Ethanol and Levan production by sequential bath using Zymomonas mobilis immobilized on alginate and chitosan beads

Current study evaluates ethanol and levan production using Zymomonas mobilis immobilized on alginate and chitosan beads and assesses the capacity of the beads for reuse in sequential fermentations. Two experiments were carried out: In the first experiment 2 design evaluated the following independent variables: sucrose concentration; pH; incubation time; temperature and agitation. In the second experiment, based on the best conditions observed in 2, the capacity of the immobilization support reuse in subsequent fermentations was evaluated for 12 days. In experiments using fractionated factorial experimental design 2 the best results by immobilized biomass (2.6 g L) and immobilization efficiency was shown by alginate and chitosan beads showed only 0.22 g L. Chitosan support provided the best rates to levan production (8.90 g L). Alginate support showed the highest values by ethanol production (93.4 g L). In batch sequential fermentation, re-using immobilization support, alginate support was efficient by maintaining the cellular viability on immobilized support (0.94 g L), levan production (21.11 g L) and ethanol production (87.21 g L). Chitosan support was inadequate for sequential batches since the beads dissolved after the second cycle.


Introduction
Zymomonas mobilis is highlighted in the literature as a promising microorganism for ethanol and other products of technological interest production such as the gum levan (or fructan) mainly due to its capacity to tolerate high ethanol and sugar concentrations, produce low biomass concentration, use the Entner-Doudoroff metabolic pathway for carbohydrate metabolism and show cell growth regardless of carbon consumption (Behera, Mohanty, & Ray, 2012;Pentjuss, Kostromins, Fell, Stalidzans, & Kalnenieks, 2013).
Despite the fermentation versatility of Zymomonas mobilis, its efficiency may be impaired by cells exposition to unfavorable conditions during conventional fermentation processes.To solve such difficulties, several researches have been carried out to improve this technology.In fact, the use of immobilized cells stands out amongst the more promising alternatives (Behera, Ray, & Monhanty, 2010).
Among the different methodologies for cell immobilization, entrapment on supports is used since it provides greater effectiveness and easier execution.In entrapment, the cells are inserted into rigid/semi-rigid matrix, and their diffusion in the reaction medium is limited by cell accumulation in porous material (Behera et al., 2010).This process confers cell protection against pH harsh conditions, temperature, inhibiting compounds, favor exchange of nutrients, metabolites and gases between the immobilized cells and reaction.The main supports include alginate, k-carrageenan, agar, chitosan and polyvinyl alcohol (El-Naas, Mourad, & Surkatii, 2013;Cheng, Ma, Deng, Xu, & Chen, 2014;Yang, 2015).
Moreover, immobilization may result in physical and chemical changes of microbial cells as modifying metabolism products, ionic charges, water activity, osmotic pressure, or surface tension (Rathore, Desai, Liew, Chan, & Heng, 2013).Due to the need to develop technologies that increase ethanol and levan yield production for the use of economically feasible, easily handled raw materials, current research evaluates the efficiency of immobilizing Z. mobilis on alginate and chitosan beads and ethanol and levan production and assesses the recycling capacity of the immobilized cell biomass by sequential batch fermentation.
Z. mobilis stock culture was seeded into tubes containing Z. mobilis broth and incubated at 30ºC for 24 hours to reactivate the culture.The tubes were added into pre-fermentation flasks containing Z. mobilis broth (50 mL) and incubated under the same conditions described below.For tests, the prefermentation medium was centrifuged at 3660 g 15 min -1 and the supernatant was discarded and the cell pellet re-suspended in 0.85% (m v -1 ) saline solution to obtain a concentrated cell suspension which was standardized by spectrophotometry at absorbance 0.7 at 570 nm.
Z. mobilis cells at the pre-established concentration were added to a previously prepared and sterilized 3% (w v -1 ) alginate solution and stirred for 10 min.The mixture was then dripped, drop by drop, using a hypodermic syringe, into a 0.2 M calcium chloride solution under constant agitation, obtaining beads with a diameter of approximately 3 mm.Immobilization on the chitosan beads was carried out in a similar way, with a 4% (m v -1 ) chitosan solution, previously dissolved in 1% (v v -1 ) acetic acid.Cell-chitosan suspension was maintained under agitation for 10 min, and then dripped into a 1 M NaOH solution under agitation, obtaining beads with a diameter of approximately 3 mm.Beads of the two supports were matured under refrigeration at 4ºC for 24 hours and then, washed with sterile distilled water before adding to flasks containing fermentation medium and a carbon source.Ten grams of beads were added to each flask which contained 50 mL fermentation medium under conditions described in Experiment 1.
Experiment 1: Optimum conditions for ethanol and levan production A 2 (5-2) fractionated factorial experimental design determined the ethanol and levan production conditions, resulting in 27 experiments, with 2 repetitions at the central point, as shown in Table 1 (Montgomery, 2001).The parameters selected were based on previous experiments using free cells (Ernandes & Garcia-Cruz, 2011).Reuse supports capacity was based on the best conditions for levan and ethanol production obtained in the 2 (5-2) experiment, using response surface methodology: 350 g L -1 sucrose; pH 4; 30ºC and agitation at 200 rpm.Supports recycling were carried out for 12 cycles with 24 hours of fermentation.

