Fermentation Optimization, Separation, Physicochemical Property And Application Of Polysaccharide-based Bioflocculants By Paenibacillus Mucilaginosus

Flocculation was thought to be an excellent approach for removing pollutants fromwastewater, and the developments and applications of new-type flocculants were the hot spotsin worldwide. The polysaccharide-based bioflocculants (PSBs) produced by microorganismswere considered as widely used flocculants due to their innocuousness and biodegradability.However, low production, low flocculating activity and narrow application scope were themain barriers for the large-scale application of PSBs. In this study, we optimized thefermentation conditions of PSB produced by Paenibacillus mucilaginosus. The efficientseparation technology of PSB was created, and a new-type PSB with high flocculatingactivity in pH3-9was also obtained.(1) The optimization of fermentation condition for PSB produced by P. mucilaginosus.Single-factor experiment and Central composite design (CCD) were carried out to evaluatethe effect of metal ions on PSB production and flocculating activity. In single-factorexperiment, comparing the PSB production in basal medium,0.144g/L MgSO4,0.055g/LCaCl2and3.25mg/L FeCl3could increase PSB production by179%,44%and109%,respectively. But the results of CCD showed that the mutual effects of metal ions on PSBproduction differed from that of individual metal ion, where observed that Mg2+(P=0.0021)played a positive role in PSB production, while Ca2+(P=0.0093) significantly inhibited thePSB production and Fe3+(P=0.9883) exerted non-significant influence when these threemetal ions mixed. Compared to the batch production of PSB in basal medium, not only thefermentation period of PSB production in the metal ion-supplemented medium (0.144g/LMgSO4,0.028g/L CaCl2and1.625mg/L FeCl3) could be shorten by12h, but also theproduction and conversion rate of PSB could be increased by218%and111%, respectively.In addition, the maximum flocculating activity of the PSB collected from the metalion-supplemented culture was also increased by27%, comparing with that in basal medium.As comparing with another common way to enhance PSB production and activity byoptimizing carbon and nitrogen sources, adding a small amount of metal ions complex aremore efficient and economical for increasing PSB production and activity. The results offurther optimizing fermentation conditions confirmed that the optimal temperature for PSB production by P. mucilaginosus GIM1.16was28°C, and the optimal initial pH was8. Theoptimal medium compositions were determined as follows: sucrose139g/L, Na2HPO43.05g/L, MgSO40.167g/L. The maximum PSB production was3.034g/L, which was increasedby127%, comparing to the metal ion-supplemented culture.(2) The separation and purification of PSB produced by P. mucilaginosus. The resultsrevealed that15%ethanol/20%Na2CO3was determined to be the best aqueous two-phasesystem (ATPS) for PSB extraction due to its maximum value of β (selective partitioncoefficient) and minimum value of K (partition coefficient). It was also confirmed that therecovery rates of PSB and cells were over98%under this condition, which implied that thisATPS not only met the basic requirement of industrial application, but also providedfeasibility for continuous extraction and fermentation. The crude PSBs from basal medium(crude P2) and metal ion-supplemented medium (crude P1) were purified by chromatographyof DEAE-52, and three independent elution fractions designated as PSB1, PSB2and PSB3,respectively, were obtained. The amount of PSB1and PSB2from crude P1was3.7times and5.0times as much as which from crude P2, while the amount of PSB3from crude P1wasslightly higher than that from crude P2. These results indicated that the PSB compositionswere changed through adding the metal ions to the medium. Fraction PSB1, PSB2and PSB3were further purified through a column of Sephadex-200. Three fractions, namely, PSB1-1,PSB2-1and PSB3-1, were obtained respectively.(3) The studies about chemical compositions and structures of the purified PSBs. Theresults of Gel Permeation Chromatography (GPC) demonstrated that the average molecularweight of PSB1-1, PSB2-1and PSB3-1were estimated to be2.53×106,7.77×106and13.2×106Da, respectively. The analysis of monosaccharide composition showed that PSB1-1was composed of fructose, galactose, sorbose, glucose and rhamnose at a ratio of1:1.08:2.45:1.66:2.78, whereas PSB2-1and PSB3-1were composed of mannose, fructose, galactose,sorbose, glucose and xylose at a ratio of1:1.16:1.12:2.4:1.72:2.88and1:1.23:1.07:3.3:1.7:3.8, respectively. The spectra of the purified PSB1-1, PSB2-1and PSB3-1revealed thatcomparing the spectra of PSB2-1and PSB3-1, the band related to O–H stretching vibrationwas shifted to a higher wave-number (from3286to3342cm-1) in the spectrum of PSB1-1.The intensity of peak at1729cm-1in PSB1-1spectrum was stronger than that of PSB3-1, and no peak was observed in the spectrum of PSB2-1at this wavenumber. The intensity of peak at1257cm-1in PSB1-1spectrum was also stronger than those of PSB2-1and PSB3-1. Inaddition, the image of scanning electron microscopy (SEM) demonstrated that theultra-structure of the purified PSB1-1, PSB2-1and PSB3-1differed from each other. Thelinear structure of PSB1-1was rather regular and dendritic, while the linear structures ofPSB2-1and PSB3-1were rather irregular.(4) The application of PSBs produced by P. mucilaginosus. The results showed that theoptimal concentration of PSB1-1for maximum flocculating activity was2mg/L, and theflocculating rate of PSB1-1was98.4%, which was higher than that of PSB2-1and PSB3-1.The results of the effect of pH on the flocculating activity showed that PSB1-1possessd thebest flocculating activities (above97%) at pH from3to9. PSB3-1showed high flocculatingactivity (above90%) in pH3-6, while the application range of PSB2-1was relatively narrow(pH3-4), and the flocculating rate was89%-95%. According to the physical-chemistryproperties and structures of the purified PSB fractions mentioned above, we inferred thathigher content of the functional groups, such as hydroxyl groups, phosphate groups and esterlinkages in PSB1-1might be responsible for the better flocculation performance of PSB1-1than PSB2-1and PSB3-1. The results of thermal stability revealed that PSB1-1wasthermostable, which was confirmed by that PSB1-1could mountain high flocculating activity(96%-98%) after incubation for30min at50-100°C. The results showed that trivalent metal(Al3+and Fe3+) and bivalent metal ions (Ca2+, Mg2+and Fe2+) played more important role inPSB1-1flocculation performance, comparing with monovalent metal ions (Na+and K+). Theresults of the effect of kaolin concentrations on the flocculating activity revealed that PSB1-1showed poor flocculation performance when the kaolin concentration was over10g/L.PSB1-1was applied in industrial wastewater treatment. The results showed that PSB1-1showed better flocculation performance than PAM and PAC in treating wastewater frombiological product factory (pH7.11). When the concentration of PSB1-1was1mg/L, theremoval rate of COD and SS was91.1%and97.5%, respectively. As for the treatment ofwastewater from the paper mill (pH8.57), the results showed that PSB1-1showed little worseflocculation performance than PAM, but still better than PAC. When the concentration ofPSB1-1was3mg/L, the removal rate of COD and SS was81.3%and93.6%, respectively. As for the treatment of wastewater from garbage incineration plant (pH6.08), the results revealedthat PSB1-1showed worse treatment performance than those of wasterwater from biologicalproduct factory and paper mill. However, PSB1-1was better at treating wastewater than PAMand PAC. When the concentrations of PSB1-1were6mg/L, the removal rate of COD and SSwas66.2%and80.1%, respectively.