International Journal of Food Microbiology 321 (2020) 108544

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Ecological diversity, evolution and metabolism of microbial communities in T

the wet fermentation of Australian coffee beans
Hosam Elhalis, Julian Cox, Jian Zhao
⁎

Food Science and Technology, School of Chemical Engineering, The University of New South Wales, Sydney, NSW 2052, Australia

ARTICLE INFO ABSTRACT

Keywords: The microbial ecology in the fermentation of Australian coffee beans was investigated in this study. Pulped
Coffee fermentation coffee beans were kept underwater for 36 h before air dried. Samples were collected periodically, and the
Microbial ecology microbial communities were analyzed by culture-dependent and independent methods. Changes in sugars, or-
NGS ganic acids and microbial metabolites in the mucilage and endosperm of the coffee beans during fermentation
Microbial metabolism
were monitored by HPLC. Culture-dependent methods identified 6 yeast and 17 bacterial species, while the
culture-independent methods, multiple-step total direct DNA extraction and high throughput sequencing,
identified 212 fungal and 40 bacterial species. Most of the microbial species in the community have been re-
ported for wet fermentation of coffee beans in other parts of the world, but the yeast Pichia kudriavzevii was
isolated for the first time in wet coffee bean fermentation. The bacterial community was dominated by aerobic
mesophilic bacteria (AMB) with Citrobacter being the predominant genus. Hanseniaspora uvarum and Pichia
kudriavzevii were the predominant yeasts while Leuconostoc mesenteroides and Lactococcus lactis were the pre-
dominant LAB. The yeasts and bacteria grew significantly during fermentation, utilizing sugars in the mucilage
and produced mannitol, glycerol, and lactic acid, leading to a significant decrease in pH. The results of this study
provided a preliminary understanding of the microbial ecology of wet coffee fermentation under Australian
conditions. Further studies are needed to explore the impact of microbial growth and metabolism on coffee
quality, especially flavour.

1. Introduction
Coffee is one of the most popular and widely consumed beverages
worldwide due to its rich and complex flavour and other sensory
characteristics (Chin et al., 2015). Postharvest processing of coffee
cherry is believed to have a significant impact on the flavour and other
quality attributes of coffee, although the exact effects are not well un-
derstood. The main objective of primary coffee processing, which can
be done either by the dry or wet method, is to remove the flesh of the
coffee cherries and preserve the beans by reducing the moisture content
of the beans to a stable level of about 12% (Borém et al., 2008; Sfredo
et al., 2005). In the dry process, whole cherries are dried for up to
30 days to reach the moisture level and then the beans are mechanically
separated from the surrounding layers (Silva et al., 2000; Silva et al.,
2008). In the wet process, the pulp is removed by machine, but part of
the sticky mucilage remains attached to the bean parchment. This sticky
mucilage is removed either by wet fermentation or mechanically by a
de-mucilager (Brando, 1999; Silva, 2014).
In the wet process, the pulped beans are kept underwater for 6–72 h
before being dried (Knopp et al., 2006; Silva, 2014). A wide range of
microorganisms have been isolated from wet coffee fermentation in
different regions, including yeasts, lactic acid bacteria (LAB), acetic
acid bacteria (AAB), Bacillus spp., Enterobacteriaceae and, occasionally,
filamentous fungi (Avallone et al., 2001; Pereira et al., 2015; Pereira
et al., 2016). Many species of microorganisms are common to fermen-
tation in different regions, but region-specific species have also been
found. For example, in Brazil, a rich diversity of yeasts was found in
both wet and dry coffee fermentation throughout the process, where
Debaryomyces hansenii, Pichia ofunaensis, P. anomala, and P. fermentans
were the predominate species (Pereira et al., 2014; Silva et al., 2000;
Silva et al., 2008; Vilela et al., 2010). In India, Saccharomyces marx-
ianus, S. cerevisiae, and Schizosaccharomyces were the most reported
isolates (Agate and Bhat, 1966; Velmourougane, 2013). Cryptococcus
and Kloeckera species were detected during wet coffee fermentation in
Mexico (Avallone et al., 2001), while Hanseniaspora uvarum and Can-
dida species were reported in East Africa (Masoud et al., 2004). In West
Africa (Cameroon), Hanseniaspora uvarum, Cladosporium sphaer-
ospermum, Candida quercitrusa, Pichia, Aspergillus, Penicillium and

