Bacterial diversity in crude oil contaminated soil of Assam: culture-dependent and independent evaluation

Biology and Fertility of Soils

Reshita Baruaha,b, Shashank Kumar Mishrab,c, Dipak Jyoti Kalitad ,Yumnam Sillaa, Puneet Singh Chauhanc, Anil Kr. Singha, Hari Prasanna Deka Boruaha*

aBiotechnology Division, CSIR-North-East Institute of Science and Technology, Jorhat, Assam-785006, India

bAcademy of Scientific and Innovative Research, New Delhi

cDivision of Plant-Microbe Interaction, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India

dDepartment of Chemistry, OIL, Duliajan

* Hari Prasanna Deka Boruah, Biotechnology Division, CSIR-North-East Institute of Science and Technology, Jorhat, Assam-785006, India

E-mail:, , Telephone: 0376-237295, Fax:03762370011

Fig S1: Comparison of Dehydrogenase, Alkaline phosphatise, Neutral phosphatise and Urease enzyme activity of contaminated sites

Data were mean of three individual observation with three replication each, Eroor bars= standard error means of observed values, error bars followed by similar letter are not significantly different from each other at P<0.01 level according to tukeys test. CC1-J-5, CC2-J-6 and GDS-J-9 had highest alkaline phosphatase and neutral phosphatise. Soil dehydrogenase activity was seen to be highest for the samples CC1-J-5, GDS-J-9 and CTF-D2-2 whereas urease activity was seen to be highest in samples NCC-J-8, Core-10 and PWS-DGU-7

Fig 2: Shannon diversity index (H) and species richness (R) for contaminated sites under study based on DGGE banding profile data

From the DGGE fingerprint, it was observed that site PWS-J-4 having the lowest degree of crude oil contamination (0.04%), showed the maximum species richness and hence the highest diversity. The values of Shannon diversity index and species richness were seen to be not significantly different from each other.

Table S1: Influence of hydrocarbon contaminated soil properties and total bacterial counts

Soil properties / Viable bacterial count in different media (cfu/log10)
NA / R2A / Pikovskaya / M1+ 2%(v/v) crude oil
r / p-value / r / p-value / r / p-value / r / p-value
TPH (%) / 0.196372 / 0.5866 / 0.118627 / 0.7441 / 0.529132 / 0.1158 / 0.674741 / 0.0323
pH / 0.21174 / 0.5570 / 0.411784 / 0.2371 / 0.418775 / 0.2284 / 0.248395 / 0.4889
Moisture content (%) / 0.335776 / 0.3429 / 0.12798 / 0.7246 / 0.029085 / 0.9364 / 0.475511 / 0.1648
Organic Carbon (%) / 0.474694 / 0.1657 / 0.01909 / 0.9583 / 0.36778 / 0.2958 / 0.714725 / 0.0202
Dehyderogenase (µg/g fresh soil/hour) / 0.318957 / 0.3690 / 0.021424 / 0.9532 / 0.161608 / 0.6556 / 0.483993 / 0.1564
Neutral Phosphatase (µg/g fresh soil/hour) / 0.279631 / 0.4339 / 0.009328 / 0.9796 / 0.036147 / 0.9210 / 0.280112 / 0.4331
Alkaline Phosphatase (µg/g fresh soil/hour) / 0.163549 / 0.6517 / 0.052565 / 0.8853 / 0.188237 / 0.6025 / 0.246112 / 0.4931
Urease (µg/g fresh soil/hour) / 0.203721 / 0.5724 / 0.075852 / 0.8350 / 0.338826 / 0.3382 / 0.000425 / 0.9991

Correlation coefficients (r) of the physico‐chemical properties with the viable bacterial counts in was done to see the influence of hydrocarbon with total count. p-values in bold are different from 0 with a significance level α=0.05 (n = 10). Among the soil properties measured, soil TPH (%) and soil organic carbon (%) showed a significant positive correlation with the viable bacterial counts on crude oil supplemented media (r = 0.67, 0.71).

