f6fb18a91f3757ad3f7bc4446678209a pmc_9573287.pdf 65ab56973c8b89b09ad97f3c242d9348a04fc84c pmc_9573287.pdf e6763dc7d3a8007e12a6d58f6d9d75be94309b35582ec068ae599375a1008ada pmc_9573287.pdf Title: Improvement of the Polyhydroxyalkanoates Recovery from Mixed Microbial Cultures Using Sodium Hypochlorite Pre-Treatment Coupled with Solvent Extraction Subject: The use of mixed microbial cultures (MMC) and organic wastes and wastewaters as feed sources is considered an appealing approach to reduce the current polyhydroxyalkanoates (PHAs) production costs. However, this method entails an additional hurdle to the PHAs downstream processing (recovery and purification). In the current work, the effect of a sodium hypochlorite (NaClO) pre-treatment coupled with dimethyl carbonate (DMC) or chloroform (CF) as extraction solvents on the PHAs recovery efficiency (RE) from MMC was evaluated. MMC were harvested from a sequencing batch reactor (SBR) fed with a synthetic prefermented olive mill wastewaster. Two different carbon-sources (acetic acid and acetic/propionic acids) were employed during the batch accumulation of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from MMC. Obtained PHAs were characterized by 1H and 13C nuclear magnetic resonance, gel-permeation chromatography, differential scanning calorimetry, and thermal gravimetric analysis. The results showed that when a NaClO pre-treatment is not added, the use of DMC allows to obtain higher RE of both biopolymers (PHB and PHBV), in comparison with CF. In contrast, the use of CF as extraction solvent required a pre-treatment step to improve the PHB and PHBV recovery. In all cases, RE values were higher for PHBV than for PHB. Keywords: polyhydroxyalkanoates; mixed microbial cultures; extraction; sodium hypochlorite; dimethyl carbonate; chloroform Author: Gabriela Montiel-Jarillo, Diego A. Morales-Urrea, Edgardo M. Contreras, Alex López-Córdoba, Edwin Yesid Gómez-Pachón, Julián Carrera and María Eugenia Suárez-Ojeda Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Sep 21 07:30:30 2022 CEST ModDate: Wed Sep 21 07:38:19 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4086240 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PRJKGQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 TSRBXL+VnURWPalladioL Type 1 Custom yes yes no 15 0 GQEHIC+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 RECMNB+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 XFEPWD+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 TLFMRE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 46 0 JBCRLF+PazoMath Type 1 Builtin yes yes no 68 0 QMDDXF+CMSY10 Type 1 Builtin yes yes no 75 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 79 0 CZKGAS+CMR10 Type 1 Builtin yes yes no 86 0 PDXFNG+PazoMath-Bold Type 1 Builtin yes yes no 145 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-07-11 02:35:19 CEST RepresentationInformation: pmc_9573287.