⁎
Corresponding author.
E-mail address: jian.zhao@unsw.edu.au (J. Zhao).

https://doi.org/10.1016/j.ijfoodmicro.2020.108544
Received 13 November 2019; Received in revised form 27 January 2020; Accepted 28 January 2020
Available online 30 January 2020
0168-1605/ © 2020 Elsevier B.V. All rights reserved.
H. Elhalis, et al.
Saccharomycetes were detected using denaturing gradient gel electro-
phoresis (DGGE) technique of the total direct DNA extracted from dry
and wet-processed coffee (Hamdouche et al., 2016).
Similarly, lactic acid bacteria also displayed high diversity for wet
coffee fermentation in different regions. Lactobacillus plantarum and
Lactobacillus sp. were the predominant isolates detected in both Brazil
and India (Silva et al., 2000; Vilela et al., 2010; Velmourougane, 2013).
In Mexico, Ethiopia, Taiwan, and Hawaii, Leuconostoc species were the
most reported isolates (Leong et al., 2014; Schillinger et al., 2008).
Weissella, Leuconostoc mesenteroides, Lactococcus lactis subsp. lactis and
Lactobacillus fermentum were reported during both dry and wet fer-
mentation processes in west Africa (Hamdouche et al., 2016). Many
bacterial genera other than LAB have also been detected in coffee fer-
mentation such as Klebsiella, Acinetobacter, Bacillus, Escherichia, Pseu-
domonas and Serratia (Agate and Bhat, 1966; Avallone et al., 2001;
Frank et al., 1965; Silva et al., 2000). Therefore, information on the
microbial ecology of coffee bean fermentation is crucial not only for
understanding the impact of microbial growth and metabolism on
coffee quality in general, but also the different characteristics of coffee
produced in different regions.
The world market of coffee production is enormous with an esti-
mated 170 million bags of coffee produced in 2018. Brazil is considered
the leading producer of coffee beans with 58 million bags of coffee,
followed by Vietnam and Columbia (International Coffee Organization,
2019). Australia has a small but growing coffee plantation and pro-
cessing industry. Coffee beans are grown in tropical areas of Queens-
land, subtropical areas in southeast Queensland and northeast New
South Wales. Coffea arabica is the species mostly grown in Australia,
where Catuai Rojo and K7 are the predominant varieties used
(Agrifutures Australia, 2017). Several factors potentially facilitate the
continuous improvement of the coffee industry in Australia; for in-
stance, the country is free from two serious coffee diseases, namely
coffee berry disease and coffee rust that can cause severe crop losses. In
addition, the coffee beans are characterized by low caffeine con-
centrations (10–15% less) and being grown in a cold environment with
a long ripening time that produces high-quality beans (Australian
Subtropical Coffee Association, 2020). Harvesting is conducted be-
tween June to October and performed mostly with a machine. The wet
method is considered the most common way to process coffee cherries
in Australia and performed in the same way as discussed above. Cur-
rently, 50% of the coffee produced in Australia is consumed locally, and
the remaining for exporting internationally (Agrifutures Australia,
2017). Virtually no information is available on the microbial ecology of
Australian coffee fermentation as no such studies have been reported.
The objective of this study is to investigate the microbial ecology and
metabolism of wet coffee fermentation under Australian conditions
(harvested mechanically, fermented for 36 h, dried mechanically to
reach 12% moisture content, the average daily temperature was 20 °C).
2. Materials and methods
2.1. Coffee cherry collection, bean fermentation, drying and sampling
Coffee fruits (40 kg) (Coffea arabica var. Bourbon) were mechani-
cally harvested from the Kahawa Estate Coffee farm in Teven, NSW,
Australia in October 2018 (latitude and longitude coordinates,
−28.816667, 153.500000; altitude, 6.6 m above sea level). Freshly
harvested coffee cherries were immediately packed with ice in a poly-
styrene foam container, airfreighted to our laboratory at UNSW Sydney
and immediately de-pulped manually under aseptic conditions, with the
whole operations completed within 24 h. The cherries were first packed
(200 g) in sterile plastic bags, then pressure applied on the bags to
