The waste coffee in the capsule container of nine different Nespresso machines operated for at least one year was sampled (Fig. 1A). In one case (CityZ model), the cup tray was also sampled independently as it does not connect with the capsule container. The high throughput sequencing and analysis of the 16S rRNA gene amplicons from all the machines revealed a significant bacterial diversity, with the total number of identified genera ranging from 35 to 67. Although relatively similar microbial profiles were detected, there was an important variation in the frequency of particular taxa. Enterococcus sp. and Pseudomonas sp. proved to be the main taxa as they were moderately to highly abundant in nine out of the ten samples analysed. Other frequent genera were Stenotrophomonas, Sphingobacterium, Acinetobacter and, to a lesser extent, Coprococcus, Paenibacillus or Agrobacterium. Dysgomonas was very frequent in the Inissia machine, accounting for 15% of the sequences (Fig. 1B). No differences were detected between machine models (Table 1) or use (domestic vs. communal).

Table 1 Nespresso machines sampled in this work. Full size table

Figure 1 (A) Schematic representation of a Nespresso machine (1) including a capsule (3) container (2), cup tray (4) and a drip tray (5), which was sampled in this work. (B) Bacterial profile of the nine Nespresso machines sampled according to 16S rRNA gene sequencing. Samples numbered in accordance to Table 1. Full size image

One of the two most frequent genera found in the coffee machines was Pseudomonas, which is also one of the few reported examples of a caffeine-degrading bacterium. Indeed, Pseudomonas sp. has been known to catabolise caffeine since the seventies13 and is reported to degrade up to 15 g/L of caffeine through an N- demethylation reaction, which along with C-8 oxydation represent the two potential catabolic pathways14. Species reported to display caffeine degradation abilities are P. alcaligenes15 and P. putida (strains C1, CBB1 or CBB5). In fact, P. putida N-demethylation genes have been used to genetically engineer a caffeine “addicted” version of E. coli16 and caffeine removal from sewage by bioremediation with P. putida has also been proposed17.

The abundance of Enterococcus spp. in caffeine-rich leach might not necessarily involve unreported caffeine degradation abilities in Enterococcus, but it might simply be a consequence of tolerance to certain caffeine levels. The same applies to other frequent taxa. Interestingly, this genus has previously been associated with coffee18, along with several others detected in this work. For example, Acinetobacter sp. has been isolated during coffee fermentation19, while Stenotrophomonas sp., Curtobacterium sp. and Pseudomonas sp. are abundant in the coffee seed20.

The colonisation process of the wasted coffee leach was studied in an experiment using a brand new Krups Inissia machine (located in a separate room within our laboratory). The experiment lasted two months, during which leach samples were taken and bacterial diversity analysed, with a significant variation in the taxonomic profiles detected. The initially high species richness was substituted by a relatively simpler, but still highly variable, species composition (species richness significantly dropped 14 days after the beginning of the experiment; t-test p-value = 0.039). During the first 11–13 days, Pantoea sp., Cloacomonas sp. and, to a lesser extent, Brevundimonas sp. were relatively abundant but amounts decreased to undetectable levels by the end of the experiment. All these taxa were largely substituted by Pseudomonas sp., Acinetobacter sp. and Sphingobium species, which reached a peak and then fluctuated (Sphingobium sp., Bacillus sp.) or reached the highest levels at the end of the experiment (Pseudomonas sp., Acinetobacter sp.) as shown in Fig. 2A. The first 30 days exhibited greater instability in the bacterial communities, as deduced by the consecutive peaks of very abundant taxa, which were substituted by a more balanced bacterial composition after one month. As in other studies on different environments21,22, these results strongly suggest a long ecological succession during the first month, in which generalist bacterial taxa, including enterobacteriaceae genera such as Pantoea, are the first colonizers but are then displaced by successive waves of other taxa. The main keyplayers observed during this succession were, in order (Fig. 2B): enterobacteria (genus Pantoea; peaking 4–11 days after the beginning of the experiment), Firmicutes (three genera of the bacillaceae family: Bacillus, Terribacillus, Paenibacillus; peaking after 14–21 days); and, finally, the sphingomonadales genus Sphingobium (proteobacteria), the actinomycetales genus Curtobacterium (actinobacteria) and the pseudomonadales genus Acinetobacter (proteobacteria), peaking after 28, 31 and 49 days, respectively. These taxa gave way to a different bacterial profile dominated by Pseudomonas sp. and Enterococcus sp. after two months of the experiment. This profile was very similar to that found in the nine other coffee machines sampled (Fig. 1B) which had been operated for a longer time, suggesting that the particular physico-chemical conditions (cycles of high temperature, constant caffeine accumulation, etc.) of coffee leach, rather than the influence of the user or the number of uses, are the main force shaping the composition of the microbial community. A mathematical modelling performed on the dynamic series of 16S rRNA gene data revealed statistically significant correlations among the detected taxa, indicating that the distribution of bacterial genera in time is not random (Fig. 3).

