Anexo 2: Denominaciones de calidad diferenciada

5.1. Introduction

As described in Chapter 3, methanogen cultivation studies have been undertaken in order to understand the types of Methanomassiliicoccales present in the ovine rumen (Jeyanathan 2010). The Methanomassiliicoccales enrichment cultures displayed a common coccoid morphology and lacked autofluorescence at 420 nm in these putative methanogen cells (Jeyanathan 2010). Previous attempts were made to isolate the Methanomassiliicoccales organisms from the ovine rumen enrichment cultures, including 10-fold serial dilutions, application of various antibiotics and heat treatments and although pure cultures of Methanomassiliicoccales were not obtained, the bacterial inhabitants in the enrichment cultures were reduced in both number and diversity (Jayanathan, 2010). Analysis of the bacterial partner organisms in the enrichments showed a single strain of Succinivibrio dextrinosolvens (NR026476, 99% 16S rRNA gene identity) was present (Jeyanathan 2010), suggesting there may be specific interactions between this organism and Methanomassiliicoccales (Jeyanathan 2010). Additionally, a recent global rumen census project indicates a correlation between rumen Succinivibrionaceae with some rumen Methanomassiliicoccales

Recent studies have provided some insights into Methanomassiliicoccales metabolism. Methanomassiliicoccales carries out H2-dependent methylotrophic methanogenesis, utilizing mono-, di-, tri-methylamines, methanol and dimethyl-sulfide (Borrel et al. 2014). Despite the understanding gained from genomes of Methanomassiliicoccales of human and termite gut origin, it is important to study the Methanomassiliicoccales from the rumen in order to understand their function in the rumen and to assess their contribution to CH4 production in this environment. Therefore, studies were carried out on the ISO4-H5 Methanomassiliicoccales enrichment culture with the aim of attempting to isolate the methanogenic archaeon from the enrichment, to enable its growth requirements to be determined and to explore its response to different levels of H2 in co-culture experiments with the cellulose-degrading, H2-producing bacterium, Ruminococcus flavefaciens.

172 5.2. Results

5.2.1. Isolation of ISO4-H5

A S. dextrinosolvens strain from the ISO4-H5 enrichment culture was isolated previously by colony picking from BY plates and designated as S. dextrinosolvens strain H5 (Cox et al., pers. comm.) and was kindly supplied for this study. During growth studies of the S. dextrinosolvens H5 culture, pectin (1% w/v) was accidentally introduced to the ISO4-H5 enrichment culture. The culture was not discarded, but was monitored, and elevated CH4 production relative to the control culture without added pectin, was observed. Further testing was conducted on supplementation of the ISO4-H5 enrichment cultures with glucose (10mM) and pectin (1% w/v). CH4 production of ISO4-H5 enrichment cultures were stimulated by both glucose and pectin supplementation, however, pectin supplementation stimulated CH4 production earlier and to a greater extent at day five (P =5.6E-05) than glucose (P =1.4E-02) (Figure 5.1.).

Figure 5.1. Stimulation of CH4 production in ISO4-H5 enrichment cultures triplicates by glucose or pectin supplementation. All replicates contain formate (60 mM), methanol (20 mM), acetate (20 mM), CoM (10 µM) and 0.1× vitamin mix. (‒◊‒) Control, (‒□‒) glucose (10 mM final concentration) or (‒Δ‒) apple pectin (1% w/v final concentration) was supplied to ISO4-H5 enrichment cultures, and CH4 production per mL of headspace was measured daily. Error bars represent +/- standard errors of the means (SEMs). ISO4-H5 growth control is the standard cultivation conditions for the ISO4-H5 enrichment culture previously described (Chapter 2, section 2.2.1.)

From these observations, it was hypothesized that pectin addition indirectly stimulated CH4 production by enhancing S. dextrinosolvens H5 growth, which in turn supplied a growth- limiting metabolite to the methanogenic archaeon in the ISO4-H5 enrichment culture. It was

0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 CH 4 pro du ct io n ( µm o le) Time (days)

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reasoned that such a growth-limiting metabolite was likely to be soluble and present in the supernatant of S. dextrinosolvens cultures grown on pectin. If so, the supernatant itself would allow the growth of the methanogenic archaeon in the absence of S. dextrinosolvens H5 cells, therefore S. dextrinosolvens H5 cultures were grown for two days in BY medium containing pectin (1% w/v). The bulk of S. dextrinosolvens H5 cells were removed by filtering through a 0.22 µm filter to remove cells, thus filter sterilising the preparation. Although previous attempts to remove S. dextrinosolvens H5 with antibiotic treatments were unsuccessful, the sensitivity of S. dextrinosolvens and the methanogenic archaeon in the ISO4-H5 enrichment culture to antibiotics was determined. The antibiotics were added to the ISO4-H5 enrichment culture, and the culture was also supplemented with glucose (10 mM final concentration) and growth of S. dextrinosolvens H5 after overnight incubation was recorded. The incubation was continued for seven days after which CH4 production from the antibiotic tested culture was measured (Table 5.1.).