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-10-27 02:16:36 CEST Size: 4086240 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 387 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Production of PHAs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Evaluation PHAs Extraction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Characterization of the Obtained Biopolymers Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Production of PHA Using Mixed Microbial Cultures (MMC) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Evaluation of PHAs Extraction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Characterization of the Obtained Biopolymers Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Quantitative 1H/13C Nuclear Magnetic Resonance (NMR) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Gel-Permeation Chromatography (GPC) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Thermogravimetry (TGA) and Differential Scanning Calorimetry (DSC) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Info: Title: Improvement of the Polyhydroxyalkanoates Recovery from Mixed Microbial Cultures Using Sodium Hypochlorite Pre-Treatment Coupled with Solvent Extraction Author: Gabriela Montiel-Jarillo, Diego A. Morales-Urrea, Edgardo M. Contreras, Alex López-Córdoba, Edwin Yesid Gómez-Pachón, Julián Carrera and María Eugenia Suárez-Ojeda Subject: The use of mixed microbial cultures (MMC) and organic wastes and wastewaters as feed sources is considered an appealing approach to reduce the current polyhydroxyalkanoates (PHAs) production costs. However, this method entails an additional hurdle to the PHAs downstream processing (recovery and purification). In the current work, the effect of a sodium hypochlorite (NaClO) pre-treatment coupled with dimethyl carbonate (DMC) or chloroform (CF) as extraction solvents on the PHAs recovery efficiency (RE) from MMC was evaluated. MMC were harvested from a sequencing batch reactor (SBR) fed with a synthetic prefermented olive mill wastewaster. Two different carbon-sources (acetic acid and acetic/propionic acids) were employed during the batch accumulation of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from MMC. Obtained PHAs were characterized by 1H and 13C nuclear magnetic resonance, gel-permeation chromatography, differential scanning calorimetry, and thermal gravimetric analysis. The results showed that when a NaClO pre-treatment is not added, the use of DMC allows to obtain higher RE of both biopolymers (PHB and PHBV), in comparison with CF. In contrast, the use of CF as extraction solvent required a pre-treatment step to improve the PHB and PHBV recovery. In all cases, RE values were higher for PHBV than for PHB. Keywords: polyhydroxyalkanoates; mixed microbial cultures; extraction; sodium hypochlorite; dimethyl carbonate; chloroform Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Sep 21 07:30:30 CEST 2022 ModDate: Wed Sep 21 07:38:19 CEST 2022 ID: 0x149b520ced363edbbe29fa3e3ca46273, 0x149b520ced363edbbe29fa3e3ca46273 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: PDXFNG+PazoMath-Bold FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: PDXFNG+PazoMath-Bold Flags: Symbolic FontBBox: -10, -266, 829, 695 FontFile: true Font: BaseFont: GQEHIC+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: GQEHIC+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JBCRLF+PazoMath FontSubset: true FirstChar: 68 LastChar: 160 FontDescriptor: FontName: JBCRLF+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: CZKGAS+CMR10 FontSubset: true FirstChar: 40 LastChar: 61 FontDescriptor: FontName: CZKGAS+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true Font: BaseFont: RECMNB+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: RECMNB+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PRJKGQ+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: