High-Throughput rRNA Gene Sequencing Reveals High and Complex Bacterial Diversity Associated with Brazilian Coffee Bean Fermentation

Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97 % of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing.


INTRODUCTION
Coffee is one of the most appreciated beverages in the world, with a consumption of more than 500 billion coffee cups per year. Surpassing a global production of 9 million tonnes, the coffee now stands as the second largest commodity in market value, after only petroleum (1). With an annual output of 3.02 million tonnes, Brazil is the main producer and exporter of coffee beans, followed by Vietnam, Colombia, Indonesia, Ethiopia, India and Honduras (2).
Coffee beans, unlike other fermented foods, require fermentation to facilitate the drying process. After harvesting and pulping, the residual mucilaginous layer that surrounds the coffee beans can be eliminated through microbial fermentation. This involves the action of complex microbial interactions, led mainly by yeasts (e.g. Pichia guilliermondii, P. anomala, Kluyveromyces marxianus and Saccharomyces cerevisae) and lactic acid bacteria (e.g. Erwinia herbicola, Klebsiella pneumoniae and Lactobacillus brevis) (3)(4)(5). These fermentation organisms utilize the bean pulp as a carbon and nitrogen source and produce significant amounts of ethanol, lactic acid and other microbial metabolites, resulting in lowered pH (from 5.5-6.0 to 3.5-4.0) (6,7). In addition, some of these microbial metabolites, which are precursors of volatile compounds formed during roasting, help in improving beverage flavour (7,8).
Culture-independent techniques have helped to change the way to study food microbial ecology, leading to consideration of microbial populations as consortia (9). The advent of the use of molecular techniques and, more specifically, the use of high-throughput January-March 2018 | Vol. 56 | No. 1 sequencing (HTS), permitted to overcome the limitations of the cultivation-associated methods, allowing a breakthrough in understanding the diversity and composition of several food microbial ecosystems (10)(11)(12)(13). Illumina MiSeq® (Illumina Inc, San Diego, CA, USA) generates shorter reads (250 bp) than other HTS systems but gives a higher throughput, providing thousands of high-quality reads of the generated amplicons and allowing a superior taxonomical analysis (14).
In this work, we report a diversity analysis aiming to characterize bacterial communities associated with coffee bean fermentation, using high-throughput sequencing, as part of a whole metagenome study of the microbiota associated with the Brazilian coffee processing chain.

On-farm coffee fermentation and sampling
Spontaneous fermentations were performed at the Fazenda Apucarana located in the Cerrado Mineiro region (18°55'59.4" S, 46°50'41.5" W) at Minas Gerais, Brazil. Freshly harvested coffee (Coffea arabica var. Catuaí) cherries were depulped using a BDSV-04 depulper (Pinhalense, São Paulo, Brazil) obtaining beans with a surrounding layer of mucilage (15). Fermentations were conducted for 24 h in cement tanks with a nominal volume of 4.5 m³, containing 20 kg of depulped beans and approx. 500 L of fresh water, in accordance with the local wet processing method. At the end of the process, fermented beans were sun-dried for 20 days to 11-12 % moisture, as measured by a moisture meter (model AL-102 ECO; Agrologic, São Leopoldo, Brazil). Environmental temperature during the experimental procedure was 24-32 °C (day) and 12-15 °C (night). Samples (fermenting coffee bean pulp mass) were collected at random at 0, 12 and 24 h for HTS and target metabolic analysis.