Calculation of kinetic parameters
The kinetic parameters were determined according to equations below as equations 1 and 2: Y P/S = g g -1 (1) where: P = productivity; P 0 = initial product mass (g L -1 ); P f = final product mass (g L -1 ); S 0 = initial substrate concentration (g L -1 ); S f = final substrate concentration (g L -1 ); Y p/s = coefficient of product yield in relation to substrate consumed; t f = time (h); t 0 = initial time (h).

Analytical methods
Final pH rate was determined by Digmed DM20 pH meter.Free and immobilized biomasses were determined by spectrophotometry at 570 nm.Alginate beads were previously dissolved in 10 mL of 0.3 M sodium citrate (pH: 5, 1 M -1 citric acid) and chitosan beads in 5% (v v -1 ) acetic acid to determine immobilized biomasses.Ethanol content was evaluated by gas chromatography (GC focus, TR-wax column 30 x 0.053, ID x1μm) and levan content was determined by technique described by Viikari (1984).Reducing sugars were evaluated according to copper-arsenate technique described by Somogyi (1952) and Nelson (1944), and total sugars by the phenol-sulfuric acid method described by Dubois, Gilles, and Hamilton (1956).Substrate consumption using alginate (SCA) or chitosan (SCC) was calculated by subtracting the initial substrate concentration (sucrose) from the final substrate concentration, with results in %.Factorial fractionated statistical design (2 k-p ), described by Montgomery (2001), was used for the optimal experiments, using Statistica 6.0.Data were evaluated by variance analysis (ANOVA) for the best parameters to ethanol and levan production, using the contour curves produced by response surface methodology (RSM).Results obtained for the sequential batch fermentations during 12 days of support recycling were also evaluated by analysis of variance and Tukey's test, with Minitab 14. Electronic microscopy scanning was determined by fixing the samples in 3% (v v -1 ) glutaraldehyde for 6 hours and 1% (v v -1 ) osmium tetroxide during 1 hour.The samples fixed in OsO 4 were washed in distilled water and treated with increasing concentrations of ethanol (30, 50, 70, 90 and 100%  (v v -1 ) for 10 min.The samples which passed through the critical point were dried (K550, Emitech) and mounted on a SEM stub with copper tape and sputter coated with gold/palladium.Images of the samples were analyzed with scanning electron microscope (LEO 435 VPi SEM, Zeiss) according Madi-Ravazzi (2007).