Figure 2 Bacterial colonisation in a brand new Nespresso Krups Inissia machine. (A) Bacterial profile in the drip tray during the two months of operation according to 16S rRNA gene monitoring. (B) Ecological succession of the main taxa during the experiment, represented as the variation of their relative frequencies. Full size image

Figure 3 Correlations among the bacterial genera detected in this work. Distances correspond to the linear statistical correlation. Sizes of the spheres are proportional to the relative abundances in logarithmic scale. Highly correlated genera are shown in the same colour. Sph, Sphingobium; Bac, Bacillus; Aci, Acinetobacter; Ter, Terribacillus; Cur, Curtobacterium; Pae, Paenibacillus; Pan, Pantoea; Rhi, Rhizobium; Chr, Chryseobacterium; Aer, Aerococcus; Art, Arthrobacter; Ste, Stenotrophomonas; Ach, Achromobacter; Pse, Pseudomonas; Can, Candidatus Cloacamonas; Agr, Agrobacterium; Bre, Brevundimonas; Ent, Enterococcus; Cau, Caulobacter; Dys, Dysgonomonas; Spb, Sphingobacterium; Ped, Pedobacter; Com, Comamonas; Oth, Other genera. Full size image

Most of the taxa we identified during the colonisation process of the coffee machine operated in our laboratory have previously been found in natural coffee-related environments. Species belonging to the genera Acinetobacter and Bacillus and also some enterobacteria, have been detected during the natural fermentation of coffee beans23,19, whereas Paenibacillus and other Bacteroidetes and Firmicutes species have proved abundant in the composting process of coffee hulls24,25. Despite some reports describing the ability of different Sphingobium species to degrade toxic molecules, such as bisphenols26 and hydroquinones27, this is the first report where Sphingobium sp. has been associated to a caffeine-rich environment.

In addition to the 16S rRNA gene monitoring, we followed up changes in the coffee leach microbial diversity through scanning electron microscopy (SEM). Figure 4 shows a dynamic series of samples taken at different time points (4, 8, 14 and 21 days after the first day of operation) during the first month. Microbial biomass increased throughout the analysis and variations in the composition and viscosity of the coffee leach were also evident. For example, a filamentous matrix was observed at days 8 and 21 (Fig. 4). At day 14, the sample was dominated by a single shape of bacterial cells, which interestingly coincided with an overwhelming relative abundance of Bacillus spp. in that sample (Fig. 2A). Further experiments are needed to determine whether microbial community changes are the cause or the effect of the variations in the composition of coffee leach as shown by SEM.

Figure 4 Chronological series of SEM images of the drip tray samples taken after 4 (A), 8 (B), 14 (C) and 21 days (D) of operation. Figure C corresponds to a sample highly abundant in Bacillus spp. Scale bars are indicated in each case. Full size image

Our results show, for the first time, that coffee leach from standard capsule machines is a rich substrate for bacterial growth; that caffeine content does not prevent a rich bacterial biodiversity from rapidly colonising coffee leach; and that microbial succession from an initial pool of generalist bacteria gives way to an apparently coffee-adapted but still highly variable bacteriome. This bacteriome is rich in species previously reported to be associated with the coffee plant and/or the coffee fermentation processes. Colonising bacteria might be of environmental origin (no cultivable microorganisms nor bacterial DNA was detected in coffee capsules, data not shown), whereas heterogeneity of bacterial composition may relate to factors such as cleaning habits and, specially, the frequency of machine use (with higher frequencies presumably correlating with increased volume and temperature of the coffee leach). Further studies comprising more coffee machines, deep genome sequencing of the microbial communities therein and even functional metagenomics, are required to contribute to shed light on the microbial ecology of coffee leach in capsule machines.

The presence of bacterial genera with pathogenic properties and the fast recovery of the communities after rinsing the capsule container, strongly suggest the need for frequent maintenance of the capsule container of these machines. Maintenance should employ bacteriostatic compounds and avoid contact of the coffee leach with other parts of the machine to avoid unintended contamination of the beverage. On the other hand, the resistant microbial communities we describe here (microbial consortia, individual caffeine degrading/tolerant species or as a source of metabolic pathways and genes) may represent a promising tool for biological coffee decaffeination processes and for environmental caffeine decontamination.