squeeze and remove the cherries pulps from the beans. Sterile gloves
were used to separate the beans from the discard pulps. The beans were
then divided into two equal parts of about 5 kg each into two plastic
boxes containing 15 L of tap water and allowed to ferment for 36 h,
2
International Journal of Food Microbiology 321 (2020) 108544
following the local standard practice used, at ambient temperatures
which were 20–30 °C during daytime and 10–15 °C during the night.
The coffee beans were mixed manually every 6–12 h during the fer-
mentation, and triplicate samples of the beans (about 30 g each) were
taken at 0, 12, 24 and 36 h for microbiological examination by the
traditional culture methods. Another set of triplicate samples (about
30 g each) was also taken and frozen (−20 °C) for molecular and
chemical analyses. Sampling was performed from three points (middle,
upper and bottom) of the tank and was mixed evenly to represent the
whole tank. A batch of mechanically de-mucilaged beans (3 kg), which
were processed by the local farmer with the coffee cherries harvested
from the same farm on the same day, were collected, kept in ice and
transported to our laboratory with the fresh coffee cherries, and was
used as control. Chemicals used in all experiments were purchased from
Sigma-Aldrich (Sydney, Australia) and are of analytical grade unless
stated otherwise. MilliQ water was used in all chemical analyses unless
stated otherwise.
After the fermentation was completed, the parchment beans were
washed three times with tap water and spread on a perforated try which
was placed inside a laboratory air dryer at 45 °C until the moisture
content reached 12% (w/w). The moisture content of the coffee beans
was measured using a vacuum oven at 70 °C for 48 h according to the
AOAC method (Dick, 1990). Dried beans were vacuum-packed in
plastic bags and stored at −20 °C until further processing or analysis.
2.2. pH measurement and total titrability acidity
The pH of the fermenting mass was measured in real-time using a
portable pH meter (pH Cube, TPS Pty Ltd., Brisbane, QLD, Australia).
The pH and total titrability acidity of the coffee beans were determined
following Angelucci et al. (1982) with some modifications. Samples
(25 g) of ground fermented and mechanically de-mucilaged beans were
mixed with 10 ml of 80 °C hot MilliQ water, cooled to room tempera-
ture and the pH was measured using the same pH meter as described
above. To determine the total titratable acidity, 10 g of ground green
coffee were mixed with 80% ethanol (v/v) and shaken for 16 h, and
2.5 ml aliquots were diluted to 10 ml with MilliQ water. The acidity
was determined by titration with 0.1 N sodium hydroxide with three
drops of phenolphthalein as the indicator. The data reported were
means of duplicate extraction and analyses with standard deviation.
2.3. Enumeration of microorganisms
Samples of coffee beans (25 g) were mixed with 225 ml of saline-
peptone water containing 0.1% w/v bacteriological peptone and 0.8%
w/v NaCl (Sigma, Sydney, Australia) in stomacher bags, which were
manually messaged for 3 min, stomachered for another 2 min at normal
speed and then serially diluted. Plate count agar (all culture media and
supplements were from Oxoid, Sydney Australia) containing 0.1% cy-
cloheximide, to inhibit fungal growth, was used to obtain the viable
aerobic mesophilic bacterial counts. Lactic acid bacteria were en-
umerated with de Man Rogosa Sharpe (MRS) agar and M17 agar with
0.2% cycloheximide to ensure the inhibition of yeast growth under
anaerobic conditions in candle jars. Filamentous fungi were cultured in
malt extract agar (MEA) and potato dextrose agar (PDA), both con-
taining 50 mg/l each of oxytetracycline and chloramphenicol to sup-
press bacterial growth, and dichloran rose bengal chloramphenicol
(DRBC) agar. The agar plates were incubated at 30 °C for 48 h for
bacteria and up to 7 days for yeasts and filamentous fungi. After in-
cubation, the microorganisms were enumerated as groups (bacteria,
yeasts, and filamentous fungi) as well as individual species based on the
colony and cell morphology. The number of colony-forming units (CFU)
was recorded and at least 3 colonies from each type were purified by
