Chapter VI Overview of rnethanogenic archaeal diversity of Loner lake Chapter VI

6.1. Introduction Diversity studies were only conferred to conventional culture based methods till 1980's. Eventhough the culture based methods are better for its own reasons, due to the difficulties in culturing every microscopic organism, the diversity studies were resulted in limited coverage. Many molecular techniques have made possible to describe the microbial diversity without using culture based studies. However, as explained in section 1.4.4, there are limitations in molecular microbial ecological studies. Hence it is worthwhile conducting culture dependent and independent studies and compiling the data while investigating any given ecological niche. Culture independent and dependent studies of methanogenic Archacal diversity en \^ Lonar sediment samples were described in the previous chapters. The culture independent studies on lake sediment samples showed the presence of eight methanogenic archaeal phylotypes from Lonar lake. The phylogenetic analysis of the methanogenic enrichment cultures show 13 methanogenic archaeal phylotypes. Most of these phylotypes found in culture independent studies on sediment samples. However, phylotypes Hke CH9, CH42, CH84, CH71 and CA41 were only found in enrichment cultures. Microscopic observation of enrichment cultures established at neutral pH showed entirely different morphological types i.e. cultures using acetate and methanol showed presence of Methanosarcina. The isolation of methanogens resulted in nine pure cultures. Molecular identification of isolates revealed that they belong to three genera i.e. , Methanoculleus and Methanosarcina. In this chapter the sequences obtained in the culture independent and dependent studies were analyzed together to find the overall diversity of the methanogenic of Lonar lake.

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6.2. Phylogenetic analysis The 16S rDNA sequences obtained during the studies on sediments, enrichment cultures and isolates were taken for the phylogenetic analysis. The designation of clones or isolates and there accession numbers were given in the table (sediments, enrichments, and isolates). The closest sequences of reference were retrieved from the GenBank and alignment was performed using the Clustal W software (www.ebi.ac.uk/ClustalW) (Thompson et al., 1994). Ambiguously aligned portions were deleted from the matrix using DMABE software (Xia and Xia, 2000). The phylogenetic trees were constructed by using neighbor-joining method (Sitou, 1987) and distance matrices were calculated by Jukes-Cantor (Jukes, 1969), using MEGA version 3.1 (Kumar et al., 1933).

Table: 6.1. Different phylotypes observed from Lonar Lake during culture independent and dependents studies. The phylotype numbers are based on the 16S rDNA libraries established with sediment and enrichments cultures. The comparison among these libraries and over all diversity of the methanogenic archaea of Lonar lake is described in the table. 6.2.

Recovered clones from sediment Recovered clones from Recovered as samples enrichment cultures Isolates Phylotype Clone Phylotype Clone Isolate (accession number) (accession number) I LR2 (DQ302470) I CH2(DQ513409) LAI (DQ987520) II LR13 (DO302471) II CHI3(DQ513410) LH2 (D0987521) III LR64 (DQ302469) III CH9(D0513411) LA4 (DQ987522) IV LR71 (DQ302473) IV CH42(D0513412) LAS (DQ987523) V LR84 (DO302472) V CH71 (DQ513413) LA7 (DQ987524) VI LRIOO (DQ302461) VI CH50(DQ513414) LA2 (DQ987525) VII LR102(DQ302462) VII ClIS4(DQ513415) LA3(D098/526) VIII LRlll (DQ302463) VIU CH95(DQ5134I6) LA6 (DQ987527) IX CA42(D0513417) LM5 (D0987528) X CA41 (D0513418) XI CA100(DO513419) XII CA52(D0513420) XIII CT3 (DQ513421)