Results and discussion
Experiment 1: Determination of best conditions for ethanol and levan production Immobilized biomass, immobilization efficiency and substrate consumption (sucrose).
Parameters that showed significant statistical influence (p < 0.05) for increasing immobilized biomass on alginate support (2.60 g L -1 ) were initial pH (approximately 7.0), sucrose (150 g L -1 ) and without agitation of fermentation medium (Tables 2 and 4).On the other hand, in tests carried out using chitosan beads as the immobilization support (0.22 g L -1 ), no variable evaluated had any significant statistical influence (p < 0.05), as showed in Tables 3 and 4.
Initial pH rate of fermentation medium was an important parameter for biomass production, with optimal rate for bacterial growth at approximately 7.0.Several authors have reported initial pH of the fermentation medium as an important factor for Z. mobilis growth, which grows at pH rates ranging between 3.5 and 7.5, although the optimum range between 5 and 7 (Swings & De Ley, 1977;Santos, Del Biachi, & Garcia-Cruz, 2014).Immobilization efficiency using cell concentration adhered on support and the free biomass in culture medium, demonstrated that alginate support was more efficient for immobilization (2.6 g L -1 ) than chitosan support (0.22 g L -1 ), as showed in Tables 2 and 3.
Also by substrate consumption the alginate support showed the highest conversion rate (90.7%) for experiments carried out at 30ºC, without agitation, 150 g L -1 of sucrose, initial pH at 7.0, and 96 hours of fermentation (Table 5).
Table 3. Mean rates for immobilized biomass, ethanol and levan production obtained in 2 (5-2) fractionated factorial experimental design for chitosan support.
The highest rates for levan production were obtained using the bigger sucrose concentration (350 g L -1 ) by Tests 2 and 5, using alginate (6.27 g L -1 ) and chitosan in Tests 6 and 8 (4.67 and 8.0 g L -1 ), as shown in Figure 1 and Table 2. Sucrose (350 g L -1 ), lowest pH rate (4), temperature (30ºC) and fermentation time provided a significant statistical interference (p < 0.05) by levan production using alginate support (Table 6).Chitosan support revealed the same behavior with sucrose concentration, although pH 7, 40ºC and agitation (200 rpm) were statistically significant (Table 6).Regarding to sucrose concentration, a similar behavior was observed by Lorenzetti, Moro, and Garcia-Cruz (2015) using immobilized Z. mobilis in alginate/PVA hybrid support.They obtained best levan production (18.84 g L -1 ) using 300 g L -1 of sucrose.Santos, Del Bianchi, and Garcia-Cruz (2014) also obtained maximum levan production (23.5 g L -1 ) by same microorganism immobilized in alginate using sucrose 350 g L -1 .
Silbir, Dagbagli, Yegin, Baysal, and Goksungur (2014) studied Zymomonas mobilis B-14023 immobilized on alginate beads by levan production with sucrose (300 g L -1 ) as substrate and initial pH 6.0 and obtained a maximum levan concentration of 40.2 g L -1 .As shown in Tables 2 and 3, levan production decreased in the two evaluated supports but the greatest production did not present the highest yield rate. Chitosan revealed the highest rates in production yield with 0.2 g g -1 in run 9C, whereas alginate gave 0.02 g g -1 in experiments 1 and 2 (Table 3).
Variations obtained in levan production rates might be due to the biochemical mechanisms that regulate the polysaccharide synthesis since it is a reserve energy substance (Alegre, Wendt, & Rigo, 2005).It has also been observed that, since greater substrate consumption did not result in a proportionally greater levan production by alginate and chitosan supports (Tables 2, 3 and 5), the substrate might have been mainly used to produce biomass and other metabolites, such as ethanol, sorbitol and fructooligosaccharides (Swings & De Ley, 1977;Madigan, Martinko, Dunlap, & Clark, 2010).

