Comparison of aerobic and anaerobic cellulose degrading microorganisms in sediments of Sippewissett Salt March

Transcription

1 Comparison of aerobic and anaerobic cellulose degrading microorganisms in sediments of Sippewissett Salt March Victoria Grießmeier Karlsruhe Institute of Technology (KIT) MBL Microbial Diversity Course

2 Introduction The microbial degradation of cellulose in plant material is a key step in the global carbon cycle. Cellulose is insoluble, therefore microorganisms require a specific set of enzymes in order to degrade extracellularly and make it available as carbon source for many other microorganisms, which are not capable to hydrolyze cellulose. This forms the basis of many microbial interactions in organic rich environments (Leschine, 1995). The key enzymes involved and excreted by cellulolytic organisms are termed cellulases. This diverse enzyme contains at least eleven different families called the superfamily of glycoside hydrolases (GH). These families comprise three classes of extracellular enzymes, catalyzing mainly three different reactions which target the polysaccharides in plants synergistically. Endocellulases are active on the internal ß-1,4 bonds in a cellulose chain; exocellulases cleave the nonreducing ends of cellulose and break it into smaller molecules of cellobiose and finally ß-glucosidases allow the hydrolysis of cellobiose to glucose (Fig.1) (Gupta, Samant and Sahu, 2012). Fig. 1: The three types of enzymes involved in the breakdown of cellulose to glucose. Source: majordifferences.com Microbial strains which only possess the genes belonging to the most abundant families GH1 and GH3 are considered to be opportunistic cellulose degraders, as these families contain mostly ß- glucosidases. Therefore, these organisms are dependent on other lineages which are capable of 2

3 degrading the longer polymer to cellobiose, which can then be utilized. Phyla with genes for families GH5 and GH48 can be considered as potential cellulose degraders, as GH5 is quite abundant and expresses mainly genes for endo- and exocellulases (Berlemont and Martiny, 2013). The degradation of cellulose, in addition to the microorganisms involved, differs between aerobic and anaerobic environments. In addition, the diversity of microorganisms in anaerobic environments is considered to be greater than those present in aerobic environment. Aerobic organisms secrete a set of cellulases with a carbohydrate binding module (CBM), whereas anaerobic microorganisms which use a large multienzyme complex called the cellulosome. Nonetheless, the enzymes produced by aerobic or anaerobic cellulose degrading microorganisms belong to the same families (Wilson, 2011), which is important for the aim of this project. The objective of this report was to find differences in the distribution in aerobic and anaerobic sediments of Sippewissett Salt Marsh with regards to the abundance of cellulases produced and the phylogenetic analysis of the microbial diversity. This was aimed to be realized via qpcr targeting genes for three different families of enzymes: GH3, GH5 and GH48. The quantification of these three families should show if there is a difference between potential cellulose degraders (GH5/GH48) and opportunists (GH3) in these two different environments. Moreover, a clone library for the cellulose degrading genes as well as isolation approaches under aerobic and anaerobic conditions was proposed to be performed to learn more about the microbial diversity of these organisms. 3

4 Material and Methods Sample Collection Sediment samples were taken in Little Sippewissett Salt Marsh, Falmouth MA at August 2 nd. Aerobic sediments were sampled using cores from the main channel inflow to upper little Sippewissett. The sample was exposed to air with algae present on top. Anaerobic samples were taken in the upper submerged side channel of little Sippewissett, where a large amount of organic material was present. For both samples ph, salinity and temperature was measured on site. Fig. 2: Sample sites at little Sippewissett salt marsh. Left the aerobic and right the anaerobic sediment samples. Sediments were taken in the afternoon at low tide with cores and stored in a saltwater tank. The aerobic sample was rinsed with seawater for aeration. DNA Isolation DNA was isolated from both samples (aerobic and anaerobic) using the QIAamp PowerFecal DNA Kit (Qiagen) with 0.25 g starting material. DNA concentration was measured with QuantiFluor One dsdna System and the Quantus Fluorometer (Promega) following the manufacturer protocols. Primer design and PCR (qpcr) In order to identify GH family 3, 5 and 48 and compare it between the two different sample sites three different primer sets were used. For the detection of family 48, the primer combination cel48_490f and cel48_920r and for the family 5, the combination cel5_392f and cel5_754r were used (Pereyra et al., 2010). For the glycoside hydrolase family 3 a new primer set was developed using the reference protein from Clostridum cellulovorans (GenBank: ADL ). An alignment BLAST search against the National Center for Biotechnology Information (NCBI) database was performed to find organisms with an acceptable conservation of the same gene. The GH3 protein sequence from seven different organisms with similarity to the reference protein were downloaded from NCBl and aligned using a 4