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6.3. Results and Discussion Twenty one partial and nine almost full length 16S rDNA sequences obtained in the present study were compared with their closest relatives. Only 430 bases length was used in the phylogenetic analysis. Less than 99% sequence similarity was considered as separate phylotype. The phylogenetic analysis revealed that 15 methanogenic phylotypes are present in Lonar lake (Table. 6.2). Phylotypes 02, 03, 04, 07, 09, 10 and 12 showed affiliation to Methanocalculus species. Phylotypes 01, 05, 06, 08 and 13 showed affiliation to Methanocidleus species. The phylotype 12 {Methanosarcina sp. LM5) belong to Methanosarcina. The phylotypes 14 (LR64) and 15 (LR84) are very distantly related to Methanocalculus species. The phylotypes 04 and 05 are also distantly related to their closest culturable species (<95% similarity). These phylotypes may belong to novel lineages. The clones CA107, CA52, CA41 and CAIOO, obtained in the sediment phylogenetic analysis, showed high similarity (98%) to the 16S rDNA of Methanocalculus chunghsingensis (98%). The isolates obtained in the present studies such as LAI, LA2, LA3, and LA6 showed 100% similarity with those of the cultures enriched on acetate (Table. 6.1). The present isolates differ from Methanocalculus chunghsingensis by being from alkaline environment and their ability to grow at higher pH and NaCl concentration. Hence these isolates or may be one of them may turn novel species. The isolates (LA1-LA7) showed much close similarity with each other, for example, isolate LAI and LA2 had shown 98.5% similarity. However, the physiological studies have shown much difference (Table. 5.3). Based on physiological and molecular variations these seven isolates have been grouped into four strains. Viz; LAI, LA2, LA3 and LA6. The affiliation of the isolates to species or strain level can be done after performing other molecular studies like, DNA-DNA hybridization. Several studies have used 3% sequence dissimilarity to find out the phylotypes in any given niches. But in the present chapter 1% sequence dissimilarity was used, because of the physiological variations between the phylotypes (Table 5.3). The clones LR-100, LR-102 and LR-111 found in sediments (Table. 6.1, also see the Table. 3.5) were not found in enrichments. Several attempts on culturing of such clones turned unsuccessful. These clones are still uncultured to date.

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Clones such as CH9, CH42, CH84, CH71 and CA41 were found in enrichment cultures (Table. 6.1, and also see the Table. 4.1) but not in sediments. The corresponding organisms of these clones might be very less number in the sediment samples. Hence, the 16S rDNA of these organisms was not amplified as it is a very common bias of PCR amplification. The number of these organisms rose, when enriched in a habitat simulating media along with organic rich components. So these were only found in enrichment cultures. Few of these organisms i.e., CH9 and CH42 (LA5 and LA7) were isolated and characterized (The physiological studies were given in the Chapter V).

As explained in the Chapter IV trimethylamine was used in the culture based studies for isolation of methylotrophic methanogens. Methanol, di-methylamine and mono-methylamine were also used in the studies. Different substrates used in combination. Results showed successful enrichment cultures. The TMA enrichment culture clones are also observed with H2:C02 enrichment culture clones. Only one unique clone was observed in TMA enrichment culture phylogeny i.e. CT3. However, phylotype CT3 is showing close affiliation with MethanocuUeus species which is a hydrogenotrophic methanogen. Jones et al., (1998) reported that methanogenic Archaea so far identified in soda lakes are obligately methylotrophic. The studies showed that most of the methanogenic archaeal community in the Lonar belongs to hydrogenotrophic methanogens (Surakasi et al., 2007). Worakit et al., (1986) reported an alkaliphilic, hydrogenotrophic methanogen, Methanobacterium alkaliphilum, from Wadi el Natrun. The present studies and Worakit's reports are evidencing the presence of hydrogenotrophic methanogens in soda lake environments apart from the methylotrophic methanogens.

Finally, Lonar lake environment accommodates acetoclastic methanogens such as Methanosarcina species and hydrogenotrophic methanogens such as MethanocuUeus and Methanocalculus species. It also harbor few unexplored methanogens such as clone LR64, LR84 and CA41. Most of the methanogenic archaeal species (phylotypes 07-12 and 13-15, Table.6.2) from Lonar lake are still uncultured. As evidenced with sediments and enrichment culture phylogenetic

Surakasi Venkata Prasad, 2007, Ph.D Thesis 106 Chapter VI analysis no obligate methylotrophic methanogens were present in Lonar soda lake environment. Methanogenic archaeal diversity of Lonar lake