PRJKGQ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QMDDXF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 14 FontDescriptor: FontName: QMDDXF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: XFEPWD+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: XFEPWD+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: TSRBXL+VnURWPalladioL FontSubset: true FirstChar: 146 LastChar: 243 FontDescriptor: FontName: TSRBXL+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CAMXTK+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: CAMXTK+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 110, 323, 152, 332 Annotation: Subtype: Link Rect: 34, 311, 137, 320 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 165, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 292, 642, 304, 655 Annotation: Subtype: Link Rect: 413, 642, 424, 655 Annotation: Subtype: Link Rect: 528, 642, 539, 655 Annotation: Subtype: Link Rect: 170, 629, 181, 643 Annotation: Subtype: Link Rect: 421, 629, 432, 643 Annotation: Subtype: Link Rect: 237, 121, 244, 130 ActionDest: 152 Annotation: Subtype: Link Rect: 118, 17, 266, 28 Annotation: Subtype: Link Rect: 403, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 444, 87, 463 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 357, 723, 364, 732 ActionDest: 152 Annotation: Subtype: Link Rect: 365, 723, 372, 732 ActionDest: 152 Annotation: Subtype: Link Rect: 429, 710, 436, 719 ActionDest: 152 Annotation: Subtype: Link Rect: 436, 710, 443, 719 ActionDest: 152 Annotation: Subtype: Link Rect: 444, 710, 451, 719 ActionDest: 152 Annotation: Subtype: Link Rect: 469, 685, 476, 694 ActionDest: 152 Annotation: Subtype: Link Rect: 286, 647, 293, 657 ActionDest: 152 Annotation: Subtype: Link Rect: 391, 623, 398, 631 ActionDest: 152 Annotation: Subtype: Link Rect: 326, 585, 333, 594 ActionDest: 152 Annotation: Subtype: Link Rect: 333, 585, 340, 594 ActionDest: 152 Annotation: Subtype: Link Rect: 472, 547, 484, 556 ActionDest: 152 Annotation: Subtype: Link Rect: 484, 547, 496, 556 ActionDest: 155 Annotation: Subtype: Link Rect: 455, 522, 467, 531 ActionDest: 152 Annotation: Subtype: Link Rect: 468, 522, 480, 531 ActionDest: 155 Annotation: Subtype: Link Rect: 331, 484, 343, 493 ActionDest: 155 Annotation: Subtype: Link Rect: 349, 447, 361, 456 ActionDest: 155 Annotation: Subtype: Link Rect: 361, 447, 373, 456 ActionDest: 155 Annotation: Subtype: Link Rect: 498, 434, 510, 443 ActionDest: 152 Annotation: Subtype: Link Rect: 510, 434, 522, 443 ActionDest: 155 Annotation: Subtype: Link Rect: 462, 409, 469, 418 ActionDest: 152 Annotation: Subtype: Link Rect: 321, 384, 328, 393 ActionDest: 152 Annotation: Subtype: Link Rect: 266, 359, 273, 368 ActionDest: 152 Annotation: Subtype: Link Rect: 376, 359, 388, 368 ActionDest: 155 Annotation: Subtype: Link Rect: 420, 334, 432, 343 ActionDest: 155 Annotation: Subtype: Link Rect: 227, 169, 239, 178 ActionDest: 155 Annotation: Subtype: Link Rect: 508, 132, 520, 141 ActionDest: 155 Annotation: Subtype: Link Rect: 461, 94, 472, 103 ActionDest: 155 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 462, 660, 474, 669 ActionDest: 155 Annotation: Subtype: Link Rect: 253, 597, 265, 606 ActionDest: 155 Annotation: Subtype: Link Rect: 488, 572, 500, 581 ActionDest: 155 Annotation: Subtype: Link Rect: 544, 484, 556, 493 ActionDest: 155 Annotation: Subtype: Link Rect: 228, 296, 240, 305 ActionDest: 