Table S2: Bacterial strains identified in the present study with the accession numbers obtained

Strains number / Strain name / Accession numbers / % identical / Identical to
R1 / Enterobacter hormaechei / KF843695 / 99 / JN993998.1
R2 / Enterobacter hormaechei / KF843696 / 100 / KC342256.1
R3 / Enterobacter sp. / KM459022 / 97 / KF864674.1
R5 / Pseudomonas putida / KF843697 / 99 / FM211694.1
R6 / Clostridium sp. / KM459023 / 99 / JF312736.1
R7 / Lysinibacillus sphaericus / KM459024 / 99 / KM070813.1
R8 / Enterobacter sp. / KF843698 / 99 / KF360068.1
R9 / Enterobacter cloacae / KF843699 / 99 / KC764987.1
R11 / Enterobacter hormaechei / KF843700 / 99 / KC342256.1
R15 / Enterobacter sp. / KM459025 / 97 / JF799886.1
R16 / Pseudomonas stutzeri / KJ499784 / 95 / HM030754.1
R17 / Bacillus sp. / KJ499785 / 99 / KM517569.1
R18 / Lysinibacillus sp. / KJ499786 / 99 / KJ878614.1
R19 / Bacillus megaterium / KJ499787 / 99 / EU221370.1
R20 / Bacillus cereus / KJ499788 / 100 / KM405334.1
R21 / Bacillus sp. / KJ499789 / 99 / JF933914.1
R22 / Bacillus cereus / KJ499790 / 99 / KC211998.1
R23 / Pseudomonas plecoglossicida / KJ499791 / 100 / KF848498.1
R24 / Bacillus sp. / KJ499792 / 100 / KF962978.1
R25 / Bacillus aryabhattai / KJ499793 / 99 / KM817271.1
R26 / Bacillus sp. / KJ499794 / 99 / KF814108.1
R27 / Bacillus sp. / KJ499795 / 96 / DQ453811.1
R28 / Lysinibacillus sp. / KJ499796 / 96 / JF693981.1
R29 / Bacillus sp. / KJ499797 / 94 / JX198550.1
R30 / Bacillus anthracis / KJ499798 / 99 / KM888109.1
R31 / Bacillus methylotrophicus / KU752799 / 99 / KT719986.1
R32 / Bacillus subtilis / KU752800 / 97 / KT441081.1
R33 / Bacillus pumilus / KU752801 / 99 / JF834071.2
R34 / Bacillus subtilis / KU752802 / 98 / KT719485.1
R35 / Bacillus methylotrophicus / KU752803 / 99 / KT441081.1
R37 / Bacillus methylotrophicus / KU752804 / 99 / KT441081.1
R38 / Bacillus subtilis / KU752805 / 98 / KT719485.1
R39 / Pseudomonas putida / KU752806 / 89 / FJ950560.1
R40 / Bacillus subtilis / KU752807 / 98 / KM884947.1
R41 / Bacillus methylotrophicus / KU752808 / 99 / KP058543.1
R42 / Bacillus subtilis / KU752809 / 99 / KT343640.1
R43 / Bacillus methylotrophicus / KU752810 / 98 / KT441081.1
R44 / Bacillus cereus / KU752811 / 98 / KX181393.1
R45 / Bacillus cereus / KU752812 / 99 / KJ554997.1
R46 / Bacillus cereus / KU752813 / 99 / KF928687.1
R47 / Bacillus methylotrophicus / KU752814 / 98 / KT441081.1
R48 / Bacillus amyloliquefaciens / KU752815 / 99 / KF465562.1
R49 / Bacillus subtilis / KU752816 / 99 / KT719485.1
R50 / Bacillus methylotrophicus / KU752817 / 99 / KX181393.1
R51 / Bacillus megaterium / KU752818 / 99 / KU862330.1
R52 / Bacillus subtilis / KU752819 / 99 / KX037158.1
R53 / Bacillus megaterium / KU752820 / 99 / KX037158.1
R54 / Bacillus altitudinis / KU752821 / 99 / KM405335.1
R55 / Bacillus subtilis / KU752822 / 99 / HQ388813.1
R56 / Bacillus pumilus / KU752823 / 97 / EU071560.1
R57 / Bacillus pumilus / KU752824 / 90 / KP895574.1
R58 / Bacillus aryabhattai / KU752825 / 91 / HF570085.1
R59 / Bacillus pumilus / KU752826 / 94 / KC771042.1
R60 / Bacillus subtilis / KU752827 / 98 / KM884947.1
R61 / Bacillus sp. / KU752828 / 78 / KF863825.1
R64 / Bacillus subtilis / KU752829 / 99 / KC834381.1
R65 / Bacillus tequilensis / KU752830 / 97 / KT945024.1
R66 / Bacillus safensis / KU752831 / 93 / KJ741254.1
R71 / Bacillus cereus / KU752832 / 99 / JX296361.1
R73 / Paenibacillus sp. / KU752833 / 99 / KU850993.1
R74 / Bacillus subtilis / KU752834 / 98 / JN366763.1
R76 / Bacillus cereus / KU752835 / 94 / KJ769216.1
R77 / Bacillus cereus / KU752836 / 99 / KM885305.1
R78 / Bacillus thuringiensis / KU752837 / 97 / KT982229.1
R79 / Bacillus cereus / KU752838 / 97 / KT982229.1
R80 / Bacillus sp. / KU752839 / 99 / KF110088.1
R81 / Bacillus thuringiensis / KU752840 / 99 / KT963447.1
