The Microbial Community Colonizing Different Varieties of ......Grant awarded to Dr. M.E. Allen, and...
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PC1 (43.5%) PC3 (6.4%) PC2 (12.8%) The Microbial Community Colonizing Different Varieties of Fresh and Dried Hop (Humulus lupulus) Cones as Determined by Next Generation DNA Sequencing N. Cole 1 , M. E. Allen 1 , A. J. Piefer 1 , S. J. Britton 2 , P. T. Benziger 3 1 Hartwick College, Oneonta, NY, 2 Duvel Moortgat NV, Puurs, Belgium, 3 Stony Brook University, Stony Brook, NY Introduction Female cones from the Humulus lupulus plant, commonly known as hops, have been used in brewing for centuries. The presence of antimicrobial resins in hops led to a belief amongst the brewing community that the cones do not support a natural community of microorganisms. This was contradicted by the discovery of a microbiome consistent across freshly harvested Cascade variety cones (Britton et al. 2016; Benziger et al. 2015). Our continued investigation into the microbiome aimed to determine whether the resident microbial community differs with variety of hop plant and whether commercial drying processes change community composition. Microbial residents on plants may contribute to plant health and potentially even plant nutrition (Vorholt 2012). Within a plant species, it has been found that variations in genotype can alter the resident microbial community (Whipps et al. 2008), which may influence plant defenses or may cause certain individuals to exhibit different traits, potentially affecting the end product of brewing. Brewers add hops that are dried and/or fresh at various stages of the brewing process. Craft brewing includes more frequent use of fresh hops and addition after the boiling stage (fresh hopping and back-hopping), which could introduce microbes to beer that could potentially benefit or hinder aroma and flavor in ways currently unrecognized. Identification of the microorganisms that colonize fresh hop cones and of those that persist following cone drying will make possible downstream investigations into the impacts microbes have on the final beer product. Methods • Hop cones from 8 varieties (Cascade, Pearl, Mt. Hood, Fuggle, Crystal, Newport, Brewer’s Gold and Northern Brewer) were collected from Brewery Ommegang, NY. • Subsamples were dried using commercial processes and equipment (not aseptically). • Microorganisms were removed from the surfaces of all hop cones with sonication and their DNA was extracted and used for Illumina sequencing of the V3 and V4 regions of the 16S rRNA genes. • Sequence data were analyzed with Quantitative Insights Into Microbial Ecology (QIIME). Pseudomonas and Sphingomonas were the most abundant taxa on fresh and dried hops of every variety, accounting for at least 60% of the sequences. Weighted Unifrac analysis also showed that drying does not appear to greatly reduce diversity; compared to one another dried (D) and fresh (F) hops microbomes were not distinct. Weighted Unifrac analysis shows similar diversity of the microbiomes of all eight varieties. Only Newport and Fuggle appeared to have microbiomes distinct from each other. Acknowledgements: Financial support for this project was provided by an ASM-URF awarded to Nate Cole, a Hartwick College Faculty Research Grant awarded to Dr. M.E. Allen, and from Duvel- Moortgat and the Hartwick College Center for Craft Food and Beverage. Thank you to Dr. Jeff Werner, SUNY Cortland, for his assistance with the data analysis and bioinformatics and to the Union College Chemistry Department for use of the Illumina MiSeq. References • Aleklett et al. 2014. Botany 92: 253–266 • Benzinger at al. 2015. NEMPET meeting, Blue Mt. Lake, NY. • Britton et al. 2016. World Brewing Congress conference, Denver, CO. • Knief et al. 2010. The ISME Journal 4: 719-728 • Otteson et al. 2013. BMC Microbiology 2013, 13:114 • Shade et al. 2013. mBio 4(2) doi:10.1128/mBio.00602-12. • Vorholt. 2012. Nature Reviews 10: 828-840. • Whipps et al. 2008. J Appl Microbiol 105(6):1744-1755. Rarefaction curves indicate that in all samples there were many rare taxa. Examples are shown here for Cascade and Brewers Gold varieties and Dried samples. Most studies of the phyllosphere that include flowers describe microbial communities associated with individual flower parts (e.g. nectar, petals, pollen, etc) and very few include next generation DNA sequencing (Aleklett et al. 2014). So the data presented here represent a rare, in depth analysis of the microbial community of whole flowers. Hops cone microbiomes are dominated by Pseudomonas, a group also common in tomato flowers (Ottesen et al. 2013) but not in apple blossoms (Shade et al. 2013). The high amount of similarity amongst the microbiomes of all eight hop varieties suggests that for hop cones any differences in microbial communities are less attributable to genotype than to geography (Knief et al. 2010). • Chloroplast and mitochondria sequences were removed prior to additional downstream analysis. Results and Conclusions
Transcript of The Microbial Community Colonizing Different Varieties of ......Grant awarded to Dr. M.E. Allen, and...