155 Annotation: Subtype: Link Rect: 218, 230, 225, 242 ActionDest: 83 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 210, 465, 222, 474 ActionDest: 155 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 495, 597, 507, 606 ActionDest: 155 Annotation: Subtype: Link Rect: 503, 585, 515, 594 ActionDest: 155 Annotation: Subtype: Link Rect: 359, 521, 371, 530 ActionDest: 155 Annotation: Subtype: Link Rect: 218, 467, 225, 479 ActionDest: 93 Annotation: Subtype: Link Rect: 196, 392, 203, 403 ActionDest: 93 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 217, 645, 224, 657 ActionDest: 99 Annotation: Subtype: Link Rect: 273, 620, 280, 632 ActionDest: 93 Annotation: Subtype: Link Rect: 528, 189, 540, 198 ActionDest: 155 Annotation: Subtype: Link Rect: 543, 189, 555, 198 ActionDest: 155 Annotation: Subtype: Link Rect: 494, 177, 506, 186 ActionDest: 155 Annotation: Subtype: Link Rect: 506, 177, 518, 186 ActionDest: 155 Annotation: Subtype: Link Rect: 212, 136, 219, 148 ActionDest: 105 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 517, 710, 524, 719 ActionDest: 152 Annotation: Subtype: Link Rect: 525, 710, 531, 719 ActionDest: 152 Annotation: Subtype: Link Rect: 226, 463, 238, 472 ActionDest: 155 Annotation: Subtype: Link Rect: 372, 436, 379, 448 ActionDest: 105 Annotation: Subtype: Link Rect: 545, 438, 557, 447 ActionDest: 155 Annotation: Subtype: Link Rect: 323, 388, 335, 397 ActionDest: 155 Annotation: Subtype: Link Rect: 362, 338, 374, 347 ActionDest: 155 Annotation: Subtype: Link Rect: 374, 338, 386, 347 ActionDest: 155 Annotation: Subtype: Link Rect: 218, 281, 225, 295 ActionDest: 108 Annotation: Subtype: Link Rect: 430, 271, 442, 280 ActionDest: 155 Annotation: Subtype: Link Rect: 332, 256, 339, 268 ActionDest: 108 Annotation: Subtype: Link Rect: 544, 256, 551, 268 ActionDest: 108 Annotation: Subtype: Link Rect: 411, 244, 419, 256 ActionDest: 108 Annotation: Subtype: Link Rect: 250, 231, 257, 243 ActionDest: 108 Annotation: Subtype: Link Rect: 479, 231, 486, 243 ActionDest: 108 Annotation: Subtype: Link Rect: 306, 218, 313, 230 ActionDest: 108 Annotation: Subtype: Link Rect: 495, 218, 502, 230 ActionDest: 108 Annotation: Subtype: Link Rect: 374, 206, 381, 220 ActionDest: 108 Annotation: Subtype: Link Rect: 525, 168, 532, 180 ActionDest: 108 Annotation: Subtype: Link Rect: 284, 156, 291, 168 ActionDest: 108 Annotation: Subtype: Link Rect: 534, 156, 541, 168 ActionDest: 108 Annotation: Subtype: Link Rect: 330, 131, 337, 143 ActionDest: 108 Annotation: Subtype: Link Rect: 545, 118, 552, 130 ActionDest: 108 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 544, 127, 556, 137 ActionDest: 155 Annotation: Subtype: Link Rect: 193, 112, 200, 124 ActionDest: 114 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 305, 733, 312, 745 ActionDest: 99 Annotation: Subtype: Link Rect: 218, 353, 225, 367 ActionDest: 117 Annotation: Subtype: Link Rect: 517, 344, 529, 353 ActionDest: 155 Annotation: Subtype: Link Rect: 529, 344, 541, 353 ActionDest: 155 Annotation: Subtype: Link Rect: 544, 344, 556, 353 ActionDest: 155 Annotation: Subtype: Link Rect: 223, 316, 230, 328 ActionDest: 117 Annotation: Subtype: Link Rect: 243, 278, 250, 290 ActionDest: 117 Annotation: Subtype: Link Rect: 447, 278, 454, 290 ActionDest: 123 Annotation: Subtype: Link Rect: 474, 266, 481, 279 ActionDest: 117 Annotation: Subtype: Link Rect: 411, 243, 423, 252 ActionDest: 155 