Consideraciones Finales

PCR of 16S rRNA gene fragments followed by clone library construction yielded DNA sequence data. The data were analyzed and cleaned by removing chimera

sequences as mentioned in the methods section (see Section 3.2.1.6). Following the chimera check, the data were submitted to RDP seqmatch query (Cole et al., 2003; Cole et al., 2007; Cole et al., 2009; Maidak et al., 2000). Interestingly it was found that many of the sequences obtained were classified under the domain Bacteria. The reason behind obtaining bacterial clone for the primers specific to the domain Archaea may be that the archaeal primers might have annealed to the bacterial genes present in the PCR product due to the lower annealing temperature used. This suggested that the PCR program used needs further fine-tuning. The clones classified under the Bacteria domain were not considered for further analysis. Table 3.2 shows the summary of the chimera-and bacteria-free archaeal clones obtained for each sample.

Table 3.2: Number of Good Archaeal Sequences Obtained DNA

Sample

Number of Clones Sequenced

Number of Good Sequences Obtained, Classified Under Domain Archaea

UCD-1 96 66

TD-10%-1 96 68

CD-10%-1 96 57

C2a 96 55

Rarefaction curves were constructed using the DOTUR program (Schloss et al., 2005). Rarefaction curves are based on empirical calculations that help to estimate if the number of samples sequenced provides an accurate estimate of the diversity present in the population. The rarefaction value increases with each new OTU obtained in the sample (Kemp and Aller, 2004).

Figure 3.3: Rarefaction Curves for The Archaeal Clone Libraries

Figure 3.3 shows that for the given library sizes, TD-10%-1 and UCD-1 clone libraries attained nearly complete coverage, whereas C2a and CD-10%-1 clone libraries did not attain complete coverage for a library size of 57 and 55, respectively. This indicates that the archaeal communities of C2a and CD-10%-1 were more diverse than the archaeal communities of UCD-1 and TD-10%-1.

SCHAO1 and SACE provide estimates of the number of clones expected in a clone

library (Kemp and Aller, 2004) and Good’s C provides another estimate of the amount of coverage obtained using the number of unique clones and the total library size (Good,

0 2 4 6 8 10 12 0 10 20 30 40 50 60 70 Nu m b er o f O T Us

Number of clones (Library Size)

UCD-1

TD-10%-1

CD-10%-1

C9C2a

1953). Richness indices like SCHAO1, SACE and Good’s C are sensitive to the number of

rare OTUs and so must be applied with caution but the confidence intervals associated with them give a better clue of accuracy of the richness estimation (Riviere et al., 2009).

Table 3.3 shows SCHAO1, SACE and Good’s C estimates for the archaeal clone

libraries. Again, all the estimates indicated that archaeal diversity of UCD and TD-10%-1 was covered by the library size of 66 and 68, respectively, whereas there were still

archaeal OTUs to be detected in samples CD-10%-1 and C2a. According to the SCHAO1

estimates, approximately 78% of the archaeal diversity of sample CD-10%-1 was covered, whereas only 36% of the archaeal diversity of sample C2a was covered. According to SACE estimates, 64% of the archaeal diversity of sample CD-10%-1 was

covered whereas 36% of the archaeal diversity of sample C2a was covered. Good’s C estimates indicated 91 and 87% coverage for samples CD-10%-1 and C2a respectively.

It is known that the SACE estimator yields smaller coverage values than SCHAO1

and Good’s C estimates since “it is derived from a larger subset of the complete frequency distribution than SCHAO1 estimator” (Kemp and Aller, 2004). Therefore, the

SACE estimator is particularly appropriate for data sets in which some phylotypes (OTUs)

occur more frequently. On the other hand, the SCHAO1 estimator is derived from the

number of phylotypes appearing either one or two times in a given library so it is particularly appropriate for data sets in which most phylotypes are relatively rare in the library (Kemp and Aller, 2004). In the case presented herewith, the SACE estimator was

found to be an appropriate choice due to skewed distribution of clones among the OTUs and because more than two clones were often associated with an OTU (explained in following paragraphs and Figure 3.4).

Table 3.3: SCHAO1, SACE and Good’s C Estimates for Archaeal Clone Libraries

Observed Number of

OTUs

SCHAO1 SACE Good's C

Predicted Number of OTUs* Predicted Number of OTUs UCD-1 5 5 (5-5) 5 (5-5) 1.00 TD-10%-1 4 4 (4-4) 4 (4-4) 1.00 CD-10%-1 11 14 (12-33) 17 (12-43) 0.91 C2a 12 33 (17-98) 33 (16-127) 0.87 *

values in the parenthesis indicate 95% confidence interval

The Shannon-Weaver index is a measure of the diversity within a sample (Shannon and Weaver, 1963). Higher Shannon-Weaver index values indicate a higher diversity of microbes in a clone library and vice versa.

Simpson’s Index gives another estimate of the diversity in a sample. The index value is the probability of two individual clones randomly selected from a sample belonging to the same OTU. A higher Simpson’s Index value indicates a less diverse sample and vice versa (Schloss et al., 2005).

Table 3.4 shows Shannon-Weaver and Simpson’s diversity indices for the archaeal clone libraries obtained by using DOTUR program (Schloss et al., 2005). Culture C2a had the highest archaeal diversity among the samples tested, followed by

CD-10%-1, whereas the DNA sample from TD-10%-1 and UCD-1 had lower archaeal diversity.

Table 3.4: Shannon-Weaver and Simpson’s Indices for Archaeal Clone Libraries

HShannon HSimpson

UCD-1 0.76 0.63

TD-10%-1 0.93 0.51

CD-10%-1 1.57 0.34

C2a 1.94 0.17

Both digesters UCD-1 and TD-10%-1 had lower archaeal diversity than CD-10%- 1 and C2a. Moreover, both UCD-1 and TD-10%-1 performed better than CD-10%-1 in terms of methane production and effluent SCOD removal. Interestingly since C2a was used as the bioaugmentation culture and was introduced in TD-10%-1 on a daily basis, TD-10%-1 was also expected to have a higher diversity of archaea similar to culture C2a, but this was not the case.

All the diversity and coverage estimated indicated that both UCD-1 and TD-10%- 1 had lower diversity than CD-10%-1 which indicates that the better function of

anaerobic digesters digesting milk waste may require less diverse, but a dedicated, archaeal community.

Functional organization (fo) of microbes in a sample can be represented by Pareto-

Lorenz (P-L) evenness curves (Marzorati et al., 2008) (see Section 1.2.2.4). Figure 3.4 represents P-L curves for the archaeal clone libraries. Interestingly, functional

organization of all the archaeal clone libraries fell into the medium category (Marzorati et al., 2008). Moreover, the fo values of C2a and TD-10%-1 were about 0.6, whereas the fo

values of UCD-1 and CD-10%-1 were 0.8 and 0.7 respectively. This means archaeal community distributions in the libraries C2a and TD-10%-1 were more even as compared to the distribution of the clones in UCD-1 and CD-10%-1.