Nisin-loaded PH-responsive Poly(4-vinylpyridine) Microspheres For Intelligent Controlling Of Lactobacillus Plantarum Contamination In Bioethanol Fermentation

The depletion of traditional fossil energy and the increased concerns about environmental pollution issues promoted the demand for alternative renewable energy sources.As a widely recognized renewable clean energy,bioethanol could contribute to alleviating of environmental pollution and easing off energy crisis.Saccharomyces cerevisiae is the most applied microorganism to produce bioethanol.However,industrial scale bioethanol fermentation is usually stressed by bacterial contamination,thereby leading to decrease of bioethanol yield and serious economic losses.Lactic acid bacteria(LAB)take a dominant proportion among the contaminant microorganisms.Various strategies have been developed to help solve this problem,of which antibiotics addition is the most widely used way to control bacterial contamination in bioethanol fermentation.However,the abuse of antibiotics could result in serious drug resistance and further aggravate the spreading of resistance gens.Nisin used in this study,which is a kind of polypeptide bacteriocin secreted by Lactococcus lactis subsp.lactis and active against many gram-positive bacteria,would avoid drug resistance problems.Different microorganisms together with the fermentative environment constitute a micro-ecosystem,in which all living organisms coexist under various and complex relationships.Potentially inhibitory contaminants are usually present in a normal,balanced and healthy microflora,and sometimes the contaminated bacteria might be even beneficial for fermentation.It is possible that stuck fermentations would occur while such balance of the microflora is disturbed by unknown factors.The micro-nutrients and living space in the closed fermentation system are relatively enough at the beginning of the process,so it is not necessary to eliminate the contaminated bacteria at the initial stage.Therefore,traditional controlling of contaminative bacteria at the beginning of fermentation needs to be improved.Based on the factors discussed above,it would be more meaningful to realize real-time monitoring of fermenting condition for delivery of antibiotics or other reagents timely at different stages of the contamination.However,such operations are labor-intensive and time-consuming.The accumulation of acid by-products secreted by LAB could partially contribute to the consistent decreasing pH value,which would decrease from 6 to 4,of the bacteria contaminated bioethanol fermentation system.The consistently decreased pH value would provide opportunity to realize the intelligent real-time controlling of bacterial contamination with pH sensitive materials.4-vinylpyridine is a widely used functional monomer whose pKa value is 5.30 and usually functions as a good electron donor.The pKa value of poly(4-vinylpyridine)(P4VP)is about 5,which is just within the range of pH value of the bioethanol fermentation process(i.e.,pH value range from 4 to 6).Using P4VP as package material to encapsulate bactericidal substances would make the drug release in a pH-responsive manner.In this study,L.plantarum was co-cultured with S.cerevisiae in yeast extract peptone dextrose(YPD)broth to simulate the L.plantarum contamination in industrial bioethanol fermentation.Nisin was embedded into pH-sensitive P4VP microspheres synthesized by suspension polymerization to realize intelligent controlling of L.plantarum contamination in bioethanol fermentation.The prepared microspheres were characterized by SEM and FTIR.The diameters of the microspheres ranged from 40 μm to 100 μm.The encapsulation efficiency of nisin into P4VP microspheres was 47.67%and the drug-loading capacity of nisin was 2.5%.Chloramphenicol was chosen as the model drug to be embedded into P4VP microspheres to test the drug release behavior.The drug release curve of chloramphenicol-loaded P4VP microspheres showed sustained and pH-responsive characteristics.Finally,Nisin-loaded P4VP microspheres were added into the simulated contaminative fermentation system,and successfully reversed the decline of bioethanol yield secondary to L.plantarum contamination.