5 MUSCLE alignment with default algorithm within the software Jalview. Conserved amino acid regions were searched manually and it was decided that position 185 be used for the forward and 276 for the reverse primer. This results in nucleotide sequence length of the gene fragment of 273 bp. The sequence of the degenerated primers is presented in Table 1. Tab. 1: Primer sequence targeting the family 3 glycoside hydrolases. Target and primer Sequence (5-3 ) Family 3 glycoside hydrolases cel3_v_185f cel3_v_276r AAYATHATHGAYATG CARTTYGARTTYGGN PCR was performed with isolated environmental DNA from the Sippewissett salt marsh and for comparison with genomic DNA from Trunk River and Cedar Swamp. For all 50 µl PCR reactions a temperature gradient for the annealing step was performed from 47 C to 60 C. Moreover, the input of DNA amount ranged between 1-3 µl. A Streptomycetes isolate form the Microbial Diversity Course from 2017 also served as a positive control for the primer pairs. Isolation of cellulose degrading microorganisms For the isolation, two different types of media were used. One with carboxymethyl cellulose (CMC) (ATCC medium 2720) containing 1 g (NH 4) 2SO 4, 1 g MgSO 4 x 7H 2O, 1 g CaCl 2 X 2H 2O, 0.2 g FeCl 3, 2 g casitone, 15 g carboxymethyl cellulose, 15 g Agar and 950 ml distilled water. The ph was adjusted to 7 after autoclaving with a sterile filtrated solution of K 2HPO 4. The second media used filter paper (Whatsman) instead of CMC as cellulose source and contained moreover 1 g K 2PO 4, 20 mg yeast extract, 14 g Agar, 1g KNO 3, 1 g MgSO 4 x 7 H 2O, 1 g CaCl 2x 2 H 2O, 0.1g MnSO 4 x 7 H 2O, 0.2 FeCl 3 and filled up to 1 l with distilled water. The ph was adjusted to 7 with 1M NaOH. Three filter paper strip were placed on the gelated plates and inoculated. A dilution series of 0.1 g starting material of the anaerobic and aerobic sediment (same as for DNA isolation) was performed up to µl of each dilution step as well as the undiluted sample were plated on each medium type and either incubated aerobically at 30 C or anaerobically at room temperature. After the appearance of colonies, plates were flushed with a sterile congo red solution (1mg/ml) and incubated for 15 minutes. Afterwards the plates were washed with a 1 M NaCl solution. Colonies showing a clearing zone were picked and plated out on new CMC plates and incubated at 30 C. A 16S rrna gene colony PCR was performed with the primer set 8F and 1391R for all isolates and sequenced afterwards. Sequencing results were aligned against the NCBI database to find the closest related organisms. 5

6 Moreover, colonies isolated aerobically were incubated anaerobically to test their capability to different environmental situations. Growth experiment Three isolates (5, 8, 11) with a slightly different appearance were chosen for growth experiments. The first experiment should show if the isolates really degrade cellulose and instead of just growing on casitone. For this a 96-well plate with different casitone concentrations was prepared (0, 0.2, 0.4, 0.8, 1 and 2 g/l). As cellulose is insoluble, cellobiose (15g/l) was used instead for this experiment. Isolate 5, 8 and 11 were picked from the plate and inoculated directly into the wells with the different casitone concentrations. Every one to two hour the OD 600 of the isolates as well as from the blank was measured with a plate reader (Promega). For the second growth experiment the same three isolates were used (isolate 5, 8 and 11) and inoculated in a 24-well plate with a basic CMC medium but without CMC and casitone. Filter paper (Whatsman) or either wood chips were used instead of CMC as cellulose source for all three organisms (Fig. 3). Next to the negative controls, without the addition of isolate, another negative control with an orange colored isolate from a actinomycetes isolation approach was performed to see if also different organisms could grow and degrade cellulose. Moreover, one sample contained all three organisms together to see if the growth and degradation of cellulose is increased or inhibit by the coexistence of these microorganisms. Fig. 3: Experimental setup for the second growth experiment with either wood chips or filter paper as cellulose substrate. After an incubation time of 48h the wood chips and filter paper were stained with DAPI and afterwards analyzed with a confocal Zeiss LSM 880 Airyscan microscope to analyze and visualize the biofilm of the isolates on the different cellulose sources. Emission was measured in the range of nm and 410 to 515 nm (DAPI). 6