streaking on suitable media at least 4 times. The reported population
data are the means of duplicate analyses within a standard deviation
of ± 0.05.
H. Elhalis, et al.
2.4. Identification of microbial isolates
2.4.1. Conventional techniques
Wet mounts were used to determine the cellular morphologies of the
isolates. Gram staining, catalase, and growth on MRS agar with CaCO3
were conducted to confirm LAB. Further identifications were performed
by rRNA sequencing as described below.
2.4.2. Culture-dependent identification of microbial isolates
2.4.2.1. DNA extraction. DNA extraction from the purified bacterial
and yeast colonies followed the protocol of Cocolin et al. (2002) with
some modifications. LAB was grown in MRS broth, other bacteria in
tryptone soya broth (TSB) and yeasts in malt extract (ME) broth
overnight at 30 °C in shaking incubators set at 120 rpm, and 1.5 ml
of the broth culture was centrifuged in an EBA12 Centrifuge (Hettich,
Newport Pagnell, Buckinghamshire, UK) at 13,000 rpm for 10 min. The
supernatant was discarded and 200 μl of lysis buffer (1% SDS, 2%
Triton X-100, 1 mM EDTA at pH 8, 100 mM NaCl, 10 mM Tris-Cl at
pH 7.6) was added and the mixture was incubated at 30 °C for 40 min.
After that, 300 mg of sterile glass beads (0.1 mm and 0.5 mm diameters
for bacteria and yeasts respectively) were added and mixed in a bead
beater (Bio Spec Products Inc., Bartlesville, OK, USA) at 5000 rpm for
1 min. Then, 500 μl of phenol/chloroform/isoamyl alcohol (50,48:2)
was added and mixed for 1 min. The homogenate was centrifugated
(17,000 ×g, 10 min). The upper phase (aqueous) was transferred to a
new centrifuge tube and the DNA precipitated by the addition of 1
volume of isopropanol or 2 volumes of absolute ethanol. The
precipitated DNA was centrifugated (14,000 rpm for 10 min) and
washed with 200 μl 70% ethanol twice then air-dried. 50 μl of TE buffer
was added to the pellet and incubated at 55 °C for 30 min to completely
dissolve and rehydrate the DNA pellet. The concentration and purity of
the extracted DNA were determined with a UV-1800 UV–Vis
Spectrophotometer (Shimadzu Scientific Instruments) at 260 and
280 nm, respectively.
2.4.2.2. Amplification of DNA by polymerase chain reaction (PCR). Bacterial
and fungal DNA group-specific loci were amplified using PCR. The V3
region of the 16S rRNA gene was amplified with the primer pairs F338fgc
(5′ GAC TCC TAC GGG AGG CAG CAG 3′) and R518 (5′ATT ACC GCG GCT
GCT GG3′) (Ovreas et al., 1997). The PCR assay conditions consisted of an
initial step at 95 °C for 3 min, followed by 40 cycles at 95 °C for 30 s, 57 °C
for 30 s and 72 °C for 60 s. A final extension was performed at 72 °C for
7 min. The 5.8S-ITS rRNA gene region of the fungal isolates was amplified
using the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4
(5′-TCCTCCGCTTATTGATATGC-3′) (Esteve-Zarzoso et al., 1999). The
PCR conditions consisted of an initial step at 98 °C for 5 min, followed
by 35 cycles of denaturation at 98 °C for 30 s, annealing at 56 °C for 30 s,
and extension at 72 °C for 50 s. A final extension was conducted at 72 °C
for 7 min. Hot Start Taq 2× Master Mix (New England Biolabs, Ipswich,
MA, USA) was used according to manufacturer instructions. The PCR was
performed in a thermocycler (C1000 Thermal Cycler, Bio-Rad, Hercules,
CA, USA) under the described conditions.
The PCR products were checked by electrophoresis on 1.5% agarose
gel, stained with ethidium bromide and photographed using a UV
transilluminator (Alpha Innotech Corporation, San Leandro, CA, USA).
The PCR products and 9.6 pmol of respective forward primers were sent
to Ramaciotti Centre for Genomics at UNSW (Kensington, Sydney,
NSW, Australia) for sequencing. The sequencing results were compared
with the microbial sequence data in the Gen Bank through NCBI
(http://www.ncbi.nlm.nih.gov/BLAST/).
2.4.3. Culture-independent analysis of microbial diversity
2.4.3.1. Total DNA extraction from coffee bean samples. Samples (25 g)
of thawed coffee beans was added to 225 ml of 0.9% sterile saline
solution (Merck, Darmstadt, Germany) and manually inverted for 3 min
then stomached for 2 min to detach the microorganisms from the bean
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International Journal of Food Microbiology 321 (2020) 108544