R82 / Bacillus amyloliquefaciens / KU752841 / 98 / HM480309.1
R84 / Lysinibacillus fusiformis / KU752842 / 99 / CP015224.1
R85 / Bacillus subtilis / KU752843 / 94 / KT720079.1
R86 / Lysinibacillus sp. / KU752844 / 99 / CP015224.1
R87 / Lysinibacillus sphaericus / KU752845 / 99 / CP015224.1
R88 / Lysinibacillus fusiformis / KU752846 / 99 / CP015224.1
R89 / Pseudomonas putida / KU752847 / 99 / KT825741.1
R90 / Lysinibacillus sp. / KU752848 / 99 / CP015224.1
R91 / Pseudomonas fluorescens / KU752849 / 98 / KP126779.1
R92 / Lysinibacillus sp. / KU752850 / 99 / CP015224.1
R93 / Exiguobacterium sp. / KU752851 / 98 / KU686690.1
R94 / Lysinibacillus sp. / KU752852 / 99 / CP015224.1
R95 / Pseudomonas koreensis / KU752853 / 98 / KT767892.1
R96 / Lysinibacillus sphaericus / KU752854 / 99 / CP015224.1
R97 / Bacillus methylotrophicus / KU752855 / 99 / KX181393.1
R98 / Bacillus cereus / KU752856 / 87 / KJ878582.1
R99 / Lysinibacillus fusiformis / KU752857 / 99 / KF228922.1
R100 / Bacillus methylotrophicus / KU752858 / 99 / KT441081.1
R101 / Bacillus flexus / KU752859 / 99 / HM755559.1
R102 / Bacillus cereus / KU752860 / 99 / HM755559.1
R103 / Lysinibacillus sp. / KU752861 / 98 / FJ696635.1
R104 / Bacillus methylotrophicus / KU752862 / 99 / KR296931.1
R105 / Bacillus subtilis / KU752863 / 97 / KT719485.1
R106 / Pseudomonas sp. / KU752864 / 99 / KR559744.1
R107 / Bacillus sp. / KU752865 / 99 / KT583542.1
R108 / Bacillus subtilis / KU752866 / 99 / CP015004.1
R109 / Bacillus cereus / KU752867 / 99 / CP014783.1
R110 / Lysinibacillus fusiformis / KU752868 / 99 / CP015224.1
R111 / Bacillus sp. / KU752869 / 99 / KJ936975.1
R112 / Lysinibacillus fusiformis / KU752870 / 99 / KT371357.1
R114 / Bacillus sp. / KU752871 / 99 / KF956606.1
R116 / Bacillus sp. / KU752872 / 99 / JX847614.1
R117 / Bacillus sp. / KU752873 / 82 / KT719610.1
R118 / Bacillus cereus / KU752874 / 99 / KT728833.1
R119 / Bacillus pumilus / KU752875 / 99 / KX037116.1
R120 / Bacillus methylotrophicus / KU752876 / 99 / KX181393.1
R121 / Lysinibacillus sp. / KU752877 / 97 / KX037123.1
R122 / Bacillus subtilis / KU752878 / 99 / KX037119.1
R123 / Bacillus subtilis / KU752879 / 99 / KX181393.1
R126 / Bacillus aerophilus / KU752880 / 96 / KM401870.1
R127 / Lysinibacillus sp. / KU752881 / 100 / KX037123.1
R130 / Bacillus badius / KU752882 / 96 / KU177224.1
R131 / Bacillus badius / KU752883 / 99 / KU177224.1
RP1 / Clostridium sp. / KU752884 / 95 / JN093130.2
RP2 / Bacillus subtilis / KU752885 / 99 / KX181393.1
RP3 / Lysinibacillus sp. / KU752886 / 99 / CP015224.1
RP4 / Lysinibacillus sp. / KU752887 / 100 / KX037123.1
RP5 / Lysinibacillus sp. / KU752888 / 100 / CP015224.1
RP6 / Lysinibacillus fusiformis / KU752889 / 94 / KT371357.1
RR1 / Bacillus thuringiensis / KU752890 / 99 / KR822283.1
RR2 / Lysinibacillus sp. / KU752891 / 100 / CP015224.1
RR3 / Bacillus sp. / KU752892 / 95 / FR821125.1
RR4 / Lysinibacillus sp. / KU752893 / 99 / CP015224.1
RR5 / Bacillus cereus / KU752894 / 96 / KU372122.1
RR7 / Lysinibacillus sp. / KU752895 / 99 / CP015224.1
RC1 / Alcaligenes faecalis / KF843701 / 99 / KF641851.1
RC2 / Bacillus subtilis / KF953985 / 100 / KF641841.1
RC3 / Enterobacter cancerogenus / KJ499799 / 99 / HQ407292.1
RC4 / Enterobacter sp. / KJ499800 / 99 / HQ246280.1
RC5 / Alcaligenes sp. / KF953984 / 99 / KF432837.1
RC6 / Bacillus aryabhattai / KM459019 / 99 / KC503992.1
RC7 / Bacillus stratosphericus / KM505010 / 99 / KM091705.1
RC8 / Bacillus flexus / KM505011 / 99 / KM076598.1
RC9 / Bacillus amyloliquefaciens / KJ499801 / 99 / HE774679.1
RC10 / Bacillus subtilis / KM459020 / 99 / JQ954840.1
RC11 / Pseudomonas stutzeri / KJ499802 / 99 / KF850545.1
RC12 / Bacillus licheniformis / KJ499803 / 99 / CP005965.1
RC13 / Bacillus pumilus / KM459021 / 99 / KJ608548.1
RC14 / Bacillus subtilis / KJ499804 / 99 / KF929418.1