Ethanol production by Zymomonas mobilis immobilized on alginate and chitosan beads
Alginate support presented highest rates for ethanol production (93.4 g L -1 ) in run 9 C and yield of 0.41 g g -1 (Table 2 and Figure 2).Maximum ethanol yield was obtained in runs 1 (0.45 g g -1 ) and 6 (0.44 g g -1 ), as shown in Table 2.However, no variable presented significant statistical interference (p < 0.05) (Table 6).Ethanol production efficiency using Z. mobilis immobilized on alginate beads was also reported by Yamashita, Kurosumi, Sasaki, and Nakamura (2008); Behera et al. (2010) and Behera, Mohanty, and Ray (2012).Chitosan support gave maximum ethanol production of 5.24 g L -1 and ethanol yield of 0.48 g g -1 , both in trial 9, and the variables sucrose, fermentation time and agitation presented significant statistical interference (Table 6 and Figure 2).
Optimal conditions for ethanol production by Zymomonas mobilis in immobilized systems are divergent in literature.Most cited parameters include initial pH between 4.5 and 7.5; temperature between 25 and 37ºC and sugar concentration between 150 and 250 g L -1 (Bandaru, Somalanka, Mendu, Medicherla, & Chityala, 2006).These parameters were evaluated in current study (Table 1).Initial pH adjusted to 5.5 proved to be the best parameter evaluated for ethanol production by alginate and chitosan beads, even though without any statistical interference (Tables 2 and 6).This behavior was also registered by Maiti, Rathore, Srivastava, Shekhawat, and Srivastava (2011); Pinilla, Torres, and Ortiz (2011).As mentioned above, agitation affects ethanol production by chitosan support (Table 6), also reported by Pinilla, Torres, and Ortiz (2011).
In current study, temperature at 30-35ºC (Tables 2 and 3 -Experiments 6 and 9) proved to be the best for ethanol synthesis on alginate and chitosan supports, although it did not statistically influence ethanol production (Table 6).This behavior was also cited by Behera et al. (2010) and by Bandaru et al (2006).
Similar to levan production, cell concentration on the support did not affect ethanol synthesis (Tables 2 and 3).This can occur because Zymomonas mobilis growth is independent of ethanol synthesis.Due to this peculiarity, Z. mobilis is considered to be an excellent ethanol producer (Yamashita et al., 2008;Pentjuss et al., 2013).
Experiment 2: Ethanol and levan production by sequential fermentation with immobilization support recycling Behavior of immobilized biomass and final pH during alginate and chitosan supports recycling were carried out during 12 fermentation cycles, using the best production conditions obtained in the 2 (5-2)  experiments (Table 7).
Immobilized biomass on alginate support remained viable throughout all the reused cycles, and was observed high affinity of this support by the microorganism.This was demonstrated in scanning electronic microscopy and the micrographs show a great number of immobilized cells on the surface and inside the beads (Figure 3A and B).
Table 7. Optimum conditions for levan and ethanol production using alginate and chitosan supports obtained by 2 (5-2) experiments using response surface methodology.Figure 3 shows that the amount of immobilized biomass during the first fermentation cycle was 0.003 g L -1 ; it increased to 0.19 g L -1 after 5 days; at the end of recycling it was 0.94 g L -1 .In fact, alginate beads remained intact from the first to the last fermentation cycle and only changed their texture and color possibly due to microbial growth or to levan presence inside the beads.Free biomass during 12 fermentation cycles ranged between 0.14 and 2.65 g L -1 (Table 9).Fermentation cycles were carried out by a hybrid system of free and entrapment cells.In fact, the above behavior has been observed in fermentative process with immobilized microorganisms.
On the other hand, chitosan beads showed 0.001 g L -1 immobilized biomass during the first fermentation cycle (Figure 3) even though they were not viable for immobilizing Zymomonas mobilis CCT4494.Chitosan beads would not withstand the fermentation conditions used or the fermentation cycles and dissolved after the second recycle.Free biomass was 1.10 and 1.30 g L -1 for first and second cycles, respectivelly (Table 8).
Zymomonas mobilis adhered to chitosan support after immobilization is showed in Figures 4C and D. Although reduced microbial adhesion there was microbial growth on the inside and on the outside of the beads, similar to alginate.Figure 4 also revealed the beads irregular external structure with the immobilized microorganism.Chitosan beads behavior during the fermentation cycles could have been due to support nature, which dissolves in acid media and too by metabolism of the microorganism those results in acids accumulation in culture medium and would have dissolved the chitosan beads (Xu, Huang, Zhu, & Ye, 2015).Final pH of fermentation media using chitosan beads was extremely basic with rates 7.55 and 8.14, respectively, in the first and second cycles (Table 8).