Table 5.1. Antibiotic application to enrichment culture

Antibiotic Final concentration (µg/mL)

S. dextrinosolvens H5 growth overnight

CH4 production after 7

days

Ampicillin 50 Yes Yes

100 Yes Yes

250 No No

500 No No

Chloramphenicol* 50 No No

Erythromycin* 20 Yes Yes

Gentamycin 20 Yes No

Kanamycin 50 Yes Yes

Rifampicin# _{1.2 Yes Yes}

Spectinomycin 100 Yes Yes

Streptomycin 100 Yes Yes

Tetracycline* 10 Yes Yes

20 Yes Yes

50 Yes Yes

100 Yes Yes

* dissolved in ethanol. # dissolved in methanol at maximum antibiotic solubility.

Only chloramphenicol and ampicillin inhibited S. dextrinosolvens H5, while only gentamycin inhibited CH4 production in the ISO4-H5 enrichment cultures. In all cases of S. dextrinosolvens H5 inhibition, there was no CH4 detected after seven days of incubation, indicating that the methanogenic archaeon did not grow, possibly due to direct inhibition by the antibiotics, or possibly because ISO4-H5 did not survive in the absence of S. dextrinosolvens H5 growth.

The ampicillin results were particularly encouraging as archaea are insensitive to ampicillin, and suggested a concentration between 100 and 250 µg/mL might be useful for inhibiting S. dextrinosolvens growth. The isolation procedure used is outlined in Chapter 2 (Section 2.2.3, Figure 5.2.), but briefly it involved supplementing ISO4-H5 enrichments with cell-free, spent growth medium filtered from pectin-grown S. dextrinosolvens H5 cultures which is termed

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Succinivibrio Spent Pectin Growth Medium Supernatant (SSPGMS). In addition to supplementing filter-sterilised SSPGMS and with ampicillin and incubating the cultures until CH4 was detected in the headspace. The culture was then sub-cultured onto fresh medium containing the same supplements, while a parallel inoculum was introduced to medium containing glucose and incubated overnight to check for the presence of remaining S. dextrinosolvens. Initial attempts using 100 µg/mL ampicillin were unsuccessful and S. dextrinosolvens persisted in the enrichment culture. The ampicillin concentration was increased to 200 µg/mL, and the isolation procedure was repeated through three cycles of SSPGMS supplementation and ampicillin treatment before the glucose supplemented test cultures remained clear and CH4 was detected in the culture headspace. The putative pure methanogen culture consisted of coccoid cells that usually do not fluoresce at 420 nm. The putatively pure cultures were screened with PCR amplification using primers specific for both bacterial and archaeal 16S rRNA genes. The bacterial 16S rRNA PCR failed to produce an amplification product, while the archaeal 16S rRNA gene PCR produced a product of the expected size. The PCR product was sequenced and identified as a methanogenic archaeon, affiliated with the order Methanomassiliicoccales, and the culture from which this amplified DNA originated was designated as strain ISO4-H5.

Figure 5.2. Isolation procedure for ISO4-H5. SSPGMS: Succinivibrio Spent Pectin Growth Medium Supernatant. Antibiotics: 200 µg/mL ampicillin. Glucose: 10 mM. Length of incubation: one week.

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The morphologies of S. dextrinosolvens H5 and methanogenic archaeon ISO4-H5 were examined under transmission electron microscopy using negative staining of cultures, and ultrathin sectioning of ISO4-H5 was also carried out. The methanogenic archaeon ISO4-H5 has a thin, bi-layer cell membrane with no visible external features (Figure 5.3A.). An unidentified structure can be seen within the cell. The negatively stained S. dextrinosolvens H5 preparations show it possesses a single terminal flagellum (Figure 5.3B.), and some cells displayed an elongated spiral morphology (Figure 5.3C.). The size of ISO4-H5 was Figure 5.3. Transmission electron micrograph (TEM) of methanogenic archaeon ISO4-H5 and

S. dextrinosolvens H5. A. TEM of negatively stained thin section of the methanogenic archaeon ISO4-H5. B and C. TEM of negatively stained S. dextrinosolvens. The samples were prepared as previously described in Chapter 2. Images were captured using a Philips CM10 Transmission Electron Microscope, using an Olympus SIS Morada camera and SIS iTEM software (Germany).