Total DNA extraction
For extraction of total DNA from the samples, 1 mL of coffee bean pulp mass was centrifuged at 12 000×g for 1 min (centrifuge model 5430; Eppendorf, Hamburg, Germany). Cell pellet was resuspended in 500 µL of Tris-EDTA, homogenized with 10 µL of lysozyme solution (20 mg/mL; Sigma-Aldrich, Arklow, Ireland) and incubated at 30 °C for 60 min. Then, 50 µL of sodium dodecyl sulphate (SDS; 10 %, by mass per volume) and 10 μL of proteinase K solution at 20 mg/mL (Sigma-Aldrich) were added to the lysis solution, followed by homogenization and incubation at 60 °C for 60 min. A volume of 150 µL of phenol/chloroform (25:24; Sigma-Aldrich) was added, homogenized by inversion and centrifuged at 12 000×g (model 5430R; Eppendorf) for 5 min. Supernatant was removed and the DNA was precipitated with 3× (by volume) absolute ethanol (Sigma-Aldrich). Pellets was washed with 80 % ethanol, dried and resuspended in Mili-Q® ultrapure water (Merck, Kenilworth, NJ, USA). Total DNA was quantified with the Nanodrop 2000 instrument (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Illumina high-throughput sequencing
A fragment of the 16S rRNA gene was amplified from the total DNA extracted using primers for the V4 region (bases 515 to 806), containing complementary adaptors for Illumina platform (16) using KlenTAQ polymerase (Sigma-Aldrich). Amplification was performed using the degenerated primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGAC- . Bar-coded amplicons were generated by PCR under the following conditions: 95 °C for 3 min, followed by 18 cycles at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 68 °C for 60 s, and final extension at 68 °C for 10 min. Samples were sequenced in the MiSeq (Illumina Inc) platform using 500 V2 kit (Illumina Inc), following standard Illumina protocols.

Bioinformatics and data analysis
Data generated by sequencing went through a rigorous quality system that involved: (i) identification and removal of sequences containing more than one ambiguous base (N), and (ii) evaluation of the presence and complementarity of primer and barcode sequences. Chimeric sequence detection, removal of noises from pre-cluster and taxonomic attribution were also performed using standard parameters of QIIME (Quantitative Insights Into Microbial Ecology) software package, v. 1.9.0 (17). Applying the UCLUST method (18), sequences presenting identity above 97 % were considered the same operational taxonomic units (OTUs) according to the SILVA database (19).

High-performance liquid chromatography
The concentration of reducing sugars (glucose and fructose), organic acids (acetic, succinic, lactic and propionic acids) and ethanol was determined during coffee bean fermentation by high-performance liquid chromatography (HPLC). Samples were centrifuged at 6000×g (centrifuge model CT-6000; Cientec, Porto Alegre, Brazil) and filtered through 0.22-µm pore size filter (Sartorius Stedim, Goettingen, Germany) in order to remove debris. Analysis parameters were determined according to de Carvalho Neto et al. (20). Filtered samples were injected into HPLC system equipped with an Aminex HPX 87 H column (300 mm×7.8 mm; Bio-Rad, Richmond, CA, USA) and a refractive index (RI) detector (model HPG1362A; Hewlett-Packard Company, São Paulo, Brazil). The column was eluted in isocratic mode with a mobile phase of 5 mM H 2 SO 4 at 60 °C and a flow rate of 0.6 mL·min. Table 1 shows the evolution of sugar consumption, metabolite formation and pH decrease during fermentation of coffee bean pulp. The observed increase in the concentration of reducing sugars (glucose and fructose) at 12 h of fermentation can be attributed to the hydrolysis of sucrose by the action of yeast invertase (21). These sugars were partially consumed after 24 h of fermentation, with a final residual content of 3.2 and 4.5 g/L of glucose and fructose, respectively. Lactic acid (0.32 g/L) was the most important organic compound formed during fermentation, followed by succinic and acetic acids (0.08 and 0.05 g/L, respectively). Lactic acid is an important organic compound for coffee bean fermentation that assists in the coffee pulp acidification without interfering with the final product quality (22). The accentuated production of lactic acid is in agreement with the strong dominance of lactic acid bacteria found in the present study (Fig. 1), resulting in pH decrease from 5.3 to 4.0 at the end of fermentation ( Table 1). The reduction of pH below 4.5 is a widely used method by coffee producers to determine the end of fermentation of coffee bean during wet processing (23).  of Fructobacillus may assist in the fructose metabolism, contributing to drying of coffee beans.

