World Journal of Microbiology and Biotechnology (2021) 37:51

https://doi.org/10.1007/s11274-021-03017-2

ORIGINAL PAPER

Microbial diversity and chemical characteristics of Coffea canephora

grown in different environments and processed by dry method
Priscila Vargas Pereira1 · Danielle Gonçalves Bravim1 · Renata Pancini Grillo1 · Larissa Diirr Bertoli1 ·
Vanessa Moreira Osório2 · Daniela da Silva Oliveira3 · Maria Gabriela da Cruz Pedrozo Miguel4 ·
Rosane Freitas Schwan4 · Samuel de Assis Silva5 · Jussara Moreira Coelho1 · Patrícia Campos Bernardes1 

Received: 23 July 2020 / Accepted: 1 February 2021 / Published online: 17 February 2021
© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021

Abstract
This study aimed to assess the microbial diversity in Coffea canephora grown in four different environments of Espirito
Santo state, Brazil. Coffee cherries of two different altitudes (300 and 600 m) and two terrain aspects (Southeast-facing and
Northwest-facing slopes) were processed by the dry method. Samples were collected during the drying/fermentation process.
Microorganisms were counted, isolated, and identified by MALDI-TOF, followed by sequencing of the ribosomal region.
Sugars and organic acids were quantified by HPLC and volatile compounds of the roasted coffees were evaluated by GC–MS.
Bacteria population presented a significant number of isolates as well as higher counts during the drying/fermentation process
with respect to the population of yeasts. The principal genera of microorganisms found were Bacillus, Pichia, Candida, and
Meyerozyma. Meyerozyma guilliermondii was the most frequent yeast in all environments. On the other hand, Pichia kluyveri
was found only in coffee cherries from the 600 m altitude. The highest concentration of acetic and succinic acids observed
was 6.06 mg/g and 0.84 mg/g, respectively. Sucrose concentrations ranged from 0.68 to 5.30 mg/g, fructose from 1.30 to
4.60 mg/g, and glucose from 0.24 to 1.25 mg/g. Thirty-six volatile compounds, belonging to the groups of pyrazines, alco-
hols, aldehydes, ketones, and furans were identified in roasted coffee, with differences between altitude and terrain aspects.
Information about microbial diversity is crucial to better understand the coffee quality and distinct characteristics of coffee
produced in different environments.

* Patrícia Campos Bernardes

patricia.bernardes@ufes.br
1
Department of Food Engineering, Federal University
of Espirito Santo, Alegre, ES 29500‑000, Brazil
2
Department of Chemistry and Physics, Federal University
of Espírito Santo, Alegre, ES 29500‑000, Brazil
3
Department of Pharmacy and Nutrition, Federal University
of Espirito Santo, Alegre, ES 29500‑000, Brazil
4
Department of Biology, Federal University of Lavras, Lavras,
MG 37200‑000, Brazil
5
Department of Rural Engineering, Federal University
of Espírito Santo, Alegre, ES 29500‑000, Brazil