surface. The mixture was centrifuged at 2000 rpm for 2.5 min to
remove the coarse impurities. The supernatant was centrifuged at
13,000 rpm for 15 min to precipitate the microbial cells. The cell
pellet was washed with 50 mM phosphate buffer (pH 6.0) and
resuspended in 200 μl of 50 mM phosphate buffer (pH 6.0)
containing various cell lysis enzymes. For fungal cells, the lysis
enzymes consisted of chitinase (500 mU/ml) and lyticase (0.2 U), and
the mixture was incubated at 30 °C for 3 h. For bacterial cells, the lysis
enzyme was lysozyme (8 mg Merck) and the mixture was incubated at
30 °C for 1 h. This was followed by chemical lysis by adding 200 μl of
breaking buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-
Cl pH 7.6 and 1 mM EDTA pH 8) to the mixtures and vortexed for
1 min. The last step was physical lysis by sonication for 1 h at room
temperature.
Protein in the samples was removed by the addition of 0.5 mg
proteinase K (Merck, Sydney, Australia) and incubated at 56 °C for 1 h.
After that, 40 μl of 20% cetyltrimethylammonium bromide solution was
added, followed by incubation of the mixture at 65 °C for 30 min to
digest the carbohydrates. Then, 500 μl of chloroform-phenol-isoamyl
alcohol solution (49.5:49.5:1.0) was added and the mixture was shaken
for 1 h. The upper phase was separated by centrifugation at 13,000 rpm
for 10 min. Finally, 200 μl of absolute ethanol was added to precipitate
the extracted DNA, which was purified by using the DNeasy Blood and
Tissue kit (Qiagen, Venlo, The Netherlands) according to the manu-
facturer's instructions. The DNA concentration was measured by a
spectrophotometer at UV 260 nm while protein contamination was
checked at 280 nm.
2.4.3.2. Amplification of group-specific loci for high throughput sequencing
(HTS). The extracted DNA was sent to AGRF Empowering Australian
Genomic for identification of amplicon profile diversity following the
manufacturer specifications and AGRF protocols. In brief, the
V3–V4 region of the 16S rRNA gene amplified using primers 341F
(5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGAC-
TACNNGGGTATCTAAT-3′) for bacteria, and ITS region using ITS1F
(5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (5′-GCTGCGTTCTT-
CATCGATGC-3′) primers for fungi. The PCR assay conditions for
bacteria consisted of an initial step at 95 °C for 7 min, followed by
29 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 60 s,
and extension at 72 °C for 60 s. A final extension was performed at 72 °C
for 7 min. The PCR assay conditions for fungi consisted of an initial step
at 95 °C for 7 min, followed by 35 cycles of denaturation at 94 °C for
30 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s. A final
extension was performed at 72 °C for 7 1 min. Thermocycling was
performed by an Applied Biosystem 384 Veriti and using AmpliTaq
Gold 360 (Life Technologies, Australia) for the primary PCR. The first
stage PCR was cleaned using magnetic beads, and samples were
visualised on 2% Sybr Egel (Thermo-Fisher). A secondary PCR to
index the amplicons was performed with TaKaRa PrimeStar MAX
DNA Polymerase (Clontech). The resulting amplicons were cleaned
again using magnetic beads, quantified by fluorometry (Promega
Quantifluor) and normalised. The equimolar pool was cleaned a final
time using magnetic beads to concentrate the pool and then measured
using a High-Sensitivity D1000 Tape on an Agilent 2200 TapeStation.
The pool was diluted to 5 nM and molarity was confirmed again using a
Qubit High Sensitivity dsDNA assay (ThermoFisher). This was followed
by sequencing on an Illumina MiSeq (San Diego, CA, USA) with a V3,
600 cycle kit (2 × 300 base pairs paired-end). Data quality control was
performed as follows. Paired-end reads were assembled by aligning the
forward and reverse reads using PEAR (version 0.9.5) (Zhang et al.,
2013). Primers were firstly identified and then trimmed. The trimmed
sequences were processed using Quantitative Insights into Microbial
Ecology (QIIME 1.8) (Caporaso et al., 2010) USEARCH (version
7.1.1090) (Edgar, 2010; Edgar et al., 2011) and UPARSE (Edgar,
2013) software. Using search sequences that were quality filtered,
full-length duplicate sequences were removed and sorted by
H. Elhalis, et al. International Journal of Food Microbiology 321 (2020) 108544

