Study On The Pilot Production Process And Quality Control Techniques For Marine Oil Degrading Bacteria Agents

In the bioremediation process of environmental pollution,bioaugmentation?addi-tion of exogenous degrading microorganisms when the native microbiota is insuffi cient or overwhelmed?is commonly used to improve the degradation efficiency.However,the restoration of oil-polluted sea areas is a complex project involving the characteristics of contaminated materials,microbial ecological structures,and various environmental conditions.The composition of degrading bacteria and its adaptability to the environ-ment will affect the bioremediation project.In order to improve the effectiveness of bi-oremediation,based on the existing study in the laboratory,this paper optimizes fer-mentation media with single factor tests and orthogonal experiments to optimize the fermentation media of the four bacteria Acinetobacter sp.Y9?Acinetobacter sp.W3?Gordonia sp.X1?Acinetobacter sp.F9?Y9,W3,X1,and F9?that constitute marine oil pollution-degrading bacteria.The results showed that the most suitable fermentation media of Y9,W3,X1 and F9 as following:Y9:1%glucose,2%peptone,0.1 mol/L phosphate buffer solution;W3:0.5%glucose,1.5%corn steep liquor,0.05 mol/L phosphate buffer solution;X1:2.5%glucose,2%corn steep liquor,0.1 mol/L phos-phate buffer solution;F9:5%glucose,2.5%Peptone,0.1 mol/L Phosphate buffer solu-tion.Other inorganic salt components include:NaCl 0.05%;CaCl₂ 0.001%;MgCl₂0.019%.To study of pilot fermentation process of the four marine oil-degrading bacteria,the pilot producing process parameters and quality control methods of the bacteria were developed.The results showed that the fermentation process parameters of fermentation tank after optimization of Y9 strain were initial glucose 40 g/L,fermentation tempera-ture 30°C,agitation speed 450 rpm,inoculation ratio 15%,pH value 7,and the fed-batch fermentation process was that fresh feeds were added for 4 times from the fourth hour,and the supplemental sugar was 40 g/L.The fermentation process parame-ters of W3 were initial glucose 5 g/L,fermentation temperature 30°C,agitation speed450 rpm,inoculation ratio 15%,pH value 7,and the fed-batch fermentation process was that fresh feeds were added for 4 times from the fourth hour,and the supplemental sugar was 20 g/L.The fermentation process parameters of X1 were initial glucose 25 g/L,fermentation temperature 30°C,agitation speed 450 rpm,inoculation ratio 15%,pH value 7,and the fed-batch fermentation process was that fresh feeds were added for 4times from the fourth hour,and the supplemental sugar was 100 g/L.The fermentation process parameters of F9 were initial glucose 50 g/L,fermentation temperature 30°C,agitation speed 550 rpm,inoculation ratio 15%,pH value 7,and the fed-batch fermenta-tion process was that fresh feeds were added for 4 times from the fourth hour,and the supplemental sugar was 200 g/L.Bagasse and calcium alginate were used to prepare a biological floating carrier of the four marine oil-degrading bacteria.The results showed that when the sodium algi-nate was 2.5%,and the bagasse was 1.5%,and CaCl₂ was 1.5%,and 12 minutes for cross-linking,the carrier had the best appearance and highest mechanical strength.Marinel oil-degrading bacterial consortium was immobilized on the carrier materi-al.The preparation process parameters were that the proportion of bacterial culture(OD₆₀₀=1)and the carrier was 12:1?volume?,and stirring speed was 110 rpm,and in-cubation time was 4 h.The number of microbial loaded on the carrier material reached approximately 5.48×10⁹ CFU/g.On these preparation process parameters,the pilot production process of marine oil-degrading bacteria agent was developed in a 150 L stirred fermentation tank.Three batches of diesel-degrading bacterial agents were developed in the 150 L tank,and they were used to study the quality control methods.The results showed that the size of the agent was between 3.5⁴.5 mm,and the shape was uniform spherical,no profile,no cracking,no adhesion,It was in light brown and can float in seawater for more than 24 h.The bacterium capacity of the carrier was above 5×10⁸ CFU/g.It was recommended to save at-20?for long-range conservation.The bioremediation simulation experiment of the bacterial agent was carried out in laboratory,and the results showed that the oil-biodegradation ratio of the bacterial agent was more than 89.4%in 5 days.The results suggested that the medium optimized by the orthogonal experiment can be applied to the fermentation tank,and the fermentation process is feasible.The ba-gasse–calcium alginate carriers has good floating performance and adsorption perfor-mance,and the bacteria agents can effectively degrade the oil pollution,it should be very useful to the bioremediation of large-scale oil spill accidents at sea.