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Graphic abstract
Keywords  Endogenous microbiota · Bacteria · Yeasts · MALDI-TOF · Volatile compounds
Introduction
Coffea canephora, commercially known as Robusta, is the
second most produced coffee species worldwide. The esti-
mate for world production of this species for the 2019/2020
harvest is 71.72 million bags of 60 kg (4.303 million tons)
(ICO 2020). Brazil is the second largest producer of this spe-
cies, accounting for approximately 20% of world production
(CONAB 2019). Robusta coffee is widely used in blends
with Arabica coffee and in the soluble coffee industry (Fer-
rão et al. 2019).
In Brazil, the coffee is usually processed by the natural
or dry method, due to the hot climate and the rain scarcity
during the harvesting period. In the dry processing, coffee
cherries are fermented and dried by solar radiation with
all the constituent parts (Silva et al. 2008; Velmourougane
2013; Evangelista et al. 2014). During dry processing, the
microorganisms, naturally occurring in the cherry, perform
a series of fermentation processes that may influence the
final quality of the product (Silva et al. 2008). These micro-
organisms degrade pulp and mucilage compounds, releasing
metabolites that may influence physicochemical and sensory
characteristics of the final product (Vilela et al. 2010; Evan-
gelista et al. 2015).
13
World Journal of Microbiology and Biotechnology (2021) 37:51
The microbiota can experience differences depend-
ing on the characteristics of the production region, such
as moisture, temperature, soil microbial population, the
composition of the coffee cherry variety, and processing
method (Batista et al. 2003; Silva et al. 2008; Leong et al.
2014). Studies that address different coffee growing envi-
ronments, such as altitude and terrain aspect, are relevant,
since they enhance the knowledge about how these fac-
tors influence productivity, physicochemical and sensory
characteristics of the coffee. The knowledge of the coffee
microbiota enables the selection of beneficial microorgan-
isms with specific features, using them as a starter culture
to perform the coffee fermentation. Such a practice pos-
sesses several processing benefits, such as reduced fer-
mentation time and reduced growth of undesirable micro-
organisms, including mycotoxigenic fungi. Moreover, it
can improve the quality of coffee, considering that these
microorganisms could produce compounds that can benefit
the quality of the final beverage (Silva et al. 2008; Evan-
gelista et al. 2015; de Carvalho Neto et al. 2017). Yeasts
and mesophilic bacteria metabolize pulp carbohydrates,
producing acids and volatile compounds that are impor-
tant for coffee quality (Masoud et al. 2004; Swiegers et al.
2005; de Carvalho Neto et al. 2017). Lactic acid bacteria
World Journal of Microbiology and Biotechnology (2021) 37:51 Page 3 of 12  51
metabolize amino acids, sucrose, and citric acid to produce
volatile compounds (Yeretzian et al. 2002; Velmourougane
2013; Leong et al. 2014; Pereira et al. 2016).
Several studies have being conducted with Coffee ara-
bica in different countries, such as Colombia, Cameroon,
Ecuador, and Brazil (Silva et al. 2008; Vilela et al. 2010;
Evangelista et al. 2015; Hamdouche et al. 2016; de Carvalho
Neto et al. 2017; Pothakos et al. 2020). These studies include
the evaluation of the microbiota and its physicochemical
changes during drying/fermentation (Silva et al. 2000; Vilela
et al. 2010; Velmourougane 2013; Hamdouche et al. 2016;
Iswanto et al. 2019; Pothakos et al. 2020).
On the other hand, studies on the microbiota present in C.
canephora are very scarce in the literature. Agate and Bhat
(1966) detected the presence of Saccharomyces marxianus
(currently Kluyveromyces marxianus), Saccharomyces bay-
anus, Saccharomyces cerevisiae, Schizosaccharomyces sp.,
Streptococcus, Pseudomonas, Flavobacterium, and Proteus
in Coffee canephora from India. Pee and Castelein (1971)
identified Candida guilliermondii (currently Meyerozyma
guilliermondii) on the surface and mucilage of cherries
and, Candida parapsilopsis, S. cerevisiae, Torulopsis
famata (currently Debaryomyces hansenii), S. marxianus
(currently K. marxianus), Candida tropicalis, and Candida
pelliculosa (currently Wickerhamomyces anomalus) on the
surface of beans from Republic of the Congo. Hamdouche
et al. (2016) identified some species of microorganisms dur-
ing the drying/fermentation of coffee cherries from Cam-
eroon, including Microbacterium, Enterobacter, Wallemia,
and Aspergillus.
To our knowledge, this is the first comprehensive study
of the microbiota of C. canephora from Brazil, one of the
world’s largest producers of this species. The objectives of
the current study were to identify the microbial diversity,
evaluate sugars, organic acids, and volatile compounds
involved in natural coffee drying/fermentation in four pro-
ducing areas of C. canephora, which present distinct envi-
ronmental characteristics.
Materials and methods
Coffee processing
Coffee cherries of C. canephora, Emcaper 8151–Robusta
Tropical variety, were collected in four different geographic
areas of Espírito Santo, Brazil: on a farm located in Cach-
oeiro de Itapemirim, 600 m above sea level and Southeast-
facing slope (S6) (20° 37′ 36.82′′ S and 41° 5′ 13.35′′ W),
in Cachoeiro de Itapemirim, 600 m above sea level and
Northwest-facing slope (N6) (20° 37′ 24.22′′ S and 41° 5′
14.50′′ W), in Jerônimo Monteiro, 300 m above sea level
and Southeast-facing slope (S3) (20° 49′ 53.56′′ S and 41°
24′ 52.64′′ W) and in Jerônimo Monteiro, 300 m above sea
level and Northwest-facing slope (N3) (20° 50′ 0.99′′ S and
41° 25′ 2.72′′ W). The cherries were processed by the dry
method. Each experimental unit was composed of 15 kg of
coffee cherries, with a total of four experimental units (2
altitudes × 2 facing slope). The cherries were harvested at the
mature stage, placed on suspended platforms for sun drying
up to ca. 18% moisture. Later, cherries were transferred to
a dryer (Marconi, Brazil) at 45 °C for 24 h until reaching
11% of moisture (Gehaka G650). Samples of coffee cher-
ries (500 g) were collected, in duplicate, at 0, 12, 24, 36, 46,
72, 118, and 190 h, stored in sterile polyethylene bags, and
immediately transferred to the laboratory for microbiologi-
cal analysis. For the physicochemical analysis, the coffees