abundance. Singletons or unique reads in the data set were discarded. Table 1
Sequences were clustered, followed by chimera filtered using the Identification of the bacteria and yeasts isolated from wet fermentation of
“rdp_gold” database as the reference for 16S analysis and “Unite” for coffee beans.
fungi. To obtain the number of reads in each OTU, reads were mapped Isolates Identity %a Accessionb
back to OTUs with a minimum identity of 97%. Qiime taxonomy was
assigned using the Greengenes database (version 13_8, Aug 2013) Yeasts
Hanseniaspora uvarum 100 MF574306.1
(DeSantis et al., 2006). The extraction and analysis were done in
Pichia kudriavzevii 99 CP021092.1
duplicate and the results were reported as an average. Candida xylopsoci 100 KJ706861.1
Candida railenensis 100 HQ438305.1
2.5. High-performance liquid chromatography (HPLC) metabolites analysis Pichia fermentans 99 KM402059.1
Wickerhamomyces anomalus 99 KY587120.1

HPLC analysis of microbial metabolites was carried out for both Bacteria
mucilage and endosperm (bean). The mucilage layer was scraped off Acinetobacter lwoffii 99 MF988732.1
Enterobacter ludwigii 100 KY474547.1
from the beans manually, frozen with liquid nitrogen and ground by Citrobacter koseri 99 CP026709.1
mortar and pestle, while the endosperm was milled with a freeze miller Pseudomonas fluorescens 100 EU822884.1
into a fine powder then sieved to remove the coarse particles. All Klebsiella pneumoniae 99 CP020847.1
samples were analyzed in triplicate and average values were reported. Erwinia soli 100 MH107054.1
Serratia marcescens 100 MF440338.1
Each sample (3 g) was mixed with 30 ml of MilliQ water and
Brevibacillus parabrevis 100 MF996770.1
blended in a domestic blender (John Morris Scientific, Sydney, NSW, Anabaena 100 JQ964321.1
Australia) for 40 min. The slurry was centrifuged in an Avanti J-E Salmonella enterica 99 MH236213.1
Centrifuge (Beckman Coulter, Indianapolis, IN, USA) at 13,000 rpm for Asaia sp. 100 LT838398.1
15 min at 5 °C, and the supernatant was retained. The sediment was Serratia marcescen 99 FM207539.1
Brevibacillus parabrevis 100 MF996770.1
washed twice, each with 10 ml MilliQ water, and all the supernatants Acetobacter persici 100 MH285266.1
were pooled and filtrated through a 0.45-micron nylon filter before Gluconobacter cerinus 100 MK049954.1
injection into HPLC. Leuconostoc mesenteroides 100 MK290364.1
Alcohols, sugars, and organic acids were determined by a Shimadzu Lactococcus lactis 99 MF623273.1
HPLC system comprising a LC-20AD pump, a SIL-20A HT auto-injector, a
The percentage of the identical nucleotides in the sequence obtained from
a SPD-M20A photodiode array detector (DAD), a RID-10A detector and
PCR products and the sequence found in National Center for Biotechnology
a Rezex ion-exclusion ROA organic acid column (300 × 7.8 mm, Information (NCBI); isolates were assumed to belong to a given species if the
Phenomenex, Torrance, CA, USA) hosted in a column heater. A Carbo-H similarity was higher than 98%.
+ guard column (4 mm × 3 mm, Phenomenex, Torrance, CA, USA) b
GenBank accession numbers of the representative isolates.
was used to protect the column. The mobile phase used was 0.06 M
H2SO4 with a flow rate of 0.4 ml/min at 50 °C for organic acids and g within 36 h. The population of Acinetobacter persici was similar to that
30 °C for other compounds. Organic acids were detected by DAD at of Erwinia soli at the beginning, but it increased somewhat over the next
210 nm while other metabolites were detected by the refractive index 24 h and then declined to an undetectable level by the end. The po-
detector. The concentration of individual metabolites was calculated by pulations of Klebsiella pneumoniae and the Pseudomonas sp. were both
