Trình tự gen 16S rARN của chủng A4.2 tương đồng 99.9% (1411/1413 bp) với đoạn gen 16S rARN của vi khuẩn Bacillus licheniformis_X68416 khi so sánh với các trình tự gen 16S rARN của các vi sinh vật trong ngân hàng gen quốc tế (Hình 3.20). Điều này cho chúng tôi kết luận: chủng A4.2 thuộc chi Bacillus. Đây là công bố đầu tiên ở Việt Nam, chủng vi khuẩn Bacillus licheniformis có khả năng xử lý photpho trong nước thải bằng việc tích lũy vào cơ thể vi sinh vật, một phần cung cấp năng lượng cho các q trình sinh hóa và trao đổi chất.
Staphylococcus aureus_X68417 Bacillus cibi_AY550276 Bacillus indicus_AJ583158 Bacillus idriensis_AY904033 100 Bacillus isabeliae_AM503357 100 Bacillus aerophilus_AJ831844 Bacillus altitudinis_AJ831842 Bacillus stratosphericus_AJ831841 99 Bacillus safensis_AF234854 Bacillus pumilus_AY876289 99 97 Bacillus aerius_AJ831843 Bacillus licheniformis_X68416 A4.2 100 Bacillus sonorensis_AF302118 100 Bacillus atrophaeus_AB021181 Bacillus malacitensis_AY603656 Bacillus axarquiensis_AY603657 Bacillus mojavensis_AB021191 59 Bacillus subtilis_AB042061 77 Bacillus vallismortis_AB021198 Bacillus nematotocita_AY820954 Bacillus velezensis_AY603658 Bacillus siamensis_GQ281299
Bacillus amyloliquefaciens_NR041455 Bacillus methylotrophicus_EU194897 6866 62 81 59 71 100 100 100 99 68 89 0.01
KẾT LUẬN
1. Đã phân lập và được 65 chủng vi sinh vật trên môi trường Winogradsky và 21 chủng trên môi trường AMM từ các mẫu nước thải ở Thanh Hóa và Hà Nội.
2. Đã tuyển chọn được 4 chủng vi sinh vật có khả năng hình thành màng sinh học và có khả năng xử lý nitơ và photpho tốt nhất là các chủng có ký hiệu B11.11, B21.10, B23.2 và A4.2. Các chủng có khả năng hình thành màng sinh học ở nhiệt độ trong khoảng từ 37 đến 50oC, pH từ 7 đến 8.
3. Đã phân loại và xác định được 3 chủng có khả năng xử lý nitơ tốt nhất, trong đó, chủng B11.11 có khả năng chuyển hóa 85.21% NH4+ sau 20 ngày nuôi cấy, chủng B21.10 chuyển hóa 97,28% và B23.2 chuyển hóa 97,14% lượng NO2- sau 20 ngày nuôi cấy.
4. Đã phân loại và xác định được chủng A4.2 có khả năng xử lý photpho tốt nhất, sau 10 ngày, hàm lượng photpho trong môi trường giảm đi 39.32% tương ứng với mơi trường có hàm lượng photpho là 18 mg/l.
5. Dựa trên trình tự gen 16S rARN, đặc điểm khuẩn lạc, tế bào, các đặc điểm sinh lý, sinh hóa, chủng B11.11 và A4.2 được xác định gần với loài Bacillus licheniformis, chủng B21.10 được xác định gần với loài Bacillus amyloliquefaciens,
chủng B23.2 xác định gần với loài Pseudomonas pseudoalcaligenes.
KIẾN NGHỊ
1. Thay đổi hàm lượng nitơ và photpho phù hợp với nghiên cứu. Kiểm tra khả năng chuyển hóa các chất trong mơi trường và sự hình thành màng sinh học khi thay đổi hàm lượng.
2. Nghiên cứu, tối ưu hóa điều kiện chuyển hóa các hợp chất nitơ và photpho, nghiên cứu và lựa chọn giá thể, chất mang phù hợp cho sự hình thành màng sinh học của các chủng nghiên cứu.