Number of methanogenic phylotypes 15

Number of acetoclastic phylotypes 01

Methanosarcina mazei, strain LM5

Number of Hydrogenotrophic phylotypes 14

Methanoculleus sp 05

Methanocalculus sp 07

Novel lineages 02

Because of the cultured dependent studies few species like Methanosarcina, Methanoculleus clones Ci-i84, CHI3 and CH71 and Methanocalculus clones CA41 and isolates LA3 and LA6 were duly recovered from Lonar lake. The culture based studies also predicted that the organisms are alkaliphilic and halotolerant. Particularly, the isolates had shown much wide tolerance to high salt and alkalinity. The isolates withstand 8% of Na2C03 (80 grams of Na2C03 was added to adjust the pH of the medium to 10.8) and 12% NaCl. Presently the NaCl and COs^concentrations are much lower than earlier (Table. 3.4). From the above two statements it can be said that the organisms are now adapting to grow at low concentrations of NaCl and NaaCOa. Rather these organisms were proper halophiles. Want et al., (2006) reported wide diversity of bacteria also. As explained earlier, (section 4.4) a serious threat is ached for the lake environment. If the lake last its uniqueness we will miss extremophilic and wide diverse micro flora from Lonar Lake. It is also needed to correlate the microbial diversity with ecology. Quantitative and qualitative estimation of not only archaea and bacteria but also algae and other eukaryotes are necessary for this purpose.

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Table: 6.2. Overall diversity of Methanogenic Archaea from Lonar Lake and recovery of phylotypes in enrichment and isolations. It describes phylogenetic affiliation of the clones from sediment and corresponding clones in enrichments and isolation.

Phylotypes Clone numbers Clone Isolates Nearest neighbor (% in sediment numbers in similarity) of the enrichment clones and isolates cultures present in the Lonar lake* 01 LR-2 CH2 LH2 Methanoculleus sp IIEl (97%) 02 LR-102 CA42, CA52 LAI Methanocalculus sp01F9702c (98%) 03 LR-111 CA41,CA52, LA4, Uncultured Archaeal CA107 LAS, clone PL-22A1 ( LA2, LA7 97%) 04 NR CH9,CH42 LAS, LA7 Uncultured Archaeal clone PL-3SA 05 NR /—iiQ ^ ^i t-t -^ NR Uncultured Archaeon clone GW70-10-35 (92.6) 06 NR CH71 NR Uncultured euryarchaeon (98.4) 07 NR CA41 NR Methanocalculus pumilus (97.7%) 08 LR-13 CH50 NR Uncultured Archaeal clone PL-9A5 (93%) 09 LR-100 CAIOO NR Uncultured Archaeal done PL-22A11 (97%) 10 NR NR LA3 Methanocalculus chunqhsingensis 11 NR NR LA6 Methanocalculus ch unghsingensis 12 NR NR LMS Methanosarcina mazei 13 LR-71 NR NR Methanoculleus submarinus (97%) 14 LR-64 NR NR Methanocalculus halotolerance (94%) 15 LR-84 NR NR Methanocalculus pumilus (94%)

* The nearest neighbor of the phylotypes was found when analyzed by BLASTn and RDPII. NR = not recovered in respeclve study.

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LR102 LA48F CA52 CA107 81 - LR100, CA100 -LAI CA41 34 LA3 LR111,LA2, LA7, LAS 66— —LA6 Methanocalculus chunghsingensis (AY234332) 92n AY026256 54- CH9 28 28 CH84, LR84 34 r "-CHQS '—Methanocalculus halotolerance (AF033672)

£6- 54 Methanocalculus taiwanensis (AF172443) Methanocalculus pumilus (AB008853) 61 Uncultured archaeon clone PI35A9 (AY570680) J8- "- Uncultured archaeon clone PI22A1 -a&- Methanocorpusculum parvum (M59147)

-CH13 00302" 71 • Methanoculleus thermophllus (AJ862839) Methanoculleus chikogeonsis (AB038795) 10 83 5^ r^£ ' Methanocu/teu s sp IIE1 (AF531178) AF028693 56 |—CH42 'Methanoculleus submannus (AB089178) 43 36 rLR71,CH71 38 '|LR2, LH2, CH2

39 h-CT3 ^^ '-CH50

-LM5

0.02

Fig. 6.1 The phylogenetic relationship of Lonar lake-methanogenic phylotypes based on distance matrix analysis of 16S rDNA gene sequences. The sequence alignment was performed using CLUSTAL W program and tree were constructed using the Neighbor-Joining with Kimura 2 parameter distances in MEGA 3.1 software (Kumar, 1994). Bootstrap values (1000 replicates) are shown at the nodes. LR, series clones are obtained from sediment analysis, LA, LH and LM series are from isolates and CA, CH and CT scries are from enrichment cultures.

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