The Study On Isolation And Identification Of Acetic Acid Bacteria And Yeast From Kombucha And The Symbiosis Of Bacterial Cellulose Production

Bacterial cellulose is an extracellular polysaccharide produced by microorganisms.It has a variety of superior properties and it is widely used in many fields.Typically,the production of bacterial cellulose is by acetic acid bacterias,however,the major challenges faced for the applications of bacterial cellulose and its up-scale production are high cost of the media and low productivity at industrial scale.Kombucha is a symbiont,which is typically formed by acetic acid bacterias and yeasts.Kombucha can ferment sweetened black tea to produce bacterial cellulose,particularly suitable for up-scale industrial production.However,the microbial community of kombucha is complex.And it is difficult to preserve,easily degrade and contaminated by other microorganisms,which can lead to unstable bacterial cellulose production.A strain of acetic acid bacteria with bacterial cellulose production capacity was isolated from Kombucha,but the yield of bacterial cellulose was far less than kombucha,so it is speculated that the yeasts in Kombucha can promote the production of bacterial cellulose.A pre-experiment was conducted in this work,an acetic acid bacteria with the ability to produce bacterial cellulose was isolated from kombucha,but the bacterial cellulose yield was much smaller than kombucha when it was cultured alone.It is speculated that the yeasts in kombucha has a great impact on the bacterial cellulose production and they are symbiont.This paper carried out the following research works in order to understand the symbiosis between acetic acid bacteria and yeasts in the synthesis of sacterial sellulose and explain the reasons that yeasts promotes the production of bacterial cellulose:(1)The acetic acid bacteria and yeasts were isolated by spread plate method,and then the 16S rDNA sequence and 26S rDNA sequence were determined to identify the strains.The results showed that one bacterial cellulose-producing acetic acid bacteria Al was isolated and identified as Komagataeibacter intermedius;three strains of yeast were isolated,identified as Brettanomyces bruxellensis,Zygosaccharomyces bisporus,Metschnikowia fructicola.(2)K.intermedius was respectively co-cultured with B.bruxellensis,Z.bisporus,and M.fructicola at different ratios,and then orthogonal experiments were performed to determine the optimal ratio of co-culture.The results showed that as the proportion of B.bruxellensis increased,the yield of bacterial cellulose increased and then decreased,but both were significantly higher than the control(P<0.05),when K.intermedius:B.bruxellensis=1:100,the yield of bacterial cellulose was the highest,with a dry basis of 4.70 g·L-1;as the proportion of Z.bisporus increased,the yield of bacterial cellulose increased and then decreased,but both were significantly higher than the control(P<0.05),when K.intermedius:Z.bisporus=1:1,the yield of bacterial cellulose was the highest,with a dry basis of 5.19 g·L-1;when M.fructicola was co-cultured with K.intermedius in different ratios,the bacterial cellulose yield was not significantly different from the control group(P>0.05).In orthogonal test,when K.intermedius:B.bruxellensis:Z.bisporus=1:10:10,the inoculation amounts of K.intermedius,B.bruxellensis,and Z.bisporus were 1 × 104 CFU·mL-1,1 × 105 CFU·mL-1,and 1 × 105 CFU-mL-1 respectively,the yield of bacterial cellulose was the highest,with the dry basis of 5.51 g·L-1.(3)The effects of yeast supernatants and yeast cells at different fermentation times on bacterial cellulose production were studied.The results showed that after adding yeast supernatant to HS medium,the yield of bacterial cellulose was significantly higher than the control(P<0.05),the highest bacterial cellulose yield was 5.42 g·L-1(dry basis),and the yeast supernatant could significantly promote bacterial cellulose production.The supernatant in the early fermentation period(1-3 days)had a strong promotion effect on the production of bacterial cellulose;after adding inactivated yeast cells to HS medium,the bacterial cellulose yield was also significantly higher than the control(P<0.05),the highest bacterial cellulose yield was 2.89 g·L-1(dry basis).Yeast cells with different fermentation times had little effect on bacterial cellulose production.It is determined that the metabolite of yeast had a positive effect on the production of bacterial cellulose.(4)The content of amino acids,volatile substances,organic acids and vitamins in the yeast supernatant were analyzed.The results showed that 16 amino acids were detected in the yeast supernatant,and cysteine was only detected in the yeast supernatant.Compared with the unfermented HS medium,the increase of isoleucine was the largest,which is 11.64 times that the control.The increase of aspartic acid and glycine are ranked second and third,the increments are respectively 60.00%and 41.67%.The main volatile substances in the yeast supernatant were alcohols,accounting for 84.89%of all volatile substances,of which the relative content of ethanol is the highest at 77.35%.The relative content of 3-methyl-1-butanol and phenethyl alcohol were also higher,the relative contents were respectively 4.13%and 3.14%.Three organic acids were detected in the yeast supernatant,which were tartaric acid,citric acid and succinic acid,respectively.Compared with the unfermented HS medium,the species did not change,but the contents were reduced.Vitamin B2 and B6 were detected in the yeast supernatant,compared with the unfermented HS medium,the species and contents did not change.It is speculated that amino acids(especially cysteine,isoleucine,aspartic acid and glycine)and alcohols(especially ethanol)may have a great positive effect on bacterial cellulose production,organic acids and vitamins had no significant effect on bacterial cellulose production.