Sugar consumption and metabolite formation
Bacterial composition and dynamics shown in Fig. 1 reveal that, despite the presence of a high bacterial diversity associated with coffee fermentation environment, several microorganisms are suppressed by the growth and dominance of LAB group. Reads assigned to LAB genera, including Lactobacillus, Pediococcus, Enterococcus, Leuconostoc, Lactococcus and Fructobacillus, corresponded to 26.32 % at the start of the process and reached a total of 97.59 % of the total operational taxonomic units (OTU) at 24 h. The high availability of fermentable sugars coupled with the low presence of dissolved oxygen creates a propitious environment for the rapid growth and colonization of these species, which promote an efficient conversion of sugars into mainly lactic acid (44).
Within the LAB group, Leuconostoc and Lactococcus shared dominance. Species of Leuconostoc, such as L. mesenteroides, L. pseudomesenteroides and L. citreum, have already been reported as dominant LAB in coffee fermentations performed in Mexico, Colombia, India and Taiwan (6,45,46), while Lactococcus species dominates coffee fermentations performed in Taiwan and Brazil (45,47). Co-dominance of LAB enables the production of a wide range of organic compounds (e.g. acetate, acetaldehyde, ethanol, short-chain fatty acids) by heterofermentation (e.g. Leuconostoc sp.) and a high production of lactic acid through homofermentation (e.g. Lactococcus sp.), which promotes yeast growth and reduces the prevalence of spoilage microorganisms.

CONCLUSION
The present study suggests that most of bacterial species involved in the coffee bean fermentation have not been determined. High-throughput 16S rRNA gene sequencing analysis allowed us to reveal in depth the presence of several microbial groups with potential applications. A strong dominance of LAB was confirmed, proving the good adaptation of this microbial group to coffee fermentation environment. Further studies should focus on the isolation of some microbial groups first reported in this study for potential biotechnological applications.
average length of 250 bp. A great coverage was obtained in all samples as demonstrated by the rarefaction curves (Fig. 2).

Bacterial diversity and dynamics
Studies evaluating the microbiology of coffee fermentation have been performed over the last 100 years in several coffee-producing regions, evidencing the dominant species during the post-harvest processing (6,7,(24)(25)(26)(27)(28)(29). On average, nine bacterial genera had been reported in previous studies using culture-dependent methods (7,15,(30)(31)(32)(33). Our work demonstrates that these findings are an underestimate, since over eighty genera of bacteria have been identified by HTS. High frequency and abundance of readings corresponding to Proteobacteria (e.g. Erwinia, Pseudomonas and Methylobacterium) and Firmicutes (e.g. Bacillus, Fructobacillus, Leuconostoc and Lactococcus) were observed. The possible habitat origins of these microbial groups are: human contact, e.g. Pseudomonas sp., Enterobacter, Erwinia and Actinobacteria (34), soil or aerial parts of coffee plants, e.g. Mesorhizobium, Methylobacterium, Stentrophomonas, Sphingobium and Sphingomonas (35)(36)(37), the water source used for wet processing, e.g. Planctomyces, Luteimonas, Devosia and Brevundimonas (38), and the air surrounding the fermentation tank, e.g. Janthinobacterium, Pedobacter, Burkholderia and Kaistobacter (39). These findings indicate the need for a program of research to understand the microbial ecology origin of coffee cherries and processing sites.
The rich and complex bacterial diversity revealed in this study demonstrates the potential of coffee terroir as a source of microorganism species with biotechnological application. An example is the first report of the presence of Fructobacillus in coffee fermentation. This LAB group has a unique biochemical metabolism when compared to other LAB, having preference for fructose consumption and the necessity of an elector acceptor when in presence of glucose (40). Fructobacillus microorganisms were found in gastrointestinal tracts of insects feeding on fructose-rich diet and presented symbiotic interactions with its hosts (41,42). A survey of previous studies demonstrates significant amount of residual pulp fructose at the end of coffee fermentations conducted under field conditions (20), even by using selected starter cultures (4,15,43). With these findings, the isolation and further implementation