7 Results and Discussion Sample site and DNA extraction The measurement of the abiotic parameters on site and the DNA isolation for the two different sampling sites brought the following results: Tab. 2: Abiotic parameters measured on site for the aerobic and anaerobic sample. DNA was extracted and measured in the lab. Aerobic site Anaerobic site Salinity Temperature ph DNA concentration [ng/µl] These abiotic parameters were important to know for the following isolation approaches and the DNA concentration for the PCR reactions. PCR All PCR reactions failed for aerobic and anaerobic sediments from Little Sippewissett for all three primer sets. Several temperature gradients for the annealing temperature were performed as well as different concentrations of genomic DNA. Beside the environmental samples, also the PCR reaction for the possible positive control (Streptomycetes) failed. The negative PCR results for the three target genes made a quantitative analysis of the cellulose degrading enzymes via qpcr impossible. Also, the attempt to make a clone library for these genes to learn more about the diversity of cellulose degrading organisms could not be pursued further. The failure of the different PCR reactions may be due to other reasons. First, the abundance of the genes in the environmental sample is not high enough or the genes may be absent. Also, the overall concentration of DNA may have not been high enough and so the GH genes could not have been captured. It also could be that there are other GH families present than GH3, 5 and 48, which are responsible for the cellulose degradation. Because the genes for the GH families are phylogenetic widely distributed and show low conservation, another reason could be that the primer sets were too specific targeting only a few organisms, which are not present in the environmental samples. Further PCR reactions with genomic DNA from Trunk River and Cedar Swamp were performed to ascertain not 7

8 only the samples from Sippewissett are inappropriate for this approach, but also that these PCR reactions failed. Moreover, the protein sequence of these three genes were aligned against the contigs of the metagenome dataset of Cedar Swamp from 2016 to see if the gene was present in these days. For the GH48 three contigs could be found and after performing an NCBI BLAST search a consensus nucleotide sequence of 100bp could be found with an identity of 87% to Paenibacillus sp., showing that at least this gene was present at this time. The search for GH3 and GH5 against the Cedar swamp metagenome contig for certain microorganisms failed, but this may also be due to the low conservation of these genes. Isolation of cellulose degrading microorganisms Overall 18 isolates could be identified from the aerobic plates, which showed a clearing zone after the congo red staining (Fig. 4). Fig. 4: Aerobic CMC plates with congo red staining. Left undiluted sample, middle 10-1 dilution and right 10-2 dilution. 18 colonies showed a clearing zone and were picked for further analysis. All 18 isolates were sequenced and for nine of them the sequencing was successful. All isolates had a 97% to 99% % sequence identity with the NCBI database to Pseudomonas species, preferentially Pseudomonas putida (Abb.5). 8

9 Fig. 5: Phylogenetic distribution of the nine sequenced and isolated organisms. Seven showed as closest related organism from the NCBI database Pseudomonas putida. The alignment was performed using MUSCLE and the tree was built with the Maximum Likelihood method. Other studies have already shown that Pseudomonas putida is a cellulolytic microorganisms, which is also capable of degrading lignin and can deal with high concentrations of toxins released from the biomass (Mulakhudair, Hanotu and Zimmerman, 2016). A cellulolytic activity of P. parafulva is not known so far. On the media with the cellulose filter no growth could be detected within the 2 week of incubation. A plausible reason for this result may be that the degradation of the filter paper needs different enzymes than using CMC. Moreover, the media contained no casitone, which could influence the growth of organisms. Before knowing the sequencing results, all aerobic isolates were also incubated anaerobically, but no growth could be detected after 7 days so far. From the anaerobic plates, only one organisms from the undiluted sample showed a clearing zone after the congo red staining, but the 16S rrna gene PCR failed several times for this isolate and therefore no phylogenetic result is available. Growth experiments The CMC media contained a high concentration of casitone (2 g/l), which exists mainly of amino acids and enhances the growth of microorganisms. Two different growth experiments should be performed to ensure that the isolated organisms are not growing with casitone instead of cellulose. Fig. 6: Growth curve for the three different isolates (5, 8, 11) with different concentrations of casitone (0, 0.2, 0.4, 0.8, 1 and 2 g/l). 9

