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Investigation of CB and SH soils with various amendments and substrates revealed the chitinolytic potential of CB to be much greater than that of SH. This may be reflected

in the observations for the number of species detected in response to a-chitin amendment

in the 16S rRNA data. Number of detected OTUs increased ⇠53% in CB but decreased

⇠50% in SH compared to the respective unamended soil, implying a large diversity of

low-abundance chitin degraders in CB.

The basal chitinase activity in both soils was very low when unamended or amended with starch, although CB was still more active than SH. It is well established in the literature that cells at rest exhibit very little enzymatic activity (Nannipieri, 2006) and these observations support the view that relatively little chitin is present in soil. With

amendment ofa-chitin or b-chitin, the measured chitinase activity increased in both soils

(Figure 27), indicating that the soil bacteria can respond to take advantage of chitin when it is present.

With respect to the responses of CB and SH to a-chitin or b-chitin, activity towards a-

chitin was generally proportionally higher in SH compared tob-chitin (Figure 27), whereas

in CB it was substrate-dependent. CB, being a site under marine influence, does come

into contact with b-chitin-containing squid pen, whereas b-chitin may be a rare source of

chitin in SH. This observation may therefore reflect the biogeography of chitinases that are specific to the sources of chitin in their environment.

One would expect higher chitinase activity to be observed in samples amended with a-

chitin and starch due to increased microbial biomass. What was striking however, was the extent to which chitinase activity was increased in CB, as measured by the GlcNAc substrate, compared to SH. SH is expected to be a relatively nutrient rich environment compared to CB, due to nutrient input from ovine excreta (Hilder, 1964), therefore starch amendment would be more influential in CB.

3.6.2.1 Retained chitinolytic activity of Test Soil post-amendment The chitinolytic potential of the Test Soil was investigated from soil sampled 1 month, 6 months, and 12 months after the last amendment with prawn carapaces. The chitinolytic potential of the soil with both substrates inversely correlated with the time since the last amendment of the soil (Figure 29).

Once can postulate three explanations for this observation. Firstly, the large quantity of chitin applied to the soil induces the autochthonous chitinolytic population, which can sur-

vive offthe chitin for many months. This has been shown to be unlikely by the observation

of chitin being rapidly degraded in 1 month post-amendment TS.

Secondly, the blooming of the predominantly mycelial autochthonous chitinolytic popu- lation allows the bacteria to widely colonize the soil and reach previously unexploited resources. These resources can then sustain the chitinolytic bacterial population for many months before the ecosystem stabilizes. Finally, allochthonous bacteria that became asso- ciated with the carapaces either from the marine environment, by aeolian deposition, or contamination at the factory during processing or storage may be being introduced into the system. These bacteria will be exploiting the proteinaceous flesh left attached to the

carapace but also the chitin in the shell. Once the shell waste has been degraded the organisms may be outcompeted in the soil removing a previously chitinolytic population. Unfortunately, an area of the site which had not been systematically amended with car- apaces was not available for sampling, when conducted, so the native soil bacteria could not be investigated.

3.6.2.2 Confidence in the result Two soils, CB and SH, were each treated in one of 5

ways, amended with 1%a-chitin, 1%b-chitin, 1% starch, 1%a-chitin and 1% starch, or left

unamended. These microcosms were performed in biological duplicate. The heterogeneity of soil could prove problematic for the highly sensitive 4-MU assay. Previous research

has shown four 100 mg replicates per soil to be representative of chitinase activity with a

cv<15%64 (Miller et al., 1998). Therefore 4 samples from each biological replicate were

taken. The cv for the CB and SH samples were generally 24–40%, with far higher values

for low-activity samples asx¯_!0₎ cv ! 1, meaning the coefficient is sensitive to small

changes in the mean. This suggests that sampling wasn’t extensive enough to capture all the variations within the soil.