were stored at − 20 °C prior to the tests.
Characterization and identification of microbiota
Quantification, isolation, and phenotypic characterization
A coffee cherry sample (10 g) was added to 90 mL of ster-
ile bacteriological peptone water (Himedia, India), homog-
enized for 2 min in a Stomacher®, and used for decimal
serial dilution. Lactic acid bacteria (LAB) were counted by
spread plating on De Man, Rogosa and Sharpe agar (MRS,
Himedia, India) with added 0.25% (v/v) of nystatin (Teuto,
Brazil). Plate count agar (PCA) was employed (Himedia,
India) for mesophilic bacteria (MB), Yeast potato dex-
trose agar (YPD, Himedia, India) which was acidified to
pH 3.5 with tartaric acid (Sigma-Aldrich, United States)
for yeasts, and Malt extract agar (MEA, Himedia, India)
with 0.01% (m/v) of chloramphenicol (Inlab, Brazil) for
filamentous fungi. MRS plates were incubated in anaero-
bic jars at 30 °C/4 days, PCA and YPD plates were incu-
bated at 35 °C/3 days, and MEA plates were incubated at
28 °C/7 days (Vilela et al. 2010; Evangelista et al. 2015).
The colonies’ morphology (cell size andshape, edge, color,
and brightness) were recorded, and the square root of each
colony type was isolated and purified by streaking on new
agar plates (same culture media used for plating). The pure
cultures were stored at − 80 °C in the same broth culture
media used for plating, with added 20% glycerol (w/w). The
phenotypic characterization of the bacterial colonies was
performed using Gram staining, catalase, oxidase activities,
motility tests, and spore formation (Holt et al. 1994).
Identification by MALDI‑TOF
All isolates were grown in plates using a specific culture
medium, previously described for each taxonomic group,
and then incubated at 28 °C for 18 h. Each strain (approxi-
mately ­1 0 7 cells) was aseptically transferred to micro
test tubes. Subsequently, 3 µL of organic solution (water/
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Page 4 of 12
acetonitrile/trifluoroacetic acid, 50:47.5:2.5) was added to
each microtube containing the bacterial isolate, and 3 µL
of formic acid/acetonitrile (25:75) for yeast isolate. The
micro test tubes were vigorously mixed for 1  min, and
then 1 µL of the resulting suspension was transferred to a
96-well MALDI flex target plate (Bruker Daltonics, Bremen,
Germany). After evaporation of most part of the liquid, 1
µL of matrix solution—saturated solution of α-cyano-4-
hydroxycinnamic acid (CHCA) in 50% acetonitrile/2.5%
trifluoro-acetic acid–was added, and the resulting mixture
was gently mixed (Oliveira et al. 2015).
The strain Escherichia coli K12 obtained from the Public
Portuguese Culture Collection from the Micoteca da Univer-
sidade do Minho (MUM, http://www.micot​eca.deb.uminh​
o.pt) was used for in situ extraction of proteins, which were
used as a standard for the MALDI-TOF MS external calibra-
tion. Cells of E. coli K12 were grown in Luria–Bertani agar
medium (LB—1% bacto tryptone, 0.5% bacto yeast extract,
1% NaCl) at 37 °C for 18 h. Briefly, approximately 1 µg
of cellular material from a single E. coli K12 colony was
transferred from the plate to the MALDI flex plate, and the
CHCA matrix solution was added, followed by gently mix-
ing. Each MALDI-TOF sample was spotted in triplicate to
evaluate reproducibility. Later, samples were analyzed in a
MALDI-TOF microflex LT spectrometer (Bruker Daltonics,
Bremen, Germany), using the database Bruker Daltonics,
MALDI Biotyper 3.0 automatic system.
Genotypic identification
The isolates with scores obtained in Biotyperlog less than
1.70 were subjected to sequence analysis, as well as repre-
sentatives of each group previously obtained by MALDI-
TOF. DNA extraction was performed using the InstaGene
Matrix kit (Bio-Rad). The primers, 27F and 1512R, were
used for amplification of the 16S rRNA gene of bacteria
(Devereux and Wilkinson 1995). For the ITS region of
yeasts, the primer ITS1 and ITS4 were used (Evangelista
et al. 2015). Reactions were made to 50 μL with 25 μL of
the TopTaq master kit (Qiagen, Hilden, Germany), 1.5 μL
of each primer at 10 mM, 5 μL of template DNA and 17 μL
of ultrapure water. The reactions were performed in a ther-
mocycler under the following conditions: 97 °C for 5 min,
30 cycles (97 °C for 1 min, 52 °C for 1 min and 72 °C for
1 min) and final extension at 72 °C for 8 min. The reac-
tions occurred in 1.0% (w/v) agarose gel to verify the qual-
ity of the amplicons. Samples were submitted to Macrogen
USA for sequencing and subsequently analyzed by the Gen-
Bank database using the BLAST algorithm (NCBI) and the
strings were deposited in the database. After identification,
the cultures were deposited in the Agricultural Microbiol-
ogy Culture Collection (CCMA) of the Federal University
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World Journal of Microbiology and Biotechnology (2021) 37:51
of Lavras, which is accredited by the World Federation for
Culture Collections.
Analysis of organic acids and sugars
Malic, lactic, acetic, butyric, propionic, citric, oxalic, suc-
cinic, and tartaric acids and sugars (glucose, sucrose, and
fructose) were evaluated during the coffee drying/fermen-
tation using high-performance liquid chromatography
(HPLC) (Shimadzu Corp., Japan) with a UV detector at
210 nm and RID, respectively. Three grams of sample were
mixed with 5 mL of Milli-Q water for 10 min, and the flu-
ids were centrifuged twice at 10,000×g for 10 min at 4 °C.
Samples were subjected to a microfiltration process using a
0.2 µm cellulose acetate filter, and a volume of 20 µL was
injected. The chromatographic column (Aminex HPX-87H)
(300 cm × 7.8 mm) was maintained at 55 °C to achieve chro-
matographic separation of water-soluble acids and sugars,
which were eluted with 5 mM of sulfuric acid solution at a
flow rate of 0.6 mL/min. Standard compounds were used for
calibrations curves employed in the quantification of organic
acids and sugars (Evangelista et al. 2014).
Volatile compounds by GC–MS
Volatile compounds were extracted from coffee beans
shortly after roasting by solid-phase microextraction using
a Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/
CAR/PDMS) 75 µm SPME fiber. The coffee was roasted in
a roaster (Probat-TP2, Curitiba, Brazil) with a capacity of
120 g, following the International Coffee Organization (ICO
2010) recommendations (medium to medium-dark roasting,
