Optimization of culture conditions for bioflocculant-producing Bacillus megaterium MBFF6 and its chemical characteristics

Abstract

At present,flocculants are prevalent in the variety of industrial processes such as wastewater treatment,drinking water purification and downstream processing in fermentation processes.In general,flocculants are classified into three groups: inorganic flocculants,organic synthetic flocculants and naturally occurring flocculants.Bioflocculants have received considerable scientific and biotechnological attention in recent years because of their biodegradability,harmlessness and lack of secondary pollution of their degradation intermediates.Compared with inorganic flocculants and synthetic flocculants,bioflocculants have not yet been produced industrially because of their low flocculating capability and high cost.In order to reduce costs of production,research is needed to identify and select new bioflocculant-producing microorganisms and strains capable of utilizing low-cost substrates,and to learn how to optimize fermentation conditions to enhance bioflocculant activity.We had previously screened a bioflocculant-producing strain identified as Bacillus megaterium MBFF6,and the strain producing bioflocculant was named as BF6.In this study,Optimization for culture conditions of strain MBFF6 and chemical characteristics of bioflocculant BF6 were studied.The effect of culture time,carbon source,nitrogen source,C/N ratio,pH,metal ions,culture temperature and inoculum size on bioflocculant production by B.megaterium MBFF6 was investigated by signal factor test.At the same time,thermal stability and chemical characteristics of bioflocculant were studied.The bioflocculant BF6 was purified from supernatant by ethanol precipitation.Whether containing α-amino acids and polysaccharide of the purified bioflocculant was analyzed by ninhydrin reaction and Molish and anthrone reaction.Proteins and nucleic acids were qualitatively determined by UV spectrophotometry.The chemical groups of purified bioflocculant were analyzed using a Fourier-transform infrared(FTIR) spectrophotometer(FTIR-8400S).The results indicated that the optimal medium for bioflocculant production was glucose 5 g/L,(NH4)2SO4 0.5 g/L,K2HPO4 5 g/L,KH2PO4 2 g/L,MgSO4?7H2O 0.2 g/L,pH 7;The optimal culture conditions were inoculum size 2 %(V/V),culture temperature 30 °C,and shaking speed 150 r/min for 24 h(Fig.1-5).This bioflocculant was stable at 60-100 °C.The production of bioflocculant BF6 from fermentation broth was 1.28 g/L and it was a white powdery substance.The results of color reaction showed that the anthrone reaction produced blue-green compounds,and Molish reaction appeared a clear violet ring on the boundary of sulfuric acid and samples,and the ninhydrin reaction produced blue compounds,which indicated that the bioflocculant contained polysaccharide and protein.The results of UV spectrophotometry revealed that there was a faint characteristic absorption peak at 280 nm,but no absorption peak at 260 nm,confirming that the bioflocculant was primarily consisted of polysaccharide and proteins but no nucleic acids.Infrared spectrophotometry analysis revealed that this bioflocculant contained hydroxyl,amino,carbonyl,and methoxyl groups in its structure and these groups were the preferred ones for flocculation process(Fig.6).The experimental result above shows that the bioflocculant-producing strain MBFF6 has such advantages as simple nutrition and short fermentation period,and it synthesises a bioflocculant with better thermal stability and high flocculating activity.It can be seen that the B.megaterium MBFF6 is a very broad application bioflocculant-producing strain.Bioflocculant BF6 may find possible application as a polymer for environmental bioremediation and other biotechnological processes.Further studies on its flocculation mechanism and scaling-up culture process are in progress in order to enhance the bioflocculant production and realize its industrial and agricultural utilization.