Table S3: Sequence similarities of prominent Operational Taxonomic Units (OTUs) obtained from DGGE for the contaminated sites under study

Operational taxonomic unit (OTU) / Accession no. of the obtained OTUs / % Identical / Accession no. of closest GenBank relative
Uncultured bacterium Duliajan oil DGGE band 2.20 / KT186590 / 99 / KC884580.1
Uncultured Sphingomonas DGGE band 2.12 / KT186591 / 100 / LC053269.1
Uncultured Ralstonia DGGE band 2.16 / KT186592 / 99 / JQ433928.1
Uncultured Sphingobium DGGE band 3.6 / KT186593 / 99 / KC884582.1
Uncultured bacterium Duliajan oil DGGE band 3.15 / KT186594 / 100 / KC884570.1
Uncultured Massilia aurea Duliajan oil DGGE band 3.18 / KT186595 / 100 / LM994750.1
Uncultured Acinetobacter DGGE band 5.19 / KT186596 / 97 / KM243756.1
Uncultured Pseudomonas DGGE band 6.1 / KT186597 / 100 / HG322886.1
Uncultured Pseudomonas mendocina DGGE band 6.2 / KT186598 / 100 / KP282824.1
Uncultured Pseudomonas alcaliphila DGGE band 6.4 / KT186599 / 99 / KJ573571.1
Uncultured Pseudomonas mendocina DGGE band 8.B / KT186600 / 100 / KP282824.1
Uncultured Pseudomonas mendocina DGGE band 6.X / KT186601 / 99 / KP282824.1
Uncultured bacterium Duliajan oil DGGE band 9.5 / KT186602 / 100 / KC521869.1
Uncultured bacterium Duliajan oil DGGE band 9.11 / KT186603 / 99 / EU491331.1
Uncultured Pseudomonas DGGE band 9.A / KT186604 / 99 / JN221506.1
Uncultured Sphingomonas DGGE band 10.9 / KT186605 / 99 / KJ958399.1
Uncultured Pseudomonas mendocina DGGE band 10.D / KT186606 / 100 / KP282824.1

Prevalence of genera Sphingomonas, Ralstonia, Sphingobium, Massilia, Acinetobacter and Pseudomonas in the contaminated sites. In addition, many of the unidentified clones were seen to have 99%-100% similarity to bacterium clones reported earlier by researchers from other oil contaminated sites like the Dagang oil fields of China.