Levan production during sequential fermentation by immobilization support recycling
Levan production decreased when chitosan beads were used after the second reuse cycles.In fact, the highest production and yield occurred in the first cycle (3.47g L -1 and 0.01 g g -1 ) with significant statistical difference (p < 0.05) when compared to that of the second cycle (1.99g L -1 and 0.007 g g -1 ), as showed in Table 8.Levan production could not be evaluated for the remaining reuse cycles, since the beads dissolved due to the culture medium pH.This behavior was not observed during the batch fermentations in which the beads remained stable during the experiments, demonstrating that chitosan is not a viable support for sequential batch fermentations.
In spite of the reduced efficiency for the immobilization of Z. mobilis reported for chitosan support (Figure 3), the highest consumption rate of the sucrose substrate was not associated with the highest production of levan for the first (56.69%)and second (76.32 %) fermentation cycles (Table 8).Zymomonas mobilis immobilized in alginate beads showed the highest production and yield in the tenth (21.11 g L -1 and 0.09 g g -1 ) and fourth (10.10 g L -1 and 0.07 g g -1 ) cycles, without significant difference between them (p < 0.05).However, in relation to other cycles, the 10 th cycle showed the highest rates with significant difference (p < 0.05), as demonstrated in Table 8.
Levan production during twelve cycles was between 3.63 and 21.11 g L -1 and was almost constant after the tenth cycle, with substrate consumption (sucrose) between 43.58 and 78.96% (Table 9).The highest sucrose consumption was accompanied by the highest levan production and also by the largest amount of free biomass in the culture medium, as highlighted in Table 9.The maximum sucrose consumption was 78.96% in run 7 and 75.75% in run 9, which was not followed by a higher levan productivity (0.07 and 0.09 g L -1 hour -1 ) respectively for runs 4 and 10, according to Table 9.
Lower production rates were observed in the first fermentation cycles, possibly due to microorganism adaptation.However, higher rates were reported for all reuse cycles, after the 2 nd recycle (8.05-12.11g L -1 ) which were higher than those results observed in batch cultivation (Table 2).Immobilized inoculum reuse demonstrated greater physiological stability and capacity to levan produce in larger amount (Table 9).Silbir et al. (2014) also obtained good stability of alginate beads during continuous fermentation using : central point; x 1 : sucrose (g L -1

Figure 3 .
Figure 3. Immobilized biomass on chitosan and alginate supports during fermentation cycles of recycling support.

Table 2 .
Mean rates for immobilized biomass, ethanol and levan production obtained in 2 (5-2) fractionated factorial experimental design for alginate support.

Table 4 .
ANOVA table for biomass concentration of Zymomonas mobilis immobilized in alginate and chitosan supports.

Table 5 .
Results for free biomass, total sugars and substrate consumption obtained using Zymomonas mobilis CCT 4494 immobilized on alginate and chitosan.

Table 6 .
ANOVA table for levan and ethanol production of Zymomonas mobilis immobilized in alginate and chitosan supports.

Table 8 .
Results for the production of levan, ethanol, substrate consumption, final pH and free biomass produced by Zymomonas mobilis CCT4494 immobilized on chitosan during sequential batch fermentation cycles., b... (column) -means followed by the same small letters do not differ according to Tukey´s test (p < 0.05).

Table 9 .
Results for the production of levan, ethanol, substrate consumption and free biomass produced by Zymomonas mobilis CCT4494 immobilized on alginate during sequential batch fermentation cycles... (column) -means followed by the same small letters do not differ by Tukey´s test (p < 0.05).L: levan production; LPT: levan productivity; YL: yield in levan; E: ethanol production; EPT: ethanol productivity; YE: yield in ethanol; SC: substrate consumption; FB: free biomass in the fermentation medium. .