Ba vi khun hiu kh phn lp c t nc knh Nhiu Lc - Th Ngh l: Thiobacillus 1 (C1); Thiobacillus 2 (C2); Thiobacillus 3 (C3).

Kh nng kh sunfua ca cc vi khun ny c kho st trn ba loi mi trng: MT1 (mi trng peptone 0, 5%); MT2 (mi trng Beijerick khng c FeSO4.7H2O) v MT3 (mi trng Beijerick c b sung 0,5% Ca (NO3)2 v 0,5% anthraquinnone). Kt qu th nghim cho thy, kh nng kh sunfua ca cc vi khun Thiobacillus din ra nhanh trong 4 n 5 ngy u kho st. Thm vo , pH ca mi trng tng nh trong 7 ngy u, v gim mnh t ngy th 8 tr i.

t vn Hydro sunfua (H2S) l cht kh khng mu, c mi trng thi, rt c, d ha tan trong nc thnh axt sunfuhydric (H2S). Hydro sunfua nu ln vo du kh d ch mt lng rt nh, n cng lm gim nghim trng gi tr thng phm ca du kh. Axt sunfuhydric (H2S) l cht n mn kim loi rt mnh, gy ra hin tng n mn l v gin kim loi do hydro, c th dn n nt v ng ng hoc bn cha. V vy, kh hydro sunfua l vn cp thit trong ngnh khai thc v ch bin du kh nc ta. C th kh hydro sunfua bng cc phng php ha hc, ha l hoc sinh hc... Trong bi bo ny, chng ti trnh by kt qu kh hydro sunfua bng vi khun phn lp c t knh Nhiu Lc Th Ngh ti thnh ph H Ch Minh (Tp. HCM). Vt liu v phng php nghin cu 1. S nghin cu Knh Nhiu Lc - Th Ngh nm trong ni thnh TP HCM, di 9035m. c ca nc knh Nhiu Lc - Th Ngh thay i t 30-50 FTU. Nng oxy ho tan thp (3,3-4,0mg/l) bo hiu knh b nhim nghim trng bi cht thi hu c. BOD trung bnh bng 78,8% COD trung bnh. S phn hy cht hu c cha lu hunh bi cc vi khun k kh, ch yu l vi khun kh sunfat (sulfate reducing bacteria), sinh ra hydro sunfua (H2S) . Trong thin nhin tn ti nhiu loi vi sinh vt c kh nng chuyn ha cc hp cht hu c v v c cha lu hunh, trong c cc vi sinh vt t dng ha nng, vi sinh vt t dng quang nng v c vi sinh vt d dng. Vi sinh vt t dng ha nng tham gia chuyn ha lu hunh bao gm cc vi khun thuc loi Thiobacillus, Beggiatoa, Thiothrix, v Thioploca, trong ng ch . nht l hai loi Thiobacillus v Beggiatoa . Chng ti phn lp loi vi khun hiu kh Thiobacillus t mu nc thi knh Nhiu Lc - Th Ngh, v nghin cu kh nng kh hydro sunfua (H2S) ca Thiobacillus theo s sau:

