Characterization of Normal and Waxy Corn Starch for Bioethanol Production

Objectives of this study were to compare ethanol production between normal and waxy corn using a cold fermentation process and to understand effects of starch structures and properties on ethanol production. Ethanol yields positively correlated (<i>p</i> < 0.01) with starch contents of kernels of the normal and waxy corn. The average starch–ethanol conversion efficiency of waxy corn (93.0%) was substantially greater than that of normal corn (88.2%). Waxy corn starch consisted of very little amylose and mostly amylopectin that had a shorter average branch chain length than normal corn amylopectin. Regression analyses showed that average amylopectin branch chain lengths and percentage of long branch chains (DP > 37) of waxy corn starch negatively correlated with the starch hydrolysis rate and the ethanol yield. These results indicated that starch structures and properties of the normal and waxy corn had significant effects on the ethanol yield using a cold fermentation process

Figure 4

<p>A. Branch chain-length distribution of glycogens from WT (), M1 (▪), and M2 (□). Glycogen samples were treated with a debranching enzyme, isoamylase, and the resulting debranched-samples were separated on an HPAEC system. The peak area was calculated and normalized from the chain-profile chromatogram. B. The comparison of difference in the normalized peak area calculated from M1-WT (□), M2-WT (○), or M1-M2 (⋄).</p

Effects of <i>sll</i>1393 and <i>sll</i>0945 deletions on glycogen accumulation and structure, using commercial rabbit-liver glycogen and waxy maize starch for comparison.

a<p>Values given are means ± standard deviation obtained from at least three independent experiments.</p>b<p>Calculated based on peak area of each chain on HPAEC chromatograms.</p><p><i>Number</i>-average degree of polymerization = Σ peak area/Σ (peak area/number of glucose of each chain). Values given are means ± standard deviation obtained from at least three independent experiments.</p>c<p>Not Applicable.</p

Oligonucleotide sequences used to construct 3 pSHK1393 and pSHK0945 plasmids.

a<p>Final constructs were used to replace endogenous <i>sll</i>1393 and <i>sll</i>0945 genes, respectively, by homologous recombination.</p><p>The position of the oligonucleotides in the nucleotide sequences of the <i>sll</i>1393 and <i>sll</i>0945 structural DNA are indicated. The restriction sites introduced by PCR amplification are underlined.</p

Effects of insertional mutation of <i>sll</i>1393 and <i>sll</i>0945 genes on GS, GBE, and AGPase activities.

a<p>Incorporation rate of [¹⁴C]glucose onto rabbit-liver glycogen primers.</p><p>Values given are means ±standard deviation obtained from three independent experiments.</p>b<p>Stimulation of glucose incorporation rate.</p><p>Values given are means ± standard deviation obtained from two independent experiments with two replicates in each.</p>c<p>Rate of ADP-glucose formation.</p><p>Values given are means ± standard deviation obtained from two independent experiments with a total of four replicates.</p>d<p>not detected.</p

Photomixotrophic growth of WT (▵), M1 (□), M2 (○), and M12 (⋄) strains of <i>Synechocystis</i> sp. PCC6803.

<p>Cells were grown in a BG-11 medium containing 5 mM glucose and the number of cells were counted in the exponential phase. The same amount of the cells were inoculated in the new media. Each culture was inoculated with an equal number of exponential-phase cells, and growth of the cultures was monitored by measuring absorbance at 730 nm at various time intervals.</p

Branch structure of glycogen and starch.

a<p>Values given were determined from at least two duplications.</p>b<p>ECL (exterior chain length) = CL (which is equal to DP_n in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091524#pone-0091524-t003" target="_blank">Table 3</a>)×β-amylolysis (%)+2.0.</p>c<p>The ratio was calculated based on the mole fraction of (G2+G3) stubs released, by isoamylase and pullulanase hydrolysis, from β-limit dextrin.</p

Gene structure of <i>sll</i>1393 (A) and <i>sll</i>0945 (B) on <i>Synechocystis</i> sp. PCC 6803 genomic DNA.

<p>The targeted replacement of each GS gene with the Km^R gene was achieved by homologous recombination. (C) PCR analysis of <i>sll</i>1393 and <i>sll</i>0945 genes using genomic DNA from wild-type, <i>sll</i>1393⁻, and <i>sll</i>0945⁻ strains as templates. For comparison, PCR products of intact genes in wild-type were loaded next (left lane) to the corresponding mutated genes. Lane1 is the size marker (1 kb plus DNA ladder, Life Technologies). DNA bands on lane 2 (WT) and 3 (M1) were amplified using prA1 and prA4; Lane 4 (WT) and 5 (M2) were amplified using prB1 and prB4.</p

Figure 3

<p>Zymogram analysis of GS activity from wild-type and mutant cells. Ten µg of each soluble extract was subjected to native PAGE. The bands showing GS activity were visible after overnight incubation in a buffer containing ADP-glucose and rabbit-liver glycogen and were stained with iodine solution. Lane 1. WT, Lane2. M1 (<i>sll</i>1393⁻), Lane3. M2 (<i>sll</i>0945⁻), and Lane 4. M12 (<i>sll</i>1393⁻/<i>sll</i>0945⁻).</p