Download Mobilis 7dyas
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Download Mobilis 7dyas
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After enzymatic saccharification, liberated acetate continues to act as a fermentation inhibitor for many microorganisms, including Zymomonas mobilis CP4 (pZB5) and E. coli KO11 [3, 8, 13, 14]. Maiorella et al. postulated that soluble acetate disrupts the transportation of nutrients, such as phosphate, across the cell membrane .
To test the effect of acetate removal prior to pretreatment on fermentation yields, we performed higher solids (25% solids) enzymatic saccharification experiments using unwashed solids. There are several reasons for selecting high solids, whole slurry enzymatic hydrolysis. First, higher solids enzymatic hydrolysis results in higher sugar concentrations in fermentation, requiring less energy demand for the distillation process for ethanol purification. Humbird et al. reported that the minimum solids loading occurred between 15% and 20% total solids in saccharification and fermentation based on their techno-economical analysis . Also, washing pretreated solids is difficult because the fines produced require a solid/liquid separation step, which is expensive when implemented at the industrial scale. Lastly, using a higher solids loading leads to higher acetate concentrations in hydrolyzate, which may lead to greater inhibition of fermentation by Z. mobilis. Therefore, enzymatic hydrolysis on whole slurry was carried out at a total solids content of 25%, which includes about 18% insoluble solids.
After saccharification, a toxicity analysis was performed on the hydrolyzates. Whole slurries were analyzed using two assays developed for toxicity analysis: growth assays using the Bioscreen C  and mini-fermentations assays, both using Z. mobilis 8b as the biocatalyst. Because growth assays are performed using spectrophotometric analysis, solids are removed by centrifugation and the supernatant liquor is filtered. Due to the opaqueness of the supernatant liquor from high solids slurries, samples were diluted to 10 wt % of the pretreatment slurry with the addition of 10 g/L yeast extract, 2 g/L K2HPO4, water, and sugars, while normalizing the concentrations for both glucose and xylose.
Growth rates of Z. mobilis 8b in whole slurries from deacetylated 34M95, 33B15, and INL feedstocks were compared with their respective non-deacetylated controls, as shown in Figure 4. In addition, two hydrolyzate liquor samples obtained from higher severity pretreated corn stover were also tested for toxicity in this study for comparison purposes. VT refers to corn stover pretreated in a pilot scale vertical reactor at 190C for one minute in 3% sulfuric acid. The details of the reactor and its relevant acid pretreatment process have been reported by Shell et al. . HT refers to corn stover pretreated in the pilot scale horizontal reactor for 158C for five minutes using 2% acid (Nagle, Kuhn, Shekiro, unpublished data). Pure sugar fermentations were conducted as positive controls for both growth and mini-fermentation assays. We have noted slightly different growth rates for Z. mobilis 8b when grown in different sugar concentrations. Other laboratories have also noted specific growth rates to change at high glucose concentrations . For this reason, glucose and xylose levels were normalized based on the concentrations of sugars in samples containing the maximum levels.
Growth rates for deacetylated and control slurry samples are not significantly different at the 10% total solids equivalent level; both are approximately 15% to 20% lower than growth rates observed with the pure sugar controls. Furfural concentrations in both control and deacetylated 10% total solid slurries are very similar (approximately 0.1 g/L) and are 78% to 86% lower than concentrations observed in the higher severity pretreated hydrolyzates experimented (0.45 to 0.7 g/L), which causes significantly more growth inhibition. Growth rate inhibitions at 0.1 g/L furfural are predicted to be minimal (< 5%) for Z. mobilis 8b when no other inhibitors are present . Likewise, the acetate levels of < 5 g/L are predicted to have minimal impact on growth in the absence of other inhibitors . Therefore, if the toxicity contribution is only from acetate or furfural, one would not expect to see much difference in fermentation yields between deacetylated and control samples at this solids loading, which was observed.
Mini-fermentations were conducted at the process relevant condition of 18.5% total equivalent solids after normalizing for glucose (83 g/L) and xylose (56 g/L), medium, and inoculum. Fermentations were carried out with Z. mobilis 8b at an inoculum loading of 1.0 (OD600) at 33C. In Figure 5, process ethanol yields and sugar utilization consumed were plotted along with the inhibitor concentrations of acetate and furfural. Glucose utilization was complete for all samples except for the higher severity samples, HT and VT, where approximately 5% of the initial glucose remained post fermentation. In pure sugars, Z. mobilis was able to ferment 75% of the initial xylose (without pH control). Xylose conversion yields in deacetylated samples were approximately 10% higher when compared to control samples and resulted in approximately 7% higher ethanol yields. When compared to hydrolyzates generated from higher severity pretreatment conditions, VT and HT, xylose utilization was approximately 25% greater, resulting in approximately 15% higher ethanol process yields.