Hnh 1. S quy trnh nghin cu Ghi ch: Mu nc thi ca knh Nhiu Lc Th Ngh c ly lc thy triu xung, 2 v tr: Trn b mt knh v di su 50cm, ti u knh Nhiu Lc v chn cu Th Ngh. 2. Phng php phn lp v gim nh vi khun kh sunfua Mi trng nui cy: Mi trng baz (Thiosunfat medium, pH = 9,5); mi trng trung tnh (Beijerick Medium, pH = 7,4); mi trng axt (s dng cho Thiobacillus thiooxidans, pH=4,4) . S dng dng lng nhn sinh khi; b sung agar thnh dng c phn lp. Mi trng trung tnh (mi trng Beijerick, Ph = 7,4): 5,0g Na2S2O3.5H2O; 1,0g NaHCO3; 0,2g Na2HPO4.H2O; 0,1g NH4Cl; 0,1g MgCl2.6H2O; 0,5g FeSO4.7H2O; nc ct cho 1 lt. nh danh vi khun bng cc phn ng: Nhum Gram, s dng b 14 phn ng sinh ho IDS 14GNR, s dng b 32 phn ng sinh ha trn b kit ID 32E, th nghim oxydase, th nghim Lysin decarboxylase, th nghim kh nng di ng. Kt qu phn ng sau khi c s tin hnh nh danh bng phn mm k thut xt nghim vi sinh lm sn (Software of Clinical Microbiology). 3. Kho st kh nng kh sunfua ca vi khun Thiobacillus Mc ch ca kho st ny l nghin cu kh nng kh sunfua ca vi khun Thiobacillus trn cc mi trng c thnh phn khc nhau, nhm xem xt kh nng s dng vi khun khi khng v khi c b sung thm mt s cht trong qu trnh x l H2S. Sinh khi vi khun c cho vo nc mui sinh l 0,8% hp tit trng. Cho 0,5ml dch vi khun ny vo ng nghim cha 10ml mi trng kho st v trng. Kho st kh nng kh sunfua cu vi khun trn 3 loi mi trng khc nhau trong 10 ngy. y l cc mi trng c cha cng mt lng H 2S, nhng c thnh phn khc nhau. - Mi trng mt (MT1) - mi trng cn bn nhn sinh khi vi khun: cha peptone 0,5% c pH =7, dung dch H2S v vi khun. - Mi trng hai (MT2) - mi trng c trng cho Thiobacillus sp., dng kim chng kh nng kh sunfua ca vi khun: Cha mi trng Beijerinck c pH = 7,4, dung dch H2S v vi khun. - Mi trng ba (MT3) - ging nh mi trng th hai l mi trng c trng cho Thiobacillus sp, nhng c b sung thm cht to kh xy l: 0,5% canxi nitrat Ca(NO3)2 v 0,5% anthraquinnone). - Mu trng l mi trng khng cho H2S vo. - Mu i chng l mi trng c cho H2S vo, nhng khng cho vi khun vo. Nng H2S c kim tra mi ngy bng phng php it. 4. Kho st s to thnh ion sunfat SO42Hm lng ion sunfat SO42- c nh tnh mi ngy bng dung dch mui bari clorua BaCl2, nh lng bng phng php o c. 5. Kho st s thay i pH S thay i pH c theo di mi ngy bng pH k. Kt qu v tho lun

1. Kt qu phn lp v gim nh vi khun kh sunfua Vi khun trong mu nc thi c nui trn 3 mi trng khc nhau: Kim, trung tnh v axit. u tin, vi khun trong mu nc thi c nui trong mi trng lng 14 ngy. Sau , cc vi khun ny c cy trn mi trng thch. Khi vi khun pht trin trn mi trng thch th tin hnh cy phn lp v nhum Gram xc nh loi trc khun Gram m. Sau , vi khun c cy tng sinh trn mi trng thch ri tin hnh nh danh. Cc vi khun phn lp c t nc knh Nhiu Lc-Th Ngh l: Thiobacillus 1 (C1); Thiobacillus 2 (C2); Thiobacillus 3 (C3). 2. Kho st kh nng kh sunfua ca cc vi khun Thiobacillus T Hnh 2 ta thy, kh nng kh H2S ca cc ging Thiobacillus trong mi trng MT1 nhanh nht trong 3 ngy u, n ngy th 6 th kh hu ht H2S. Trong mi trng MT1, tc kh H2S gim dn theo dy: Thiobacillus C1 v Thiobacillus C2 > Thiobacillus C3.