comparison with standard curves constructed from standard solutions low in the initial fermenting mass at about 2 log CFU/g and remained
of known concentrations of glucose, sucrose, fructose, mannitol, gly- essentially unchanged during fermentation.
cerol, citric, lactic, malic, acetic, quinic and succinic acids. Analyses of There were two isolates of lactic acid bacteria (LAB), Leuconostoc
individual samples were done in triplicate and average values were mesenteroides and Lactococcus lactis, identified in the fermenting mass of
reported within a standard deviation of 0.05. coffee beans. They had an initial population of 3.1 and 2.6 log CFU/g,
which steadily increased in the first 24 h to reach 5.5 and 5 log CFU/g,
3. Results and then dropped to 5 and 4.7 log CFU/g, respectively, at end of fer-
mentation (Fig. 2).
3.1. Microbial ecology of wet fermentation of Australian coffee beans Six yeast species were identified in the fermenting mass of the coffee
beans with an initial total population of approximately 4 log CFU/g,
3.1.1. Evolution of microbial population during fermentation which increased to 5.5 log CFU/g by the end of the fermentation in 36 h
Seventeen bacterial and 6 yeast isolates were identified in the fer- (Fig. 3). Hanseniaspora uvarum, Pichia kudriavzevii, and Candida rail-
menting mass of coffee beans by the culture-dependent methods enensis were the predominant yeasts at .the start of fermentation with a
(Table 1). Figs. 1–3 show the total bacterial and yeast count during the population of 3.6, 3.1 and 3.6 log CFU/g, respectively. Hanseniaspora
wet fermentation. The initial total aerobic mesophilic bacterial (AMB) uvarum led the fermentation, followed by Pichia kudriavzevii, with their
count was about 5 log CFU/g, which grew to 5.3 log CFU/g in 12 h, and levels reaching 5.2 and 4.6 log CFU/g, respectively, after 36 h. The
eventually reached a maximum population of 7.2 log CFU/g by the end initial population of Candida railenensis was similar to that of Hanse-
of the fermentation (Fig. 1). The dominant AMB species present at the niaspora uvarum, which, however, declined quickly in the first 12 h,
beginning of the fermentation were Acinetobacter and Enterobacter reaching a level less than 2 log CFU/g, and thereafter continued a slow
species with 20 and 16 isolates, respectively. The population of Acine- decline to about 1.5 log at the end of fermentation. The initial popu-
tobacter lwoffii was slightly over 4 log CFU/g at the start of fermentation lation of Candida xylopsoci was low at about 2 log CFU/g, which in-
but declined continuously right from the beginning to an undetectable creased slowly over the fermentation to about 2.5 log CFU/g. The re-
level by the end. The population of Enterobacter was similar to that of maining two yeasts, Pichia fermentans and Wickerhamomyces anomalus,
Acinetobacter at the beginning, which remained relatively stable during both had a low initial population of less than 2 log CFU/g, which re-
the first 12 h, but then declined slightly over the rest of fermentation. In mained relatively unchanged for the former but decreased slightly for
contrast, the population of Citrobacter was lower than those of Acine- the latter during the fermentation (Fig. 3).
tobacter and Enterobacter at the start, but it increased steadily over the
fermentation, became the dominant species after 12 h and reached a
maximum level of 7.1 log CFU/g by the end. The level of Erwinia soli 3.1.2. Diversity of microbial communities in coffee bean fermentation
also increased during the fermentation from less than 2 to 5.5 log CFU/ The diversity of the microbial community in the fermenting mass of