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PHỤ LỤC
1. Trình tự gen mã hóa 16S rARN của vi khuẩn B11.11 được đọc trên máy đọc trình tự tự động ABI PRISM 3100 Avant (Hoa Kỳ) .
GTGCCTAATACATGCAAGTCGAGCGGACCGACGGGAGCTTGCTCCCTTA GGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGA CTGGGATAACTCCGGAAACCGGGGCTAATACCGGATGCTTGATTGAACC GCATGGTTCAATCATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGA CCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACG ATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACA CGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGG ACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGAT CGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGG CACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCA GCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTA AAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCA ACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAG AGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAAC ACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGA AAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTA AACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGC AAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAA TTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAAC CCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATG GTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTG ACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCA TGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAG
GGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTT CGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACC GCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACC TTTTGGAGCCAGCCGCCGAAGGTGGGACAGA
2. Trình tự gen mã hóa 16S rARN của chủng B21.10 được đọc trên máy đọc trình tự tự động ABI PRISM 3100 Avant (Hoa Kỳ).
AGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAG ATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTG GGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATA CCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGG CTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAA CGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGC CACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTA GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGA GTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAG TGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCAC GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTG TCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGA TGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAA CTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATG CGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGT AACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATAC CCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTC CGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTA CGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGT CTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCA GAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG
GGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTC AGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGG GGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGC TACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATC CCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTG AAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT TCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACAC CCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGGGA CAGA
3. Trình tự gen mã hóa 16S rARN của chủng B23.2 được đọc trên máy đọc trình tự tự động ABI PRISM 3100 Avant (Hoa Kỳ).
AGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGA AGGGAGCTTGCTCCTGGATTTAGCGGCGGACGGGTGAGTAATGCCTAGG AATCTGCCTGGTAGTGGGGGATAACGTTCCGAAAGGAACGCTAATACCG CGTACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCA GATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAG GCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTG AGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGAC AATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTT CGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGTTGTACGTTAATACC GTGCAATTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCC AGCAGCCGCGGTAATACGAAGGGTGCAAGCGTTAATCGGAATTACTGG GCGTAAAGCGCGCGTAGGTGGTTCAGTAAGTTGGAAGTGAAATCCCCGG GCTCAACCTGGGAACTGCTTTCAAAACTGCTGAGCTAGAGTACGGTAGA GGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGG AACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGT GCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGC CGTAAACGATGTCAACTAGCCGTTGGGATCCTTGAGATCTTAGTGGCGC AGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAA
ACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT AATTCGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAAC TTTCCAGAGATGGATTGGTGCCTTCGGGAACTCAGACACAGGTGCTGCA TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACG AGCGCAACCCTTGTCCTTAGTTACCAGCACCTCGGGTGGGCACTCTAAG GAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCA TCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACA AAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGT AGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAG TAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACA CACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCT AACCTTCGGGGGGACGGTACCACGGAG
4. Trình tự gen mã hóa 16S rARN của chủng A4.2 được đọc trên máy đọc trình tự tự động ABI PRISM 3100 Avant (Hoa Kỳ).
CTCTGCTCAGGACGAACGCCGGCGGCGTGCCTAATACATGCAAGTCGAG CGGACCGACGGGAGCTTGCTCCCTTAGGTTAGTGGCGGACGGGTGAGTA ACACGTGGGTAACCTGCCTGTAAGATTGGGATAACTCCGGGAAACCGGG GCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATCATAAAAGGTG GCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGG TGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGG TGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG CAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACG CCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGA AGAACAAGTACCGTTCGAATAGGGCGGCACCTTGACGGTACCTAACCAG AAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGG CAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCT TAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAA CTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCG GTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCT
CTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGG ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTA GAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCC TGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCC CGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAAC CTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCT TCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGT GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTG CCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAG GAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTAC ACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTA AGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCG ACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGT GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTT TGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCCGAAGT GGACAGAATGACTCA