Hnh 2. S thay i nng H2S (ppm) trong mi trng MT1 ph thuc vo ging vi sinh v s ngy th nghim Cc thc nghim c kho st trn mi trng MT2 - mi trng c cc dng cht cho vi khun Thiobacillus - khng nh v so snh s kh H2S ca cc ging vi khun. Hnh 3 cho thy, kh nng kh H2S ca cc ging Thiobacillus trong mi trng MT2 nhanh nht trong 3 ngy u, n ngy th 6 th kh hu ht H2S. Trong mi trng MT2, tc kh H2S gim dn theo dy: Thiobacillus C1 > Thiobacillus C2 v Thiobacillus C3. Mi trng 2 - mi trng Beijerinck c cha cc dng cht phc v cho s pht trin ca vi khun, ng thi cng cha cc cht h tr vic gip vi khun nh hng kh H2S. Chnh v vy, trong mi trng ny qu trnh kh H2S cng d xy ra hn, vi khun cng dng cht tn ti lu hn. Kho st trn mi trng MT3 nhm mc ch xem xt tc dng ca cc cht to oxy: Canxi nitrat Ca(NO3)2 v Anthraquinnone (9,10-dioxoanthracene (C14H8O2) n s pht trin ca vi khun. T Hnh 4 ta thy, kh nng kh H2S ca cc loi Thiobacillus trong mi trng MT3 nhanh nht trong 3 ngy u, n ngy th 5 th kh hu ht H2S. Trong mi trng MT3, tc kh H2S gim dn theo dy: Thiobacillus C1 > Thiobacillus C2 > Thiobacillus C3. Kh nng kh H2S (ppm) ca Thiobacillus C1 trong cc mi trng khc nhau c th hin qua Hnh 5. Chng vi khun Thiobacillus C1c kh nng kh H2S tt hn trong MT3. iu ny khng nh tc ng ca cc cht to oxy ln chng ny. Vi mi trng MT1 v MT2, tuy c khc bit nhng khng ng k. Hnh 6 cho thy, chng vi khun Thiobacillus C2 c kh nng kh H2S tt hn trong mi trng MT3, nhng s khc bit khng ng k so vi mi trng MT1 v MT2. Vy vic s dng cht to oxy, khng tc ng nhiu n kh nng kh H2S ca Thiobacillus C2. Tng t nh chng Thiobacillus C1, chng vi khun Thiobacillus C3 c kh nng kh H2S tt hn trong MT3 (Hnh 7). iu ny khng nh tc ng ca cc cht to oxy ln chng ny. Vi mi trng MT1 v MT2, tuy ti mi trng MT1 vi khun ny c kh nng kh H2S tt hn, nhng s khc bit khng ng k.

Hnh 3. S thay i nng H2S (ppm) trong mi trng MT2 ph thuc vo ging vi sinh v s ngy th nghim

Hnh 4. S thay i nng H2S (ppm) trong mi trng MT3 ph thuc vo ging vi sinh v s ngy th nghim

Hnh 5. Kh nng kh H2S (ppm) ca Thiobacillus C1 trong cc mi trng khc nhau theo thi gian

Hnh 6. Kh nng kh H2S (ppm) ca Thiobacillus C2 trong cc mi trng khc nhau theo thi gian So snh cc kt qu ta thy, Thiobacillus C2 c kh nng kh H2S tt trong c 3 mi trng MT1, MT2 v MT3; cn ging Thiobacillus C1 v ging Thiobacillus C3 th kh H2S tt hn trong MT3. Trong mi trng MT3 c cc cht to oxy, cc cht ny lm tng lng oxy trong mi trng, khin cho vi khun pht trin tt hn, nht l vi nhng chng hiu kh c yu cu nhiu xy. Nh vy, trong mi trng MT3 tc kh H2S ca cc vi khun Thiobacillus cng nhanh hn. Kt qu cng cho thy, vi khun Thiobacillus ging Thiobacillus C1 v ging Thiobacillus C3 c nhu cu xy cao hn so vi Thiobacillus C2. 3. Kho st s thay i pH Trong c 3 mi trng kho st, ta thy s thay i pH tng t nhau. u tin, trong cc mi trng MT1, MT2 v MT3, pH u tng nh (t 7,1 ln khong 7,4-7,5); sau vi ngy - ty theo chng loi vi khun - th. pH gim dn. iu ny c th gii thch nh sau: - Trong nhng ngy u, pH mi trng tng nh, v. vi khun chuyn ha hydro sunfua v oxy thnh lu hunh v nc theo phn ng: 2H2S + O2--> 2S + 2 H2O - Song song , khi mc cn i gia cc cht c trong mi trng thay i, lu hunh s b xi ha thnh axt sunfuric: 2S + 3O2 + 2H2O --> 2H2SO4