10 Fig. 6 shows a considerably increased growth (OD close to 0.4) for all three organisms with a casitone concentration of 2 g/l. Organisms growing without casitone showed a decreased growth rate and did not reach an OD of 0.1 after 22h. This could lead to the assumption that casitone is essential for the growth of these isolates, because of the obvious enhanced growth. The organisms do not have to produce every single amino acid individually, as it is already present in the media. Another explanation could be possible, why there was no growth without casitone. For this experiment the cellulose substrate was changed from CMC to cellobiose, because of its solubility in water. It could be, that the organisms are not able to use cellobiose, because of a lack of the ß-glucosidase enzyme and hence grow instead on casitone. Moreover, the HPLC results confirmed that the cellobiose was not used, because after 22h the starting concentration of cellobiose (15 g/l) was still present and no amounts of glucose or galactose were produced. For this reason, another growth experiment was started to observe if the isolates could better deal with crystalline cellulose like from filter paper or wood chips. This time the OD could only be measured at the end of the experiment, because the substrate had to be taken out of the well, because of the insolubility of filter paper and wood in the media (Tab. 2). The highest growth could be observed for all three isolates together with the wood chips substrate. Because no casitone was added to this experiment it confirms that these three organisms can also grow with wood as cellulose source and do not grow alone on casitone. Nevertheless, the HPLC results showed no available amounts of glucose or galactose which should be produced after cellulose degradation, meaning that one possibility could be, that it is already further metabolized. The growth experiment with filter paper instead showed only a little growth, meaning that this is not the appropriate cellulose source for these organisms. The wood chips offer a wider range of usable polysaccharides than the filter paper and therefor offer more organisms the possibility to grow. Tab. 2: OD of all isolates after 48h with different cellulose substrates. The sample with all three organisms showed the best growth. The DAPI staining of the cells and microscopy with the confocal microscope should whether the isolates are in fact present and growing in the sample and also if they are attached to the surface of the cellulose substrate. 10

11 Cellulose exhibits autofluorescence with excitation maximum at 365 nm and emission maximum at 440 nm within the same wavelength as DAPI. Nevertheless, the wood chip shows also an emission between 490 nm and 562 nm (green) (Fig. 7). Fig. 7: Autoflourescence of the wood chips in the wavelength of 490 to 562 nm. The inoculated and DAPI stained samples should show an emission for cells only around 410 nm to 515 nm (blue). The wood material instead exhibits in the blue and green light. Therefore everything, which appears blue and at the same time green is wood material, whereas bacterial cells should only appear in blue. The DAPI staining brought suitable results for the wood chip samples, especially for the sample with all three isolates together (Fig. 8 top right). This picture shows a lot of DAPI stained cells and leads to the assumption that a decent growth of the isolates occurred. Besides, it appears that the cells are attached to the surface of the wood chips. The negative control (Fig. 8 top left) shows how the inoculated sample appears. Everything here appearing in blue or green should be wood material. The sample with only one isolate showed fewer DAPI stained cells, which would be conform with the OD measurement. The same applies to the sample with the cellulose filter paper for all three organisms. The OD in this sample was only 0.03 and so was it also difficult to find DAPI stained cells under the microscope. 11

12 Fig. 8: Top left: Negative control, DAPI stained but no inoculation with isolates. Top right: Sample with all three isolates. Bottom left: Sample with one isolate. Bottom right: Cellulose paper with all three organisms. Conclusion This investigation showed that Pseudomonas putida could be isolated frequently from sediments of Sippewissett salt marsh and shows an enhanced growth with CMC and casitone but at the same time it can also grow with wood material and without castione. The lack of growth of P. putida strains on filter paper showed that they possess a quite specific substrate range for cellulose using either wood material or CMC. The investigation of the microbial diversity of cellulose degrading organisms in this approaches relies mainly on the isolation approach, because of the failure of the PCR reaction for the genes encoding GH families. Because of the short time range the main selection focused on fast growing organisms. In further investigations, the design of primer pairs for GH families could be increased to detect a wider 12

13 range of celluase-encoding genes. Moreover, a next generation sequencing approach for functional GH genes is appropriate to detect a greater diversity of cellulose degraders. The isolation approach could be expanded using more variant media types with higher and lower nutrient concentrations and different types of cellulose. Acknowledgments I want to thank all of the TAs, especially Matthew Tien for the helpful discussions. Scott Dawson for the support in the molecular room, Kyle Costa for the primer design and Georgia Squyres for the help on the confocal microscope, as well as Kurt Hanselmann for taking me out to the sampling site and all the other interesting ideas belonging the isolation. And of course all of the other TAs, staff and students for helping me with my project. References Berlemont, R. and Martiny, A. C. (2013) Phylogenetic distribution of potential cellulases in bacteria, Applied and Environmental Microbiology. doi: /AEM Gupta, P., Samant, K. and Sahu, A. (2012) Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential, International Journal of Microbiology. doi: /2012/ Leschine, S. B. (1995) Cellulose degradation in anaerobic environments, Annu. Rev. Microbiol., 49, pp Mulakhudair, A. R., Hanotu, J. and Zimmerman, W. (2016) Exploiting microbubble-microbe synergy for biomass processing: Application in lignocellulosic biomass pretreatment, Biomass and Bioenergy. doi: /j.biombioe Pereyra, L. P. et al. (2010) Detection and quantification of functional genes of cellulosedegrading, fermentative, and sulfate-reducing bacteria and methanogenic archaea, Applied and Environmental Microbiology. doi: /AEM Wilson, D. B. (2011) Microbial diversity of cellulose hydrolysis, Current Opinion in Microbiology. Elsevier Ltd, 14(3), pp doi: /j.mib