SENTENCIA EN PRIMERA INSTANCIA N° 01-2015: RESOLUCIÓN N° 25-2015

lignin-degrading bacterial strain, the ability to degrade a variety of aromatic compounds was examined by carrying out growth experiments in M9 salt solution supplemented with an aromatic compound or cellulose as the sole carbon source (0.02–0.1%w/v). The aromatic compounds used (Fig. 3.4) were biphenyl, vanillic acid, veratryl alcohol, m-cresol, p-cresol and ferulic acid. Growth in vanillic acid (2.0 % w/v) was initially monitored in liquid media, though growth was severely limited with only very slow growth of some of the strains in glucose after 336 hours. By comparison, growth on agar plates of identical media composition was faster with the appearance of colonies after 168 hours, therefore the remainder of the experiments were performed on agar plates.

! Figure 3.4. Structures of carbon sources: biphenyl (1), vanillic acid (2), veratryl alcohol (3), m- cresol (4), p-cresol (5), ferulic acid (6) and cellulose (7).

OH CH₃ CH₃ OH OH O OH O CH3 O O OH 1 ₂ 3 4 5 O O O HO OH HO OH O OH OH HO H₃CO COOH 7 6

The ability of the strains to grow on biphenyl and vanillic acid was of significant interest since biphenyl is a component of lignin [77] and R. jostii RHA1, a recently studied lignin-degrading strain, has been identified as a degrader of biphenyl [177]. Vanillic acid is an intermediate on the "–aryl ether cleavage pathway in S. paucimobilis SYK-6 [137]. Growth on veratryl alcohol was also of interest since an enzyme purified from Sphingobacterium sp. ATM was found to be specific towards this compound [170]. Growth on cellulose was monitored in order to examine the selectivity of the strains and find out whether they grow selectively on the lignin-related aromatic compounds in preference to cellulose, or they are also capable of metabolizing cellulose.

Another reason for carrying out the growth experiments is to examine the reliability of the sequence data by examining the growth patterns of different isolates that have identical BLAST results, e.g. the two M. phyllosphaerae strains A1.1 and A1.2. Note that strains A4.3 and C4.1 were not characterized further since O. anthropi, which has a sequence is highly similar to O. pseudogrignonense was discovered to be a Category 2 organism that is a potential opportunistic pathogen.bbbbbbbb………...bbbbbbbbb

Table 3.3.!Level of growth of environmental lignin-degrading bacterial strains on different carbon sources biphenyl (1.0gl-1), m-cresol (0.22gl-1), p-cresol (0.22gl-1), ferulic acid (1.0gl-1_{), vanillic acid (1.0gl}-1_{), veratryl alcohol (0.84gl}-1_{) and cellulose (0.5gl}-1_{), after 240hr at 30 or 45ºC. += no growth, ++= poor growth, +++=}

moderate growth and ++++= good growth. ND= not determined.

Strain biphenyl (1) vanillic acid (2) veratryl alcohol (3) m-cresol (4) p-cresol (5) ferulic acid (6) cellulose (7)

Microbacterium phyllosphaerae A1.1 ++ ++++ + + + +++ ++

Microbacterium phyllosphaerae A1.2 ++ ++++ +++ + + +++ ++

Microbacterium marinilacus A2.1 ++ +++ + + + ++ +++

Microbacterium marinilacus A3.1 ++ + + + + ++ +++

Ochrobactrum pseudogrignonense A4.3 + ++ +++ +++ + ND ND

Rhodococcus erythropolis A5.1 ++++ +++ +++ +++ +++ + ++++

Microbacterium oxydans A5.2 + + + + + ++ ++

Micrococcus luteus B5.3 ++ +++ +++ +++ +++ +++ +

Ochrobactrum rhizosphaerae C4.1 +++ +++ + + + ND ND

Microbacterium marinilacus C5.1 +++ +++ +++ + + +++ +++

Ochrobactrum rhizosphaerae D5.1 +++ +++ +++ + + + ND

Micrococcus luteus E1.1 +++ +++ +++ +++ +++ +++ +

Sphingobacterium sp. ++++ ++++ +++ ++ + ++ +

After 240 hours of incubation at 30 or 45 °C all strains had grown on one or

more of the aromatic carbon sources (Table 3.3), though most were selective for specific compounds. Good growth on biphenyl was observed for Sphingobacterium sp. and R. erythropolis, whilst growth of the Microbacterium strains was limited. A different pattern of growth was observed on vanillic acid, which was readily metabolized by the Microbacterium strains and Sphingobacterium sp. in addition to R. erythropolis. The good growth of Sphingobacterium sp. on biphenyl, vanillic acid and veratryl alcohol but not on cellulose suggests that this organism is selective for metabolism of the lignin component of lignocellulose. It is not surprising that R. erythropolis grew well on vanillic acid and biphenyl since R. jostii RHA1 is known to degrade biphenyl [177] and Rhodococcus strains are reported to degrade phenolic compounds [175, 176]. The moderate growth of the Ochrobactrum strains on biphenyl is also consistent with previous studies, which found that Ochrobactrum strains are capable of degrading polychlorinated biphenyls and polycyclic aromatic hydrocarbons [178, 179].

The identical M. phyllosphaerae strains A1.1 and A1.2 exhibited almost the same level of growth, the only difference being the moderate growth of A1.2 on veratryl alcohol in comparison with A1.1. The same degree of similarity in the results was observed with the two M. luteus strains B5.3 and E1.1 and the two M. marinilacus strains A2.1 and A3.1. However, the other M. marinilacus strain, C5.1, grew more readily on biphenyl and veratryl alcohol than A2.1 and A3.1. This slight difference in growth rate could be due to experimental error, since growth was monitored qualitatively, and/or a result of the difference in environmental conditions; C5.1 was isolated from heathland soil in a different

geographical region to A2.1 and A3.1, which were both isolated from soil in woodland locations.

It is clear that all strains have difficulty in degrading m-cresol and p-cresol; this observation is not striking since cresol-degradation pathways have been characterized in only a small number of isolates from environmental samples, mainly Pseudomonas species [162]. It is not surprising that R. erythropolis showed moderate growth on the cresols since Rhodococcus strains have also been reported to degrade phenolic compounds [175, 176].