- T s liu ca Bng 1, ta c th nhn thy, pH ca MT1 > MT2 v MT3. iu ny l do trong mi trng MT2 v MT3 cn c s to thnh H2SO4 t Na2S2O3 (t mi trng Bejerinck)

5Na2S2O3+H2O+4O2--> 5Na2SO4+2S2+H2SO4+Xkcal S2 + 3O2 + 2H2O --> 2H2SO4 + Xkcal Theo thi gian, vi khun kh ht dn lng H2S c trong mu, kh nng s dng H2S ca vi khun nhanh hn tc phn ng ca cc phn ng v c nh vo lng enzym c trong vi khun. Sau qu trnh hnh thnh H2SO4 tng dn dn n vic lm gim pH mi trng. 4. Kho st s to thnh ion sunfat: Kt qu kho st c 3 mi trng u khng thy s c mt ca ion sunfat trong thi gian u. Sau lng inon sunfat bt u xut hin nhng khng ng k. Ch t ngy th 8 tr i, s xut hin ca ion sunfat SO42- trong mi trng mi tr nn r rt. iu c nhn bit nh s to thnh kt ta trng c ca mui bari sunfat, khi cho vo mi trng dung dch bari clorua BaCl2: BaCl2 + H2SO4 2HCl + BaSO4 (Trng c) Nhng kho st trnhby trn chng t rng, c 3 loi vi khun Thiobacillus C1, C2, C3 u c kh nng kh hydro sunfua thnh axt sunfuric, lm cho pH mi trng gim mnh.

Hnh 7. Kh nng kh H2S (ppm) ca Thiobacillus C3 trong cc mi trng khc nhau theo thi gian

Hnh 8. Phn ng theo di s hnh thnh ion sunfat trong mu Kt lun 1. phn lp c vi khun hiu kh Thiobacillus t mu nc thi ca knh Nhiu Lc Th Ngh, gm 3 ging: Thiobacillus C1, Thiobacillus C2, Thiobacillus C3. 2. C 3 ging vi khun hiu kh thuc chi Thiobacillus phn lp c, u c kh nng kh hydro sunfua H2S trong 3 mi trng kho st: - Tc kh H2S ca cc vi khun Thiobacillus nhanh nht trong 4 - 5 ngy u. - Trong thi gian u, pH trong 3 mi trng kho st u tng nh v khng to thnh ion sunfat t H2S.

- T ngy th 8 tr i, pH ca mi trng gim mnh, do cc vi khun Thiobacillus kh H2S thnh axt sunfuric H2SO4 trong qu trnh trao i cht ca chng vi mi trng. 3. C th nhn sinh khi vi khun Thiobacillus kh cht H2S c mt trong cc mi trng nc thi v du kh. Cn sc thm kh oxy hoc cho thm mui sinh oxy nh Ca(NO3)2 vo mi trng, to iu kin thun li cho 3 ging vi sinh vt hiu kh Thiobacillus C1, C2, C3 pht trin v kh H2S c nhanh hn v trit hn. TS. m Sao Mai Trng i hc Cng nghip TP.HCM KS. Cao Hng Ngc, GS. TSKH. V nh Huy Trng i hc Bch Khoa Tp.HCM (Theo TCDK)

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