180 °C for 8–9 min). A bean (2 g) was macerated with liquid
nitrogen and placed in a 15 mL hermetically sealed vial.
After equilibration at 60 °C, for 10 min, the volatile com-
pounds were extracted at 60 °C for 30 min. The desorption
time in the column was 5 min. After extraction of the vola-
tile compounds, the analysis was conducted using gas chro-
matography combined with mass spectrometry (GC–MS)
(QP-PLUS-2010, Shimadzu) and a silica column Rtx®-5MS
(30 m × 0.25 mm × 0.25 μm). The programming of the col-
umn temperature occurred as follows: the oven temperature
was maintained at 40 °C for 5 min, then increased to 125 °C
in increments of 3 °C/min and subsequently kept at 125 °C
for 1 min. The temperarute of the injector and detector were
maintained at 245 °C for 3 min, respectively. The carrier gas
(He) was injected at a flow rate of 1.67 mL/min. Volatile
compounds were identified by comparing the retention times
with those of standard compounds injected under the same
conditions of the samples and compared with data from
the spectra library (NIST11) (Evangelista et al. 2014). The
volatile compounds were analyzed by Principal Component
World Journal of Microbiology and Biotechnology (2021) 37:51 Page 5 of 12  51
Analysis (PCA) using SensoMaker software (Nunes and
Pinheiro 2012).
Results
Population counts of microbial groups
The mesophilic bacteria (MB) population was higher than
the other microbial groups for coffees harvested at an alti-
tude of 300 m, which will be referred here as 300 m coffees
(Fig. 1). Filamentous fungi were the second microbial group
with the highest count in 300 m coffees followed by yeasts
and LAB. In 600 m coffees, that is those harvested at an
altitude of 600 m, populations of bacteria and filamentous
fungi presented the highest counts, and yeasts and LAB the
lowest counts during drying/fermentation. Regarding the
count in the final drying time, the 600 m coffees presented
the higher amounts for the populations of LAB, yeasts, and
filamentous fungi for both terrain aspects, Southeast-facing
and Northwest-facing (Table S1).
Identification of isolates
A total of 234 bacteria and 89 yeasts were isolated and iden-
tified by MALDI-TOF. In the current study, a special focus
was given to bacteria and yeasts aiming at the development
of a collection for the application of bacteria and yeasts on
fermentations of biotechnological interest in future research.
For this reason, the filamentous fungi were not identified.
Some isolates, representatives of each species identified by
MALDI-TOF, were submitted to DNA sequence analysis
(Tables 1 and 2). A total of 27% of isolates were sequenced
and presented sequence similarity between 97 and 100% to
sequences deposited in the database Genbank. Gram-posi-
tive bacteria represented 89.75% of bacterial isolates. The
genus Bacillus and Staphylococcus sp. were present in all
samples. The most substantial number of isolated bacteria
belonged to the genera Bacillus (24%), Kosakonia (21%),
and Staphylococcus (13%). Bacillus licheniformis, Bacillus
cereus, and Raoultella ornithinolytica were present at six
of the seven assessed time intervals in the samples N3, N6,
and S6, respectively. Bacillus pumilus, Stenotrophomonas
maltophilia, Lactobacillus oligofermentans, Lactobacillus
oris, and Lactobacillus paracasei were identified only in
sample S3. Bacillus altitudinis, Bacillus safensis, Derma-
coccus nishinomiyaensis, Enterobacter hormaechei, Entero-
bacteriaceae bacterium, Salmonella sp., and Streptomyces
variabilis were observed in sample S6; Bacillus shackleto-
nii, Cellulosimicrobium cellulans, Micrococcus luteus, and
Micrococcus yunnanensis in sample N3; and Acinetobacter
pittii, Enterococcus pallens, and Pseudomonas putida in
sample N6.
The 89 isolated yeasts belong to the genera Candida
(39%), Meyerozyma (35%), Hanseniaspora (18%) and
Pichia (8%). Meyerozyma caribbica and M. guilliermondii
were present in samples from all environments studied. M.
guilliermondii was the most frequently found yeast during
drying/fermentation (Tables 1 and 2). Some yeast species
were present in only one sample studied. Candida glabrata
and Pichia cecembensis were found only in samples from
S3, C. tropicalis, and Pichia guilliermondii in samples from
S6, Candida dubliniensis and Candida parapsilosis in sam-
ples from N3, Candida dendronema, Hanseniaspora opun-
tiae, and Hanseniaspora uvarum in samples from N6.
Organic acids and sugars
The organic acids and sugars detected during drying/fer-
mentation of coffee are shown in Figs. 2 and 3. Acetic and
succinic acids were identified in all samples and at all times
of drying/fermentation. In the last time interval assessed,
a higher concentration of acetic acid was observed in the
samples from the altitudes of 300 m, for both terrain aspects,
compared with the samples harvested at the altitude of
600 m. The highest concentration of acetic acid observed
was 6.06  mg/g, and the highest concentration of  suc-
cinic acid was 0.84 mg/g. Sucrose concentrations varied
from 0.68 to 5.30 mg/g, fructose from 1.30 to 4.60 mg/g,
and glucose from 0.24 to 1.25 mg/g throughout the drying/
fermentation processes.
Volatile compounds
A total of 36 volatile compounds were detected in samples of
roasted coffee beans (Fig. 4), including pyrazines (11), alco-
hols (3), aldehydes (3), ketones (2) and furan (1). The results
of volatile compounds showed that the 600 m coffee sam-
ples are arranged on the positive side PCA1 (which explains
47.87% of the results), and the coffee samples from 300 m
on the negative side. Some compounds were found only in
300 m samples, such as pyridine, n-undecane, 2,6-diethyl-
3-methylpyrazine, and n-dodecane (Table S2). Other com-
pounds were identified only in samples from 600 m, such
as 2,3,5-trimethylpyrazine, 2,3-dimethylpyrazine, 2-ethyl-
5-methylpyrazine, and ethylpyrazine.
Discussion
To the best of our knowledge, this is the first report about
microbial diversity during the drying/fermentation process
of C. canephora from Brazil. Some studies with this coffee
species were previously reported (Agate and Bhat 1966; Pee
and Castelein 1971; Velmourougane 2013; Hamdouche et al.
2016) in other countries (India, Cameroon and Republic of
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Page 6 of 12 World Journal of Microbiology and Biotechnology (2021) 37:51

