Mechanism Of Accelerating Bioleaching Process Of Sludge With Rhodotorula Sp.R30

Sewage sludge,a sediment formed during the sewage treatment process,contains many plant nutrients and heavy metals.But many improper sludge disposal would threaten environment.Bioleaching have been performed using Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans isolated from sewage sludge with the main aims to explore the optimum parameters of sludge bioleaching.But many results from other workers revealed that the bioleaching periods would be long if only Acidithiobacillus were incubated to the bioleaching system.The main reason for the long bioleaching periods was the slow growth rate of Acidithiobacillus during the bioleaching process.Especially the dissolved organic matter(DOM) in sludge is toxic to Acidithiobacillus.To overcome the handicap for bioleaching,we tried to isolate heterotrophic microorganism assimilating sludge DOM as energy source and carbon source.The effect of combined cultures of heterotrophic microorganism and Acidithiobacillus on bioleaching was studied,and the mechanism of accelerating the bioleaching process by combined cultures was also explored. The main results of this study are listed as follows:A heterotrophic microorganism,isolated from sewage sludge collected from Kasen Tannery Ltd.Company,Haining,Zhejiang Province,P.R.China,was identified as Rhodotorula sp.R30.When R30 was inoculated in the medium containing dissolved organic matter derived from sewage sludge and incubated for 96 h,dissolved organic carbon in the medium declined from 1485 mg/L to 345 mg/L,and the yeast number was 4.8×10⁷ cell/mL.It indicated that yeast R30 could eliminate the toxicity of sludge DOM to Acidithiobacillus through assimilating some sludge DOM.To examine the positive effect of yeast R30 on tannery sludge bioleaching,bioleaching tests of tannery sludge with addition of yeast R30 were performed.It was found through the batch trial that the period of bioleaching could be apparently shorted to 4 days if yeast R30 was inoculated in the bioleaching system.Furthermore,the removal efficiency of Cr in sewage sludge could be enhanced by 39%compared to the control without inoculating yeast R30. The tolerance abilities of A.ferrooxidans LX5,A.thiooxidans TS6 and Rhodotorula sp.R30 to Cr³⁺ were also investigated.Results showed that 700mg/L of Cr³⁺ has no obvious impact on the growth of LX5 and TS6,but 500mg/L of Cr³⁺ could restrain the growth of R30.It was also observed that the co-cultures of LX5,TS6 and R30 could significantly accelerate the bioleaching process.The optimum inoculation ratio was 10%for A. ferrooxidans LX5 and A.thiooxidans TS6 and 2.0%for Rhodotorula sp.R30.Treating tannery sludge with bio-acidified sludge containing yeast R30 as inoculum exhibited that the bioleaching efficiency was not obviously higher compared with the controls.With re-inoculation of 2.0%yeast R30 in the beginning of bioleaching,the period of bioleaching could be apparently shortened by 36 hours.So the amounts of yeasts in sludge were very important during the bioleaching when the other conditions were similar.It is very critical for accelerating bioleaching process through adding R30 to the bioleaching system at the initial stage to ensure the amounts of yeasts in sludge achieve 10⁶ CFU/mL.A.ferrooxidans LX5 and A.thiooxidans TS6 are strict chemoautophilic bacteria.They grow very slowly and require 2-3 weeks to form colony on solid medium with low plating efficiency.Therefore,the two-layer plate culture system to promote plating efficiency was developed in this study.The two-layer assay was performed as following:0.1mL of R. sp.R30 solution was spread onto the underlayer made of 2%of agar in plate.As superstratum,the mineral-salt synthetic medium for A.thiooxidans TS6 and A.ferrooxidans LX5 were spread and superimposed on the underlayer.The suspension containing A. thiooxidans TS6 or A.ferrooxidans LX5 were spread evenly on the superstratum.The colonies of A.thiooxidans TS6 were found to appear on the double plates for five days earlier than on the single plate without the underlayer composed of 2%of agar and the yeast strain.Furthermore,the colony numbers of A.thiooxidans TS6 shown on the double-layer plate was 3.6 times as many as that on the single-layer plate.Likewise,for A. ferrooxidans LX5 strain,the plating efficiency was enhanced by 2.1 folds.It was illuminated that R.sp.R30 lived in underlayer consumed a lot of dissolved organic matter in medium and released from the toxicity to chemoautotrophic Acidithiobacilli bacteria and improved the colony count efficiency for double-layer plate.Furthermore it was testified that dissolved organic matter in medium is toxic to chemoautotrophic Acidithiobacilli bacteria and one of detoxification mechanism of R.sp.R30 is that R.sp.R30 utilized dissolved organic matter as carbon and energy sources,consequently accelerated the growth of Acidithiobacilli bacteria.To study the else mechanism of accelerating bioleaching process of tannery sludge with co-cultures of A.ferrooxidans LX5 and A.thiooxidans TS6 and R.sp.R30,the bioleaching process was performed in a bioreactor which could monitor dynamics of CO₂, pH,DO of sludge by some electrodes.It was found that the bioleaching of sludge added with yeast R30 shorten 36h less than the control.And the concentration of CO₂ in sludge added with R30 was enhanced 3 times than control.But the dynamics of DO concentration in both trial and control sludge were similar.To test the effect of CO₂ concentration on the growth and activity of A.thiooxidans TS6 which is the main microorganism responsible for the bioleaching process, A.thiooxidans TS6 was cultivated in CO₂-rich bioreactor through the addition of CO₂. Results showed that the growth of A.thiooxidans TS6 was significantly accelerated,as exhibiting that its fermentation time was shorten by 34h in comparison with the control without the addition of CO₂.Moreover,the amounts of A.thiooxidans TS6 were doubled at the end of fermentation due to the addition of CO₂.It is concluded that strain R30 could assimilate sludge DOM,produce CO₂ and increase CO₂ concentration in sludge medium to accelerate the growth of Acidithiobacillus may be another mechanism for strain R30 facilitating bioleaching process.R.sp.R30 could excrete many extra cellular Polymeric Substances(EPS) in its growth process,so the effect of EPS on the bioleaching of tannery sludge was studied.It was found through the experiments of EPS complexion with Fe³⁺ and oxidation abilities of Fe³⁺and EPS—Fe³⁺ that EPS could band Fe³⁺ in medium produced from Fe²⁺oxidation by A.ferrooxidans LX5 and consequently produce EPS—Fe³⁺complex with a maximum banding capacity of 7.2mg Fe³⁺/mgTOC.Therefore,Fe³⁺ concentration was maintained in medium and didn't form a great deal of ferric hydroxysulfate precipitation in acidic sulfate-rich environment.It was observed that EPS—Fe³⁺complex was,to a certain extent, capable of oxidation ability for Fe²⁺,although its oxidation ability was lower than Fe³⁺ under same concentration condition.Generally,the more the amount of Fe³⁺ capable of oxidation activity in medium,the higher bioleaching efficiency.This also illuminated that EPS produced from R.sp.R30 could,to a small extent,accelerate bioleaching process when R.sp.R30 was introduced into bioleaching system.Obviously,it was concluded that three mechanisms involved in the facilitated bioleaching process of tannery sludge due to the addition of R.sp.R30,which including sludge DOM assimilation,the increase of CO₂ concentration in sludge medium and the inhibition of Fe³⁺ precipitation owing to the formation of EPS-Fe³⁺ formation facilitated by R.sp.R30.