The liquid phase separated from whole slurry enzymatic hydrolysis as described in the last sections were subjected to the fermentability test. Samples were analyzed using two assays developed for toxicity analysis: growth and mini-fermentation assays using Zymomonas mobilis 8b as the biocatalyst. Toxicities were also tested for previously generated hydrolyzate liquors (after acid pretreatment with the biomass separated in the absence of saccharification enzymes). Two hydrolyzate liquor samples were also tested for toxicity for comparison purposes. The vertical reactor (VT) samples were generated from corn stover pretreated at 190C, 3 wt % sulfuric acid for one minute in a pilot scale Sunds (Metso USA, Norcross, GA, USA) one metric ton/day vertical reactor. The conditions used to generate the horizontal reactor (HT) samples were of lower severity, 158C, 2% sulfuric acid for five minutes in a pilot scale Metso (Metso, Norcross, GA) 200 kg/day horizontal screw reactor.
Z. mobilis 8b was used for the evaluation of hydrolyzate toxicity. It was revived from frozen glycerol stocks for approximately 6 to 8 hrs in 10 mL of RMG (2% glucose) at 33C prior to inoculating overnight seed cultures in RMG8X2 (8% glucose, 2% xylose) using Blank volume shake flasks filled to 80% capacity at 33C at 120 rpm. When the glucose concentration decreased from approximately 80 g/L to approximately 20 to 40 g/L, cells were spun down at 3,840 g for 10 minutes at room temperature and re-suspended in RMG (2% glucose, 10 g/L yeast extract, 2 g/L K2HPO4) at a 10-fold concentration and used as inocula for Bioscreen C growth assays or fermentation studies.
Mini-fermentation assays were conducted with whole slurries conducted at higher biomass loadings in miniature vials vented with 18 gauge needles capped with 0.2 μM filters, using a total 4 mL volume. Yeast extract, K2HPO4, water, glucose and xylose were added, while normalizing the total glucose and xylose to the same concentration for each sample. Mini-fermentations were conducted by inoculating log phase cultures of Z. mobilis 8b at an initial cell OD of OD600 nm 1 (approximately 1 108 cells/mL). Samples were taken at 0, 24, 48, and 72 hours for OD600 nm readings and HPLC analysis. Fermentation assays were conducted at 33C at 120 rpm.
The possible producers of levan were also identified among gram-negative halophilic bacteria of the Halomonas genus [2,21], including Halomonas smyrnensis AAD6T . Their ability to grow in high concentrations of NaCl can be used to resolve the problem of sterility in an industrial setting. Zymomonas mobilis also produces levan as originally reported in the 1960s . The original strains ZAG-12  and ATCC 31821  were able to produce levan with approximate yields of 14.67 and 21.69 g/L respectively. It was reported that during continuous cultivation of Z. mobilis CCT4494, when immobilized in Ca- alginate gel the amounts of levan can range from 18.84 up to 112.53 g/L dependent on the incubation time . Komagataeibacter xylinus, the producer of bacterial cellulose can also produce levan [27,28]. Several recombinant E.coli strains have been developed to study the biochemistry of levan synthesis by cloning and expression of levansucrase genes from Leuconostoc mesenteroides and Васillus amyloliquefaciens [29,30].
The yield, structure and physical-chemical properties of P. polymyxa EPSs depend on many factors such as cultivation condition, source of carbon, C/N ratio, pH of media and time of cultivation [38,45] Levan biosynthesis occurs extracellularly from sucrose under the influence of the enzyme levansucrase (EC 18.104.22.168) [2,46,47]. The enzyme breaks down sucrose into glucose and fructose, with the remainder of the latter transferred to the sucrose acceptor molecule . It has been reported that levansucrase, with strong sucrose hydrolysing activity, is involved in many P. polymyxa strains and may be responsible for the high yield of EPS with sucrose as a carbon source [33,38]. Thus, the strain P. polymyxa 92, isolated from the roots of wheat forms 38.4 g/L of levan in 10% sucrose media . Sugar-containing waste from food production, such as molasses, was used to reduce the cost of levan production . It has been reported that levan was obtained on molasses media with the help of bacteria such as Azotobacter vinelandii VKPM B-5787 , Bacillus lentus V8 , Microbacterium laevaniformans PTCC 1406 , Paenibacillus polymyxa NRRL B-18475  and Zymomonas mobilis ATCC 31821 . The release of the polysaccharide, formed by Bacillus licheniformis NS032, in a molasses-containing medium with an estimated sucrose concentration of 62.6% lead to 53.2 g/L levan yield . 59ce067264