Fig. 1  Population count of
mesophilic bacteria (full A
line), filamentous fungi (filled 8
square), yeasts (filled triangle), 7
and lactic acid bacteria (filled 6
diamond) during drying/fer- 5

Log cfu/g
mentation in the coffee cherries. 4
a Samples Southeast-facing 3
slope/300 m, b Northwest- 2
facing slope/300 m, c South- 1
east-facing slope/600 m, and d 0
Northwest-facing slope/600 m 0 12 22 36 46 70 118 190
Drying/fermentaon me (h)

Bacteria Filamentous Fungi Yeasts LAB

B
8
7
6
5

Log cfu/g
4
3
2
1
0
0 12 22 36 46 70 118
Drying/fermenta
on
me (h)

Bacteria Filamentous Fungi Yeasts LAB

C
8
7
6
5
Log cfu/g

4
3
2
1
0
0 12 22 36 46 70 118 190
Drying/fermenta
on
me (h)

Bacteria Filamentous Fungi Yeasts LAB

D
8
7
6
5
Log cfu/g

4
3
2
1
0
0 12 22 36 46 70 118 190
Drying/fermenta
on
me (h)

Bacteria Filamentous Fungi Yeasts LAB

13
World Journal of Microbiology and Biotechnology (2021) 37:51 Page 7 of 12  51

Table 1  Species of bacteria and yeasts identified during drying/fermentation of coffee at the altitude of 300 m
Bacteria % of Population of bacteria and yeasts (log CFU/g)
similar-
ity Access code S3 N3
or Drying/fermentation time (h) Drying/fermentation time (h)
MALDI
score 0 12 22 36 46 70 118 0 12 22 36 46 70 118

Bacillus cereus 97 MW490698 5.41 – – – – – – – – – – – – –

Bacillus licheniformis 99 MW490693 4.95 – – – – – – 4.00 – 4.48 3.30 3.00
3.30 3.00
Bacillus pumilus 99 MW490687 – – – 5.36 – – – – – – – – – –
Bacillus shackletonii 97 MW490692 – – – – – – – – – 4.00 – – – –
Bacillus subtilis 97 MW490690 – – – – 4.00 – – – – – – – –
Cellulosimicrobium cellulans 2.10 MT – – – – – – – – – – 3.00 – – –
Citrobacter freundii 1.83 MT – – – – 4.00 4.60 – – – – – – – –
Enterobacter cloacae 97 MW490697 – – – – – – – – – – – – 3.00 –
Escherichia vulneris 1.74 MT 4.00 – – 4.95 – 4.30 – – – – – – – –
Kosakonia cowanii 97 MW490691 – – – 4.60 4.95 4.00 4.30 4.00 – 4.30 3.00 3.00
– 3.30
Lactobacillus oligofermentans 1.73 MT – 4.36 – – – – – – – – – – – –
Lactobacillus oris 1.75 MT 5.30 – – – – – – – – – – – – –
Lactobacillus paracasei 1.73 MT – 4.34 – – – – – – – – – – – –
Leuconostoc mesenteroides 100 MW490689 – – – – – – 4.30 – – – – – 3.00 –
Micrococcus luteus 98 MW490695 – – – – – – – – – – – 3.00
– –
Micrococcus yunnanensis 98 MW490694 – – – – – – – – – – 3.00 – –
Pantoea agglomerans 98 MW490686 – – – – – – – 4.00 4.00 – – 1.00 – –
Pantoea ananatis 97 MW490685 4.00 – 4.70 – – – – – – – – – – –
Staphylococcus cohnii 98 MW490684 – – – – – – – – – – – – – 3.00
Staphylococcus epidermidis 99 MW490696 – – – – – – – – – 4.00 3.00 – –
Staphylococcus haemolyticus 1.84 MT – – – – – 4.00 – – – – – – – –
Staphylococcus saprophyticus 98 MW490688 – – – – – – – – 4.00 – – – – 3.00
Staphylococcus xylosus 1.78 MT – – – – – – – 4.30 – – – – – –
Stenotrophomonas maltophilia 1.76 MT – – – – 4.00 – – – – – – – – –
Yeasts Access code 0 12 22 36 46 70 118 0 12 22 36 46 70 118
Candida glabrata 100 MW465982 – – – 2.53 – – 4.01 – – – – – – –
Candida orthopsilosis 99 MW465983 1.78 1.00 1.60 2.53 2.95 – 2.00 – – – – – – –
Candida dubliniensis 1.74 MT – – – – – – – – 2.95 – – 2.85 – –
Candida parapsilosis 1.78 MT – – – – – – – – – – – – 2.00 –
Meyerozyma guilliermondii 98 MW465984 1.30 1.30 – – – 1.30 2.00 2.00 – – 2.70 – 2.30 –
Pichia cecembensis 97 MW465985 – – – – 2.00 – – – – – – – – –

– not detected, S3 Southeast-facing slope/300 m, N3 Northwest-facing slope/300 m, MT identification by MALDI-TOF with a score greater than
1.7

the Congo). In both terrain aspects and altitudes, a predom-
inance of mesophilic bacteria and filamentous fungi was
observed over populations of yeasts and lactic acid bacte-
ria during the drying/fermentation period. In samples from
300 m, the count of mesophilic bacteria was higher through-
out the drying/fermentation process. The filamentous fungi
counting was close to the mesophilic bacteria and, in some
points, higher (specially at the end of drying/fermentation)
for 600 m samples (Fig. 1).
In contrast, Velmourougane (2013) observed that the pop-
ulation of yeasts was dominant compared to the population
of bacteria in freshly pulped coffee beans obtained by the
semi-dry method in India. The same study also observed a
growth of filamentous fungi population during the fermen-
tation. Several factors, such as humidity, temperature, pH,
oxygen availability, sugar content, water activity, and pro-
duction of extracellular pectinase, can explain the growth of
microorganisms (Silva et al. 2008).
Coffees from 600 m presented the highest counts of yeast
and LAB at the end of drying/fermentation (Table  S1).
Yeasts and LAB can favour the production of compounds
that have a positive impact in the coffee beverage quality.

