NMR and MALDI-TOF MS based characterization of exopolysaccharides in anaerobic microbial aggregates from full-scale reactors

Anaerobic granular sludge is composed of multispecies microbial aggregates embedded in a matrix of extracellular polymeric substances (EPS). Here we characterized the chemical fingerprint of the polysaccharide fraction of EPS in anaerobic granules obtained from full-scale reactors treating different types of wastewater. Nuclear magnetic resonance (NMR) signals of the polysaccharide region from the granules were very complex, likely as a result of the diverse microbial population in the granules. Using nonmetric multidimensional scaling (NMDS), the 1H NMR signals of reference polysaccharides (gellan, xanthan, alginate) and those of the anaerobic granules revealed that there were similarities between the polysaccharides extracted from granules and the reference polysaccharide alginate. Further analysis of the exopolysaccharides from anaerobic granules, and reference polysaccharides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that exopolysaccharides from two of the anaerobic granular sludges studied exhibited spectra similar to that of alginate. The presence of sequences related to the synthesis of alginate was confirmed in the metagenomes of the granules. Collectively these results suggest that alginate-like exopolysaccharides are constituents of the EPS matrix in anaerobic granular sludge treating different industrial wastewater. This finding expands the engineered environments where alginate has been found as EPS constituent of microbial aggregates.


Extraction of crude EPS
Granular sludge was briefly washed twice in phosphate buffer saline (pH 7) to remove nongranular material. Then, 10 mL of 0.1 M NaCl was added to 3-5 g of granular sludge and the granules were mechanically disrupted using a pestle to obtain a slurry. Immediately after adding 10 mL of water, formaldehyde was added to a final concentration of 0.4% (v/v) and the slurry was incubated at 4°C for 1 h. Then, 10 mL of 0.1 M NaOH was added and the slurry was incubated at 4°C for 3 h. The sample was then centrifuged (12,000 × g, 20 min at 4°C) and the supernatant filtered (0.45 m) to recover the (crude) extracted EPS. This EPS solution was neutralized with 1M HCl.

DNA extraction from the granules
Prior to DNA extraction, a slurry of the granules was prepared by mechanical disruption in PBS buffer. Genomic DNA was extracted, in duplicate, from 0.3 to 0.4 ml of slurry using the Power Soil DNA isolation kit (MoBio, Inc.) according to the manufacturer's protocol except that two cycles of bead beating for 30 s (each cycle followed by 30 s on ice) were used to improve cell lysis. The duplicate DNA extracts were then pooled and the DNA concentration was measured using a NanoDrop-1000 spectrophotometer (Thermo Scientific, Waltham, MA).

Quantitative PCR
Quantitative PCR was conducted in triplicate using a CFX96 real time detection apparatus (Bio-Rad Laboratories, Hercules, CA). Each 25 l reaction contained 12.5 l of iQ SYBR Green Super Mix (Bio-Rad Laboratories, Hercules, CA), 300 nM of each forward and reverse bacterial or archaeal primers as defined below and 2 l of template DNA. Cycling conditions were: 3 min at 95 o C, 40 cycles of 30 s at 94 o C, 30 s at 56 o C (53 o C for archaea) and 30 s at 72 o C. After cycling, a melting curve analysis from 50 to 95 o C was conducted to verify the specificity of the reactions. To quantify copy numbers of bacterial and archaeal 16S rRNA genes, standard curves for bacteria and archaea were constructed for each qPCR run by using dilution series of known concentrations of purified bacteria and archaea amplicons amplified using the same primer sets (described below) and genomic DNA from Geobacter sulfurreducens and Methanosarcina barkeri, respectively.

DNA amplification and 454 pyrosequencing of 16S rRNA gene amplicons
Bacterial and archaeal 16S rRNA gene fragments were amplified using primers B-341F (5'-CCTACGGGNGGCWGCAG -3') and B-805R (5'-GACTACHVGGGTATCTAATCC -3') for Bacteria, and A-519F (5'-CAGCMGCCGCGGTAA -3' ) and A-1017R (5'-GGCCATGCACCWCCTCTC-3') for Archaea. These primer combinations were selected as they result in high domain coverage and specificity based on a recent study. 1 Extracted DNA was amplified in triplicates using platinum PCR SuperMix High Fidelity Taq DNA Polymerase reagent, primer concentrations were 300 nM each and DNA template was 20 to 40 ng. The thermocycling consisted of an initial step at 94°C for 2 min, followed by 25 cycles at 94°C for 30 s, 56°C (53 o C for archaea) for 40 s, and 72°C for 60 s, with a final extension at 72°C for 7 min. Products were checked for correct length by gel electrophoresis.
For pyrosequencing, a second round of PCR 2 was performed for which the 454adaptor sequence A (5'-ccatctcatccctgcgtgtctccgactcag-3') followed by a sample specific 8 nt barcode sequence were attached to the 5' end of the forward primers, while the 454-adaptor sequence B (5'-cctatcccctgtgtgccttggcagtctcag-3') was attached to the 5' end of the reverse primers. The barcodes we used 3 met the following criteria 4 : (i) differed by at least 3 nt, (ii) did not contain homopolymers, (iii) the end nt of the adapter differed from the start nt of the barcode, (iv) the end nt of the barcode differed from the start nt of the primer and (v) barcodes had a similar GC content. The 50 l PCR mixture contained 45 l of Platinum PCR SuperMix High Fidelity Taq DNA Polymerase reagent (Invitrogen, Carlsbad, CA) and 500 nM of forward primer, 300 nM of reverse primer and 2 l of template. The thermocycling consisted of an initial step at 94°C for 2 min, followed by 12 cycles at 94°C for 30 s, 56°C for 40 s, and 72°C for 60 s, with a final extension at 72°C for 7 min. Products of triplicate reactions were pooled for each granule sample and then purified using the QIAquick kit PCR purification kit The 16S rRNA gene fragments were pyrosequenced at the Biosciences Core Laboratory at KAUST using a Roche 454 GS FLX sequencer and Titanium reagents according to the manufacturer's protocols.