Annotation: Subtype: Link Rect: 423, 243, 435, 252 ActionDest: 155 Annotation: Subtype: Link Rect: 436, 243, 448, 252 ActionDest: 155 Annotation: Subtype: Link Rect: 481, 205, 493, 215 ActionDest: 158 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 543, 137, 550, 149 ActionDest: 117 Annotation: Subtype: Link Rect: 357, 124, 364, 136 ActionDest: 117 Annotation: Subtype: Link Rect: 378, 127, 390, 136 ActionDest: 155 Annotation: Subtype: Link Rect: 537, 74, 545, 86 ActionDest: 123 Annotation: Subtype: Link Rect: 241, 61, 248, 73 ActionDest: 133 Page: Sequence: 11 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 486, 323, 498, 332 ActionDest: 155 Annotation: Subtype: Link Rect: 524, 308, 531, 322 ActionDest: 108 Annotation: Subtype: Link Rect: 246, 295, 253, 309 ActionDest: 117 Annotation: Subtype: Link Rect: 193, 285, 205, 294 ActionDest: 158 Annotation: Subtype: Link Rect: 298, 285, 310, 294 ActionDest: 155 Annotation: Subtype: Link Rect: 311, 285, 323, 294 ActionDest: 158 Annotation: Subtype: Link Rect: 332, 235, 344, 244 ActionDest: 155 Annotation: Subtype: Link Rect: 345, 235, 357, 244 ActionDest: 158 Annotation: Subtype: Link Rect: 342, 210, 354, 219 ActionDest: 158 Annotation: Subtype: Link Rect: 355, 210, 367, 219 ActionDest: 158 Annotation: Subtype: Link Rect: 238, 185, 250, 194 ActionDest: 155 Annotation: Subtype: Link Rect: 251, 185, 263, 194 ActionDest: 158 Annotation: Subtype: Link Rect: 432, 173, 444, 181 ActionDest: 155 Annotation: Subtype: Link Rect: 445, 172, 457, 181 ActionDest: 155 Annotation: Subtype: Link Rect: 457, 172, 469, 181 ActionDest: 158 Annotation: Subtype: Link Rect: 212, 157, 219, 171 ActionDest: 139 Annotation: Subtype: Link Rect: 277, 97, 289, 106 ActionDest: 155 Annotation: Subtype: Link Rect: 290, 97, 302, 106 ActionDest: 155 Annotation: Subtype: Link Rect: 468, 59, 480, 69 ActionDest: 158 Annotation: Subtype: Link Rect: 480, 59, 492, 69 ActionDest: 158 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 552, 504, 559, 516 ActionDest: 139 Annotation: Subtype: Link Rect: 361, 481, 373, 490 ActionDest: 158 Annotation: Subtype: Link Rect: 373, 481, 385, 490 ActionDest: 158 Annotation: Subtype: Link Rect: 387, 361, 399, 370 ActionDest: 158 Annotation: Subtype: Link Rect: 208, 298, 220, 307 ActionDest: 155 Annotation: Subtype: Link Rect: 221, 298, 233, 307 ActionDest: 158 Annotation: Subtype: Link Rect: 284, 286, 296, 294 ActionDest: 158 Annotation: Subtype: Link Rect: 492, 260, 504, 269 ActionDest: 158 Annotation: Subtype: Link Rect: 280, 223, 292, 232 ActionDest: 158 Annotation: Subtype: Link Rect: 267, 185, 279, 194 ActionDest: 155 Annotation: Subtype: Link Rect: 280, 185, 292, 194 ActionDest: 158 Annotation: Subtype: Link Rect: 292, 185, 304, 194 ActionDest: 158 Annotation: Subtype: Link Rect: 300, 160, 312, 169 ActionDest: 158 Annotation: Subtype: Link Rect: 544, 97, 556, 106 ActionDest: 158 Annotation: Subtype: Link Rect: 366, 69, 373, 81 ActionDest: 139 Annotation: Subtype: Link Rect: 438, 59, 450, 69 ActionDest: 152 Annotation: Subtype: Link Rect: 451, 60, 462, 69 ActionDest: 155 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 219, 717, 226, 729 ActionDest: 142 Annotation: Subtype: Link Rect: 500, 670, 512, 679 ActionDest: 155 Annotation: Subtype: Link Rect: 512, 670, 524, 679 ActionDest: 155 Annotation: Subtype: Link Rect: 525, 670, 536, 679 ActionDest: 158 Annotation: Subtype: Link Rect: 214, 416, 