PC1 (43.5%)
PC3 (6.4%)
PC2 (12.8%)
PC2 (3.5%)
PC1 (3.8%)
PC3 (3.0%)
The Microbial Community Colonizing Different Varieties of Fresh and Dried Hop (Humulus lupulus) Cones as Determined by Next Generation DNA Sequencing
N. Cole1, M. E. Allen1, A. J. Piefer1, S. J. Britton2, P. T. Benziger3
1Hartwick College, Oneonta, NY, 2Duvel Moortgat NV, Puurs, Belgium, 3Stony Brook University, Stony Brook, NY
IntroductionFemale cones from the Humulus lupulus
plant, commonly known as hops, have been usedin brewing for centuries. The presence ofantimicrobial resins in hops led to a beliefamongst the brewing community that the conesdo not support a natural community ofmicroorganisms. This was contradicted by thediscovery of a microbiome consistent acrossfreshly harvested Cascade variety cones (Brittonet al. 2016; Benziger et al. 2015). Our continuedinvestigation into the microbiome aimed todetermine whether the resident microbialcommunity differs with variety of hop plant andwhether commercial drying processes changecommunity composition.
Microbial residents on plants maycontribute to plant health and potentially evenplant nutrition (Vorholt 2012). Within a plantspecies, it has been found that variations ingenotype can alter the resident microbialcommunity (Whipps et al. 2008), which mayinfluence plant defenses or may cause certainindividuals to exhibit different traits, potentiallyaffecting the end product of brewing.
Brewers add hops that are dried and/orfresh at various stages of the brewing process.Craft brewing includes more frequent use of freshhops and addition after the boiling stage (freshhopping and back-hopping), which couldintroduce microbes to beer that could potentiallybenefit or hinder aroma and flavor in wayscurrently unrecognized.
Identification of the microorganisms thatcolonize fresh hop cones and of those that persistfollowing cone drying will make possibledownstream investigations into the impactsmicrobes have on the final beer product.
Methods• Hop cones from 8 varieties (Cascade, Pearl, Mt. Hood,
Fuggle, Crystal, Newport, Brewer’s Gold and NorthernBrewer) were collected from Brewery Ommegang, NY.
• Subsamples were dried using commercial processes andequipment (not aseptically).
• Microorganisms were removed from the surfaces of allhop cones with sonication and their DNA was extractedand used for Illumina sequencing of the V3 and V4regions of the 16S rRNA genes.
• Sequence data were analyzed with Quantitative InsightsInto Microbial Ecology (QIIME).
Pseudomonas and Sphingomonas were the mostabundant taxa on fresh and dried hops of everyvariety, accounting for at least 60% of thesequences.
Weighted Unifrac analysis also showed that drying does notappear to greatly reduce diversity; compared to one anotherdried (D) and fresh (F) hops microbomes were not distinct.
Weighted Unifrac analysis shows similar diversity of themicrobiomes of all eight varieties. Only Newport and Fuggleappeared to have microbiomes distinct from each other.
Acknowledgements: Financial support for thisproject was provided by an ASM-URF awarded toNate Cole, a Hartwick College Faculty ResearchGrant awarded to Dr. M.E. Allen, and from Duvel-Moortgat and the Hartwick College Center forCraft Food and Beverage. Thank you to Dr. JeffWerner, SUNY Cortland, for his assistance withthe data analysis and bioinformatics and to theUnion College Chemistry Department for use ofthe Illumina MiSeq.
References• Aleklett et al. 2014. Botany 92: 253–266 • Benzinger at al. 2015. NEMPET meeting, Blue Mt. Lake, NY.• Britton et al. 2016. World Brewing Congress conference, Denver, CO.• Knief et al. 2010. The ISME Journal 4: 719-728• Otteson et al. 2013. BMC Microbiology 2013, 13:114 • Shade et al. 2013. mBio 4(2) doi:10.1128/mBio.00602-12. • Vorholt. 2012. Nature Reviews 10: 828-840.• Whipps et al. 2008. J Appl Microbiol 105(6):1744-1755.
Rarefaction curves indicate that in all samples there weremany rare taxa. Examples are shown here for Cascade andBrewers Gold varieties and Dried samples.
Most studies of the phyllosphere that include flowers describemicrobial communities associated with individual flower parts(e.g. nectar, petals, pollen, etc) and very few include nextgeneration DNA sequencing (Aleklett et al. 2014). So the datapresented here represent a rare, in depth analysis of themicrobial community of whole flowers. Hops cone microbiomesare dominated by Pseudomonas, a group also common in tomatoflowers (Ottesen et al. 2013) but not in apple blossoms (Shade etal. 2013). The high amount of similarity amongst themicrobiomes of all eight hop varieties suggests that for hopcones any differences in microbial communities are lessattributable to genotype than to geography (Knief et al. 2010).
• Chloroplast and mitochondria sequences were removedprior to additional downstream analysis.
Results and Conclusions