Analysis of 16S rRNA gene pyrosequencing data
Sequence analyses were conducted using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (http://qiime.org) as described by Caporaso et al. 5 Briefly, sequences were quality filtered by eliminating reads that were shorter than 200 bp, had a quality score lower than 25, and did not perfectly match the PCR primer and the barcode sequence. After denoising, sequences were clustered into operational taxonomic units (OTUs) at ≥97% sequence similarity using Uclust 6 as implemented in QIIME. After selecting a representative sequence for each OTU, sequences were aligned using PyNAST 7 and their taxonomic identity was assigned using the RDP classifier (Wang et al. 2007) with the Greengenes (v 10-12) as reference database. Chimeric sequences were detected using ChimeraSlayer 8 and removed from further analysis. Then, a phylogenetic tree was constructed using the chimeric-free representative sequences and a table of OTU counts per sample was generated.

Metagenomic analysis
DNA extracted as specified above was sequenced using the Roche GS-FLX titanium sequencer. After removing low quality reads and ambiguous bases, unassembled sequences were uploaded into the MG-RAST server for annotation 9 . The annotation of reads was done using the IMG database of MG-RAST, using the default parameters i.e., cutoff expectation value (E) of 10 -5 , a minimum alignment length of 15 aminoacids or base pairs and a minimum identity cutoff of 60%. # A proteomic analysis as described by Thomas et al. 10 of the EPS extracts, did not show the presence of the cytosolic enzyme glucose-6-phosphate-dehydrogenase The presence of this enzyme in EPS extracts has been associated to a high extent of cell lysis during the extraction procedure. 11 The absence of this enzyme thus suggests that limited or no cell lysis occurred during the EPS extraction from the granular sludges. Also P/C ratios < 2 and low contents of extracellular DNA (e.g. < 0.6 mg DNA (gVSS) -1 ), as measured in this study, exclude significant occurrence of cell rupture during EPS extraction. 12,13 Figure S1. 1 H NMR spectra of reference polysaccharides and from exopolysaccharides extracted from anaerobic granules.

Multivariate analysis of MALDI-TOF MS spectra
The MALDI-TOF MS spectra peak list data was used for analysis. For the alginate and granules samples the following manual filter was used due to the noisy signals from 600 to 1600 m/z range. In this region, and for these samples, only the peaks with the highest intensities were retained. From the rest of the m/z range, all peaks were retained. Spectra were normalized related to the maximum peak of each spectrum. However, in further studies, more refined and automated processing of raw spectra should be investigated. Other studies have demonstrated the use of MALDI-TOF MS combined with multivariate analysis (e.g., MDS) to identify bacterial species 16 , characterize the proteolysis of cheese 17 and keratin in furs 18 .

Microbial community of the anaerobic granules
Bacteria belonging to the phylum Proteobacteria, and to the classes Clostridia and Bacteroidia are known to produce exopolysaccharides. [19][20][21] All these bacterial types were present in the granular sludges ( Figure S5a). -Proteobacteria (33%) and specifically the family Geobacteracea (17%) were most abundant in the AnG B granules. The identity of exopolysaccharides material produced by this family is not known. It was postulated that Geobacter species can directly (i.e., cell-to-cell) transfer electrons to methanogens during the anaerobic conversion of ethanol. 22 , which may explain its high abundance in these granules treating brewery wastewater, although this physiological trait warrants further investigation. Regarding the paper-mill AnG E granules, Bacteroidia (29%) and Clostridia (16%) were among the main bacterial types. The first bacterial class may degrade polysaccharides and other complex substrates to sugars 23 , while the latter may be involved in fermentation reactions 24 and their abundance may correspond with starch being the main carbohydrate in the wastewater of the paper-mill reactor. 25 Conversely, the main bacteria in the thermophilic AnG W granules were closely affiliated to clones within Thermodesulfovibrio (Class Nitrospira, Figure S5a), which are thermophilic sulfate reducing bacteria. 26 The abundance of archaea in the anaerobic granules, based on quantitative-PCR, was about 10 to   Exopolysaccharide biosynthesis protein-like 2 Exopolysaccharide production CDP-diacylglycerol-serine O-phosphatidyltransferase protein 1 Exopolysaccharide production negative regulator ExoR 2 Exopolysaccharide production negative regulator precursor 2 Exopolysaccharide production protein ExoQ 1 Exopolysaccharide production protein ExoY 3 Exopolysaccharide synthesis, ExoD 1 3 Exopolysaccharide transport protein Exopolysaccharide transport protein, putative 1 Exopolysaccharide xanthan biosynthesis chain length determinant protein GumC 1 Exopolysaccharide/PEPCTERM locus tyrosine autokinase 1 Putative exopolysaccharide biosynthesis protein 3 3 3 Putative exopolysaccharide biosynthesis protein (protein-tyrosine kinase) 1 Putative exopolysaccharide production negative regulator precursor 2 Putative exopolysaccharide production protein (ExoQ-like); putative membrane protein 3 Putative exopolysaccharide regulatory protein exoR 4 Putative membrane protein of ExoQ family involved in exopolysaccharide production 1