifugation, the frozen ice fractions were thawed and the total dissolved salt (TDS)

162

was measured at ambient temperature using a conductivity meter (model 09-326-2,

163

Fisher Scientific). The volume of the solutions was also determined. The desalination

164

rate was calculated using the following equation:  =

 −  × 100% 

165

Where Rd is the desalination rate (%); C0 is the TDS of the original seawater; Cice is

166

the TDS in the melt ice fraction.

167

To evaluate the effect of INPs on the efficiency of freeze concentration, the freeze

168

concentration procedures above were performed using different variables to determine

169

the desalination rates. The experimental variables tested in our study were INP

170

concentration, freezing temperature, and centrifugal time and speed, all of which were

171

identified, by other studies, as the main factors that could affect concentration

172

efficiency.4,

173

freeze concentration experiment are listed in Table 1. For example, the tested variable

174

of the experiment group 1 was INP concentration. The controlled variables of group 1

175

were freezing temperature at -18 oC and centrifugation condition at 500 rpm for 5

28

The levels of tested variables and controlled variables during each

8

ACS Paragon Plus Environment

Page 8 of 42

Page 9 of 42

Journal of Agricultural and Food Chemistry

176

mins. The salt concentration of seawater in nature (i.e. 33.3 g/L) was used as the

177

initial salt concentration during the experiments of these four variables (i.e.

178

experiment groups 1 to 4). For desalination cycles (i.e. experiment groups 5&6), both

179

control and INP samples started at the concentration of seawater in nature (i.e. 33.3

180

g/L). The initial concentration of each subsequent cycle was based on the salt

181

concentration in melted ice of its previous cycle. The desalination cycles ended, when

182

the salt concentration in the melted ice fraction was less than the Environmental

183

Protection Agency (EPA) standard of drinking water (i.e. 0.5 g/L). All experiments

184

were performed in triplicates.

185

Ice crystal size determination. The evaluation of ice crystal structure was conducted

186

using a 10× Olympus lens (0.25 N.A.) (Olympus, Tokyo, Japan) and a Q imaging

187

2560 × 1920 pixel CCD camera (Micropublisher, Surrey, Canada) equipped with a

188

Linkham temperature-controlled imaging stage (LTS120, Linkham, Surrey, England).

189

In a typical experiment, samples of seawater containing INP concentrations ranging

190

from 10-7 mg/mL to 10-2 mg/mL were frozen in petri dishes. The frozen preparations

191

were placed on the microscope stage setting at -18 oC, and digital images of the ice

192

crystal structure were collected by focusing on the surface ice layer. The average size

193

of the ice crystals was determined with ImageJ software (version 1.46r, NIH,

194

Bethesda, MD), using Feret’s diameter calculation.29-30

195

X-ray computed tomography image analysis. Three dimensional imaging analysis

196

of frozen and centrifuged seawater samples was obtained using the Albira PET/CT

197

Imaging System (Bruker, Billerica, MA) at standard voltage and current settings (i.e., 9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

198

45kV and 400µA) at the Molecular Imaging Center at Rutgers University. A set of

199

400 image projections was then captured throughout a 360o rotation of the sample.

200

Reconstruction of X-ray data produced 3D images in which the air, ice and brine

201

pockets could be differentiated based on differences in X-ray attenuation properties.

202

The morphology of ice structures within different growth heights was studied to

203

determine interface evolution in both control and INP samples. The samples were

204

frozen on a cold surface at -18 oC until the vertical length of frozen matrix reached

205

our targeted growth height. The frozen samples were then removed from the cold

206

surface to pour out the remaining liquid and kept in the freezer at -18 oC before

207

imaging analysis. A KI contrast agent was included in the solutions prior to freezing

208

the samples for better differentiation between ice and brine phases. The morphology

209

of ice crystals in both horizontal and vertical directions was characterized by the

210

measurement of crystal dimension and brine inclusion width. The hydraulic diameter

211

in the cross sections at different growth heights of the entire frozen sample was

212

calculated based on flow mechanic theory, in order to compare the brine flow rate in

213

control and INP samples.31-32 The hydraulic diameter with different INP

214

concentrations was determined at the same growth height. The hydraulic diameter of

215

different channel shapes is given by: D =

216 217 218

4 × Cross Section Area 4A = Wetted Perimeter P

For rectangular cross section: "#$

%#$

D = %(#'$) = #'$

(1)

For triangular cross section: 10

ACS Paragon Plus Environment

Page 10 of 42

Page 11 of 42

Journal of Agricultural and Food Chemistry

*

D =

219

) ) "× * ×(#* + , ) .*

%#'$

(2)

220

Where Dh = hydraulic diameter; a = ice crystal dimension in the cross section; b =

221

maximum width of brine inclusion between two ice crystals.

222

Frozen samples for the brine distribution study were prepared in centrifugal tubes

223

as previously described in the centrifugal freeze concentration procedures and placed

224

in a cooling bath at the subzero temperature of -18 oC until completely frozen.