13

51 

Table 2  Species of bacteria and yeasts identified during drying/fermentation of coffee at the altitude of 600 m
Bacteria % of similarity or Population of bacteria and yeasts (log CFU/g)
MALDI score

13
Access code S6 N6
Page 8 of 12

Drying/fermentation time (h) Drying/fermentation time (h)

0 12 22 36 46 70 118 0 12 22 36 46 70 118

Acinetobacter pittii 2.28 MT – – – – – – – – – – – – 4.00 –

Acinetobacter radioresistens 2.18 MT – – – 4.00 – – – – – – – 4.30 – –
Bacillus altitudinis 96 MW490699 – – – – – 3.00 – – – – – – – –
Bacillus cereus 97 MW490700 4.60 – – – – – – 4.48 5.85 5.48 - 4.60 4.00 3.60
Bacillus safensis 96 MW490702 4.00 – – – – – – – – – – – – –
Bacillus subtilis 97 MW490701 – – 4.00 – – – – – 5.00 – – – – –
Citrobacter braakii 1.79 MT – – – – – – – – – – 4.30 – – –
Citrobacter freundii 1.83 MT – – – – – – – – – – – 4.00 – –
Dermacoccus nishinomiyaensis 98 MW490703 – – 4.00 – – – – – – – – – – –
Enterobacter asburiae 97 MH071322.1 – – – – – – – – – 5.00 – – 4.00 –
Enterobacter hormaechei 98 MW490704 – – – – 3.00 – – – – – – – –
Enterobacter ludwigii 97 MW490705 – – – – 4.00 – – – – – – – 4.00 –
Enterococcus pallens 98 MW490706 – – – – – – – 3.00 – – – – – –
Enterobacteriaceae bacterium 97 MW490707 – – – – 4.00 – – – – – – – – –
Escherichia vulneris 1.73 MT 4.00 – – – – 3.00 3.00 – – – – – – –
Leuconostoc mesenteroides 98 MW490708 – – – – – – – – 5.00 – – 4.00 – –
Pantoea agglomerans 98 MW490709 – – – 4.00 4.00 3.30 3.30 3.00 – – – – – 3.00
Pectobacterium parmentieri 97 CP003415.1 – 4.00 – – – – – – – – – – – –
Pseudomonas putida 1.70 MT – – – – – – – – – – 4.00 – 4.00 –
Raoultella ornithinolytica 1.76 MT 4.00 4.00 4.00 – 4.30 3.48 3.48 – – – – – – –
Salmonella sp 2.27 MT – 4.00 – – 4.30 – – – – – – – – –
Staphylococcus epidermidis 99 MW490710 – 4.48 4.60 4.00 – 3.30 – – – – – – – –
Staphylococcus warneri 1.83 MT – – – – – – – 4.30 – – – – – –
Streptomyces variabilis 97 MW490711 – – – – – 4.00 – 4.00 – – – – – –
Yeasts Access code 0 12 22 36 46 70 118 0 12 22 36 46 70 118
Candida tropicalis 1.70 MT – – 2.00 – – – – – – – – – – –
Hanseniaspora opuntiae 1.81 MT – – – – – – – – – – – – – 3.00
Hanseniaspora uvarum 97 MW465986 – – – – – – – – – – – – – 3.70
Meyerozyma caribbica 99 MW465987 – – – – – – – – – – 3.00 3.20 3.15 –
Meyerozyma guilliermondii 99 MW465988 2.00 2.90 2.00 2.48 – – 3.15 2.41 3.71 2.66 3.30 3.18 3.61 4.54
Pichia kluyveri 98 MW465989 – 2.00 – – – – 3.20 – – – – 2.00 – –

– not detected, S6 Southeast-facing slope/600 m, N6 Northwest-facing slope/600 m, MT identification by MALDI-TOF with a score greater than 1.7
World Journal of Microbiology and Biotechnology (2021) 37:51
World Journal of Microbiology and Biotechnology (2021) 37:51 Page 9 of 12  51

1.6
8

A 1.4
A
7

1.2
6
1

Glucose (mg/g)
Acec acid ((mg/g)

5
0.8
4
0.6
3
0.4
2
0.2
1
0
0 0 20 40 60 80 100 120
0 20 40 60 80 100 120 Drying me (h)
Drying me (h)
6

1 B
0.9 B 5

0.8
4
0.7

Sucrose (mg/g)
Succinic acid (mg/g)

0.6 3

0.5
2
0.4

0.3 1

0.2
0
0.1
0 20 40 60 80 100 120
0 Drying me (h)
0 20 40 60 80 100 120
Drying me (h)
5