For the CB soil, variability between biological replicate microcosms amended with chitin alone was greater than the variation within the microcosms measured by subsampling (Figure 28). This suggests that the distribution of chitinolytic organisms within the soil is uneven and that small stochastic variation in the starting conditions of the mi-

crocosm are amplified during the incubation, resulting in differences in the relative abund-

ance of chitinolytic organisms that can be detected at the enzyme level. The activity of soils amended with starch exhibited little variation between microcosms but large vari- ation between subsamples. Starch can be metabolized by many soil organisms, so almost all will benefit from the amendment, despite uneven distribution within the microcosm. Chitinolytic bacteria not proximal to chitin could proliferate on the starch, increasing the likelihood that they encounter and hydrolyse the chitin. The subsampling variability is therefore a reflection of the uneven distribution of bacteria within the soil. There is less confidence in the interpretation of the SH soil data due to the comparatively small levels

64_c

of activity detected. Unlike CB, the activity for the biological duplicate microcosms and subsampled replicates are in general agreement.

3.6.2.3 Alternative methods Many alternative methods exist for the quantitative es- timation of chitinase activity in samples, including methods that employ coloured sub- strates: such as chitin azure (Remazol Brilliant Violet 5R) (Wirth and Wolf, 1990) and 4- Nitrophenol [Chitinase Assay Kit (CS0980), Sigma-Aldrich, MO, USA], and those that de- tect the breakdown products of a chitin-based substrate spectrophotometrically (Ghauharali-

van der Vlugt et al., 2009). The fluorometric assay used in this thesis employes 4-MU-

labelled chitinooligosaccharides and has been widely used in the literature with environ-

mental samples such as aquatic systems, peatlands, and soil (Milleret al., 1998).

Crude extracts from soil are not necessarily transparent at the wavelength absorbed or emitted by the assay substrate. Extract colour can vary between soils, but also within a soil depending on amendment (Figure 41). Because extracts may absorb non-specifically across a broad spectrum, assays that rely on the detection of a liberated dye or breakdown product spectrophotometrically or fluorometrically can introduce bias.

Figure 41: Different coloured extracts from SH soil during the metaXP extraction

There are drawbacks associated with assays based on small artificial substrates which can

both overestimate and underestimate chitinase activity. (GlcNAc)3 can act as accept-

ors in transglycosylation catalysed by chitinases. At high concentrations of substrates, transglycosylase activity by chitinases can link the substrates together to form longer chitinooligosaccharides which are then hydrolysed in a futile cycle that generates no new products and does not release the 4-MU—resulting in an underestimation of chitinase

isms can cleave the ester linkages between the 4-MU and chitinooligosaccharide using N-

acetylhexosaminidases, resulting in an overestimation of chitinase activity (Ferreiraet al.,

1993; Haran et al., 1995).

The substrates themselves are simpler than the presentation of chitin in nature, resulting in an overestimation of activity (Lindahl and Finlay, 2006). More physiological substrates

for chitinases are (GlcNAc)4-8(Ghauharali-van der Vlugtet al., 2009), but these substrates

are not commercially available in a 4-MU-labelled form and so were not used. The small substrate size also introduces the potential for lack of enzyme specificity. Several hydrolytic enzymes can cleave the glycosidic linkages in small chitinosaccharides such as cellulase,

hemi-cellulase, lysozyme, papain and pectinase (Overdijket al., 1999; Lianget al., 2007).

The larger substrates may also be sequentially cleaved, e.g. 4-MU-(GlcNAc)2 can be

cleaved byN,N�-diacetylglucosaminidases, but also twice by b-N-acetyl-glucosaminidases

to release the fluorophore, with the latter, the fluorophore is only released upon the second cleavage, thus underestimating activity.

Because of the heterogeneous nature of chitin, detailed enzyme kinetics are scarce in the

literature and kcatand Kmvalues65cannot be determined (Bokmaet al., 2000). The causes

of bias, both positive and negative, should be present in all samples; therefore the chitinase activity obtained using the 4-MU assay can still be used for comparison of the chitinolytic potential between soils.

The preferred assay was that developed by Molano et al. (1977). Radioactive chitin is

prepared by the acetylation of chitosan using tritiated acetic anhydride. By exploiting the insolubility of chitin and the solubility of the reaction’s products in water, activity against crystalline high molecular weight chitin can be measured over a desired period by recov- ery of the supernatant and removal of the radioactive unreacted chitin by centrifugation. Unfortunately, tritiated acetic anhydride was no longer commercially available in small quantities and would have required synthesis at great cost.

65_k

cat is the catalytic constant defined as Vmax/_Et or maximum rate achievable in the system over the

concentration of enzyme sites in the reaction. Kmis the substrate concentration at which the reaction