225

Segmentation for imaging processing is done using thresholding techniques where the

226

volume is partitioned into voxel groups of each region of interest (ROI) inside the

227

sample. Volumes of the brine inside frozen samples were determined using VivoQuant

228

image analysis software (version 1.23, inviCRO LLC, Boston MA). In a typical study

229

volumes of the entrapped brine liquid were resolved and determined using a threshold

230

range of 80 to 150 Hounsfield Units.21, 23

231

Statistical analysis. One-way ANOVA was performed using Data Processing System

232

software v.9.50.

233

significant difference (LSD) at 5%.

234

Results & Discussions

235

Effect of INPs on the supercooling point of seawater. The effect of INPs isolated

236

from E. herbicola on the supercooling point of seawater was investigated using

237

differential scanning calorimetry (Fig. 2A). The supercooling point was determined as

238

the lowest temperature that the supercooled solution could reach before the phase

239

transition from water to ice took place, with the release of latent heat (as indicated in

240

Figure 2B). With the addition of INPs at final concentrations of 1mg/mL, the

Differences among mean values were established by the least

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

241

supercooling point of the seawater samples was elevated to -6.24 oC, as compared to

242

the supercooling temperature of -21.38 oC for the control samples. Even at the lowest

243

INP concentration of 10-6 mg/mL, the supercooling point increased to -11.36 oC. As

244

suggested by Jung et al., the nonlinear relationship between INP concentrations and

245

supercooling point might mainly be due to the dependence of ice nucleation activity

246

on the degree of protein aggregation.33 In the theory of heterogeneous ice nucleation,

247

a larger nucleating site leads to a higher threshold temperature of ice nucleation

248

activity.34 Therefore, the variation of ice nucleation activity at the supercooling point

249

(threshold temperature), shown by the DSC measurement, is likely to be the result of

250

protein aggregation into different sizes of ice nuclei. Such aggregation process was

251

suggested to be limited by stochastic chain-terminating events in the growing ice

252

nucleus rather than the availability of INP concentration range.35 The results indicate

253

that INPs can function as effective ice nucleators for controlling the supercooling

254

level of seawater even at low concentrations. The elevated nucleation temperature

255

also suggests significant energy savings for the freezing step, which will be further

256

discussed in the energy saving section.

257

Effect of INP concentration on desalination rate. The effect of INPs on freeze

258

concentration efficiency as a function of desalination rate was investigated at different

259

INP concentrations using block freeze concentration assisted by centrifugation (Fig.

260

3). In this technique, concentrated solute drains through the porous ice block and is

261

separated from the ice under centrifugal force. The addition of INPs at a concentration

262

of 10-6 mg/mL increased the desalination rate by 14% as compared to the control 12

ACS Paragon Plus Environment

Page 12 of 42

Page 13 of 42

Journal of Agricultural and Food Chemistry

263

sample. This rate continued to increase from 48% to 53% with increases in INP

264

concentrations from 10-6 mg/mL to 10-2 mg/mL. The results demonstrate that INPs

265

can increase desalination rates, indicating that INPs can improve concentration

266

efficiency and thus reduce related energy cost.

267

Desalination rate by INPs at different freezing temperatures and centrifugation

268

conditions. To examine the practical application of INPs to the production of drinking

269

water from seawater, the effect of these agents on desalination rates at different

270

conditions, including freezing temperature and centrifugation speed or time, were

271

investigated. INP concentration of 10-2 mg/mL was utilized in the subsequent

272

experiments.

273

The effect of INPs on desalination rate at different freezing temperatures was

274

determined (Fig. 4). The results show that INPs can improve concentration efficiency

275

at freezing temperatures of -13 oC and -18 oC compared to the control samples. At the

276

temperature of -8 oC, INPs were able to freeze samples while seawater controls

277

remained liquid. This elevation in the freezing temperature induced by INPs is

278

associated with a 36% increase in the desalination rate, which was two-fold higher

279

than the effect of INPs on the desalination rate of samples frozen at -18 oC. This

280

increased desalination rate observed at the higher subzero temperature (-8 oC) is

281

thought to be due to the higher diffusion rate of solute under warmer temperature that

282

then leads to less solute entrapment as compared to samples with INPs at lower

283

freezing temperatures.

284

The effect of INPs on desalination rate at different centrifugation conditions was 13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

285

also characterized. Here, desalination rates were determined while varying

286

centrifugation times (Fig. 5A). For these studies, frozen samples of the control

287

seawater and seawater containing INPs were centrifuged at 500rpm for increasing

288

periods of time (i.e., 5, 10, 15, 20 and 30 mins). The results show that INPs exhibit

289

marked effect on desalination rates at the lower centrifugation times of 5, 10 and 15

290

minutes, with increases ranging from 16% to 20% as compared to control samples

291

under the same conditions. Since there was no significant difference (P