4.5 C
Fig. 2  Acetic (a) and succinic acids (b) concentration (mg/g) dur- 4
ing drying of coffee cherries. Southeast-facing slope/300  m (filled
3.5
triangle), Northwest-facing slope/300 m (filled diamond), Southeast-
Fructose (mg/g)

facing slope/600  m (filled circle) and Northwest-facing slope/600  m 3

(filled square) 2.5

The same samples from the present study were submitted to 1.5

sensory analysis and we observed that the 600 m coffees had 1

higher scores in the cup tasting (around 85), carried out by 0.5

trained tasters, when compared to the 300 m coffees (around 0

77) (Pereira et al. 2020).
A difference in species diversity was observed between
altitudes. This microbial succession is observed during the
fermentation of coffee of all varieties. Vilela et al. (2010)
and Evangelista et al. (2015), also observed these differ-
ences in species and describe that these changes occur due
to environmental changes and sequences of reactions that
take place during coffee fermentation. Both authors mention
that yeasts are important during fermentation because they
hydrolyze the mucilage and pectin present in coffee beans,
in addition to being precursors of some volatile compounds.
Also, bacteria play a role during the process because they
have pectinolytic and cellulolytic activity, in addition to
being organic acid producers.
In the present study, the genus Bacillus presented
the highest number of bacterial isolates. This genus is
0 20 40 60 80 100 120
Drying me (h)
Fig. 3  Glucose (a), sucrose (b), and fructose (c) concentration (mg/g)
during fermentation and drying of coffee cherries. Southeast-facing
slope/300  m (filled triangle), Northwest-facing slope/300  m (filled
diamond), Southeast-facing slope/600  m (filled circle), and North-
west-facing slope/600 m (filled square)
commonly found in soils. It can secrete enzymes, such as
cellulases, amylases, proteases, which may be necessary
for the fermentation process (Soriano et al. 2000) and has
already been reported as predominant in Arabica coffee
(Silva et al. 2008). R. ornithinolytica (formerly Klebsiella
ornithinolytica) was described by Iswanto et al. (2019) as
a caffeine-degrading bacteria. Enterobacter, Enterobacte-
riaceae, Pantoea ananatis, and Pantoea sp. were the bacteria