221, 428 ActionDest: 142 Annotation: Subtype: Link Rect: 335, 393, 347, 402 ActionDest: 158 Annotation: Subtype: Link Rect: 350, 393, 362, 402 ActionDest: 158 Annotation: Subtype: Link Rect: 291, 368, 303, 377 ActionDest: 155 Annotation: Subtype: Link Rect: 300, 343, 312, 352 ActionDest: 155 Annotation: Subtype: Link Rect: 312, 343, 324, 352 ActionDest: 158 Annotation: Subtype: Link Rect: 315, 184, 322, 196 ActionDest: 142 Annotation: Subtype: Link Rect: 522, 162, 534, 171 ActionDest: 155 Annotation: Subtype: Link Rect: 535, 162, 547, 171 ActionDest: 158 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 536, 659, 560, 670 Annotation: Subtype: Link Rect: 165, 647, 391, 658 Annotation: Subtype: Link Rect: 377, 302, 415, 313 Annotation: Subtype: Link Rect: 398, 279, 435, 290 Annotation: Subtype: Link Rect: 464, 256, 501, 267 Annotation: Subtype: Link Rect: 397, 210, 435, 221 Annotation: Subtype: Link Rect: 303, 164, 340, 175 Annotation: Subtype: Link Rect: 312, 141, 349, 152 Annotation: Subtype: Link Rect: 416, 118, 454, 129 Annotation: Subtype: Link Rect: 519, 95, 557, 106 Annotation: Subtype: Link Rect: 59, 84, 95, 94 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 131, 711, 168, 721 Annotation: Subtype: Link Rect: 163, 676, 201, 687 Annotation: Subtype: Link Rect: 505, 652, 542, 664 Annotation: Subtype: Link Rect: 372, 629, 410, 640 Annotation: Subtype: Link Rect: 117, 607, 155, 617 Annotation: Subtype: Link Rect: 161, 607, 197, 617 Annotation: Subtype: Link Rect: 90, 573, 128, 583 Annotation: Subtype: Link Rect: 395, 527, 432, 537 Annotation: Subtype: Link Rect: 438, 527, 474, 537 Annotation: Subtype: Link Rect: 503, 503, 540, 514 Annotation: Subtype: Link Rect: 268, 469, 306, 479 Annotation: Subtype: Link Rect: 473, 445, 511, 456 Annotation: Subtype: Link Rect: 243, 422, 280, 433 Annotation: Subtype: Link Rect: 520, 399, 557, 410 Annotation: Subtype: Link Rect: 313, 376, 351, 387 Annotation: Subtype: Link Rect: 201, 342, 239, 353 Annotation: Subtype: Link Rect: 381, 319, 418, 330 Annotation: Subtype: Link Rect: 424, 319, 460, 330 Annotation: Subtype: Link Rect: 131, 285, 168, 295 Annotation: Subtype: Link Rect: 175, 285, 211, 295 Annotation: Subtype: Link Rect: 182, 251, 219, 261 Annotation: Subtype: Link Rect: 226, 251, 262, 261 Annotation: Subtype: Link Rect: 375, 227, 413, 238 Annotation: Subtype: Link Rect: 104, 193, 141, 203 Annotation: Subtype: Link Rect: 250, 135, 287, 146 Annotation: Subtype: Link Rect: 471, 112, 508, 123 Annotation: Subtype: Link Rect: 458, 89, 495, 100 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 59, 711, 96, 721 Annotation: Subtype: Link Rect: 370, 687, 408, 698 Annotation: Subtype: Link Rect: 175, 664, 213, 675 Annotation: Subtype: Link Rect: 59, 642, 96, 652 Annotation: Subtype: Link Rect: 102, 642, 138, 652 Annotation: Subtype: Link Rect: 493, 618, 531, 629 Annotation: Subtype: Link Rect: 510, 595, 548, 606 Annotation: Subtype: Link Rect: 281, 573, 319, 583 Annotation: Subtype: Link Rect: 517, 549, 555, 560 Annotation: Subtype: Link Rect: 287, 527, 325, 537 Annotation: Subtype: Link Rect: 519, 503, 557, 514 Annotation: Subtype: Link Rect: 59, 492, 95, 502 Annotation: Subtype: Link Rect: 168, 458, 205, 468 Annotation: Subtype: Link Rect: 211, 458, 247, 468 Checksum: 709f5395 Type: CRC32 Checksum: f6fb18a91f3757ad3f7bc4446678209a Type: MD5 Checksum: 65ab56973c8b89b09ad97f3c242d9348a04fc84c Type: SHA-1