13
51 
Page 10 of 12
Fig. 4  Principal component analysis of the volatile compounds of
roasted coffee beans. Southeast-facing slope/300 m (S3), Northwest-
facing slope/300  m (N3), Southeast-facing slope/600  m (S6), and
Northwest-facing slope/600 m (N6). (1) 1,4-Dimethoxy-2-methylben-
zene; (2) 1-Acetoxybutan-2-one; (3) 2,2,4,6,6-Pentamethyleptane; (4)
2,3,5-Trimethylpyrazine; (5) 2,3-Dimethylpyrazine; (6) 2,5-Dimeth-
ylpyrazine; (7) 2,6-Diethyl-3-methylpyrazine; (8) 2,6-Dimethylpyra-
zine; (9) 2–3 Methyltetrahydrofuran; (10) 2-Ethyl-3,6-dimethylpyra-
zine; (11) 2-ethyl-3-methylpyrazine; (12) 2-Ethyl-5-methylpyrazine;
(13) 2-Furanylmethanol; (14) 2-Hexyl-oct-1-ene; (15) 2-Methyl-
pyrazine; (16) 2-Pyrrolecarboxaldehyde; (17) 3-Methyltridecane;
(18) 4-Ethenyl-2-methoxyphenol; (19) 5-Methylfurfural; (20) Ace-
toxyacetone; (21) alpha-Copaene; (22) bis (2-ethylhexyl) ether; (23)
Caffeine; (24) Dioctylether; (25) Dodecan-1-ol; (26) Salicylic Ether;
(27) Ethylpyrazine; (28) Furfural; (29) Heptan-2-ol; (30) Hexame-
thyl-pyranoindane; (31) n-Dodecane; (32) N-Furfurylpyrrole; (33)
n-Undecane; (34) Pentadecane; (35) Pyrazine; (36) Pyridine
genera identified by Hamdouche et al. (2016) in dry pro-
cessed Robusta coffee beans. C. cellulans presents a high
capacity to release enzymes, such as cellulases and xyla-
nases (Song and Wei 2010).
Yeasts can produce secondary metabolites such as esters,
ketones, aldehydes, and organic acids, thus affecting coffee
aroma and flavor (Silva et al. 2013). M. guilliermondii was
present since the beginning of drying/fermentation process
and remained until the end it. This species was previously
isolated from Robusta coffee (Hamdouche et al. 2016) and
cocoa (Meersman et al. 2013) and has been studied as a
fungicide (Coda et al. 2013).
Pichia kluyveri was present only in samples from 600 m
altitude, which may suggest that these species are more
adapted to such environments. P. kluyveri has also been iso-
lated during C. arabica fermentation in Brazil (de Carvalho
Neto et al. 2017). The predominance of P. kluyveri was also
observed during the processing of Arabica coffee from East
13
World Journal of Microbiology and Biotechnology (2021) 37:51
Africa (Masoud et al. 2004). Vilela et al. (2010) evidenced
that in Arabica coffee cultivated at 750–800 m of altitude,
one of the most frequently occurring species belonged to
the Pichia genus. C. tropicalis and C. parapsilosis were also
identified in dry processed Robusta coffee from the Republic
of the Congo (Pee and Castelein 1971). The presence or
absence of specific species were observed during drying/
fermentation (Tables 1 and 2). This fact could be related to
the low population at one or more stages of the process and
the limits of cultivation-dependent techniques used. Besides
that, environmental factors can also alter the microbiota dur-
ing coffee fermentation.
The organic acids identified were acetic and succinic.
Lactic acid was not detected, despite the presence of LAB,
probably due to the limit of detection of the HPLC tech-
nique. The concentration of organic acids and sugar varied
during drying/fermentation in the four environments studied.
Acetic acid was produced in higher concentration in samples
from 300 m. The production of acetic acid is an aerobic
metabolic process that may be the product of ethanol oxida-
tion produced by yeasts and heterofermentative LAB, such
as Leuconostoc mesenteroides (Leong et al. 2014). Acetic
acid is greatly accumulated during fermentation at warmer
ambient temperatures (> 22 °C) (Evangelista et al. 2014).
The daily temperature during drying/fermetation of the cof-
fees varied from 18 and 31.5 °C for S3, 20.5 and 26 °C for
N3, 18.8 and 26 °C for S6 and, 18.8 and 26 °C for N6. In the
last days of drying/fermentation of S3, the average tempera-
ture was around 30 °C, which probably influenced the accu-
mulation of acetic acid. The bacteria present on the skin of
coffee cherries produce this acid, which can then diffuse into
the pulp and mucilage, and may interfere with the sensory
quality of the final beverage (Silva et al. 2013). Succinic acid
is naturally present in the coffee cherry or can be produced
by Bacillus spp. (Silva et al. 2013) and by heterofermenta-
tive lactic acid bacteria (Swiegers et al. 2005), which were
identified in this study. The simple presence of organic acids
is not responsible for the interference with the final quality
of the beverage. However, the concentration of such acids
may interfere on it. Natural coffees of good quality favor
the occurrence of lactic and acetic fermentation, which are
desirable (Ferrão et al. 2019). The diffusion of these acids
into the coffee bean could influence the beverage flavor and
quality (Silva et al. 2013; Di Donfrancesco et al. 2014).
Sugars are precursors of flavor and aroma as they are key
participants of Maillard and caramelization reactions. In the
present study, an increase in the sugars concentration was
observed in the first 12 h of fermentation. Such increase
could be explained by the presence of microorganisms that
degrade pulp and mucilage, thus transforming them into
sugar. In the sample N3, for example, glucose concentra-
tion increased, whereas sucrose concentration decreased at
22 h of drying/fermentation.
World Journal of Microbiology and Biotechnology (2021) 37:51 Page 11 of 12  51
The volatile compounds identified in the roasted cof-
fee beans differed between the samples S3, N3, S6, and
N6 (Fig. 4). Some species of microorganisms may have
influenced this variation that appeared only in some sam-
ples (Tables 1 and 2), such as the genus Lactobacillus sp. in
sample S3, B. altitudinis, B. safensis, D. nishinomiyaensis,
E. hormaechei, E. bacterium, Salmonella sp., and S. vari-
abilis in sample S6, B. shackletonii, C. cellulans, M. luteus,
and M. yunnanensis in sample N3, and A. pittii, E. pallens,
and P. putida in sample N6. Some volatile compounds were
strongly associated with a specific sample (Fig. 4), such
as S6 with the compounds 2,6-dimethylpyrazine, 2-meth-
ylpyrazine, and caffeine. In samples N6, the compounds
2,5-dimethylpyrazine, 2-furanylmethanol, 5-methylfurfural,
N-furfurylpyrrole and dioctylether were related, while for S3
the compound pyridine was found to be associated and for
N3 the compound 2-ethyl-3,6-dimethylpyrazine. Volatiles
belonging to the pyrazine class were the most commonly
compounds found in roasted coffees. Regardless of the
sample, a compound from the pyrazine group was present.
Pyrazines were the main volatile compounds found in the
samples and can provide the flavor notes of nutty, chocolate,
cocoa, and coffee-like (Caporaso et al. 2018). These com-
pounds can be produced by bacteria of the genus Bacillus
(Müller and Rappert 2010) and also by the process of roast-
ing the grains (Caporaso et al. 2018). Aldehydes and furans
were detected and are involved in the production of flavors
observed in cup tasting, for both Arabica and Robusta coffee
(Yeretzian et al. 2002; Andueza et al. 2006; Caporaso et al.
2018). A clear separation between the coffees from 300 to
600 m was observed by the first principal component (PC1).
Some compounds related to 600 m coffees as 2,6-dimethyl-
pyrazine, 2-methylpyrazine, 2,5-dimethylpyrazine, 5-meth-
ylfurfural have chocolate, cocoa, roasted nuts, nutty, spice,
caramel and maple as sensory descriptors. On the contrary,
pyridine, that was related to 300 m coffees, have sensory
descriptors of sour, putrid, fishy, amine, bitter and roasted
(Caporaso et al. 2018).
Conclusion
Information about microbial diversity is crucial to better
understand the coffee quality and distinct characteristics
of coffee produced in different environments. The coffees
from different environments presented a diverse and specific
microbiota. This microbial diversity influenced the composi-
tion of organic acids and volatile compounds present in the
samples. The differences were more pronounced in samples
from different altitudes. The 600 m coffees showed higher
counts in the yeasts and LAB populations at the end of dry-
ing/fermentation. Another difference observed was the pres-
ence of the yeast P. kluyveri only in samples from 600 m.
The volatile compounds identified in the highest altitude
samples are linked to desirable sensory descriptors in cof-
fee. Future researches are planned to use the microorganism
isolated here as starter cultures for C. canephora fermenta-
tion to investigate if it can help in the obtention of coffee
beverages with differentiated sensory profiles.
Supplementary Information  The online version of this article (https​://
doi.org/10.1007/s1127​4-021-03017​-2) contains supplementary mate-
rial, which is available to authorized users.
Acknowledgements  The authors are also grateful to the producers of
coffee.
Funding  This work was supported by the Brazilian Federal Agency
for Support and Evaluation of Postgraduate Education  (CAPES),
with granting scholarships; and by the Foundation for Research Sup-
port of Espirito Santo State (FAPES) (Grant Numbers 730/2016 and
108/2019).
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
Ethical approval  This article does not contain any studies with human
participants or animals performed by any of the authors.
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