4
H. Elhalis, et al. International Journal of Food Microbiology 321 (2020) 108544

7
Total aerobic bacteria
6

Log10 ( CFU/g beans)

Acinetobacter lwoffii
5 Acetobacter persici
4 Gluconobacter cerinus

3 Enterobacter
Citrobacter sp.
2
Pseudomonas sp.
1
Klebsiella pneumoniae
0 Erwinia soli
0 5 10 15 20 25 30 35 40
Fermentaon me (h)

Fig. 1. Changes in the population of aerobic bacteria during the wet fermentation of coffee beans.

6 During fermentation, the OUTs showed an increase in the relative

abundance of Streptococcaceae (especially Lactococcus) and
5
Leuconostocaceae (Leuconostoc) but followed by a significant decline by
the end of fermentation. Furthermore, the abundance of
Log10 ( CFU/g beans)

4
Total LAB count Enterococcaceae (Enterococcus) was low at the start of the fermentation,
3 then a slight rise in the relative abundance was observed during fer-
Leuconostoc mentation progress followed by a decrease at the end.
AABs showed a similar increase in the first 24 h, but then a slight
mesenteroides
2
decrease near the end of fermentation. Serratia was the prevalent read
Lactococcus lac s

among the Enterobacteriaceae family, while Erwinia spp. decreased in

1

0
relative abundance in the first 24 h and disappeared by the end of
0 10 20 30 40 fermentation. Overall, Enterobacteriaceae presented a high relative
Fermentaon me (h) abundance especially in the first 24 h, followed by Leuconostocaceae
(genus Leuconostoc and others) and Streptococcaceae (genus Lactococcus)
Fig. 2. Changes in the population of lactic acid bacteria during the wet fer-
(Fig. 4). Moreover, other bacterial OTUs appeared mainly at the initial
mentation of coffee beans.
stage of the fermentation, sporadically and in relatively low abundance
during the whole fermentation process, such as Pseudomonadaceae
6 (especially Pseudomonas), Paenibacillaceae (Saccharibacillus), Nocardia-
ceae (Rhodococcus) and Methylobacteriaceae (Methylobacterium).
5 Total yeast count Regarding the fungal community, the average number of raw ITS
sequences per sample was 200,000 with estimated sequencing coverage
Hanseniaspora uvarum between 98.3 and 99.7% (Fig. 5). Members of both Ascomycota and
4
Basidiomycota dominated the fungal community in the fermentation.
Log10 CFU/g beans

3 first 24 h but their abundance declined as the fermentation progressed
2 Candida xylopsoci
1
0
0 12
Fermenta on me (h)
Fig. 3. Changes in the population of yeasts during the wet fermentation of
coffee beans.
the coffee beans was examined by cultural independent methods
(Figs. 4 and 5). For the bacterial community, the average number of
raw V3–V4 sequences per sample was 350,000 with estimated se-
quencing coverage between 98.5 and 99.7%. At the beginning of fer-
mentation, the bacterial operational taxonomic units (OTUs) were be-
longing to the Enterobacteriaceae (e.g., Enterobacter, Erwinia, and
Serratia) and Acetobacteraceae (e.g., Gluconobacter spp.). Lactic acid
bacteria species occupied a small portion of the reads with Leuconostoc,
Lactococcus, and Enterococcus being the main genera. The remaining
reads comprised mostly of soil associated bacteria and coffee plant DNA
(Fig. 4).
Pichia kudriavzevii At the initial stage, unclassified Nectriaceae were predominant in the
Candida railenensis further. Other members of phylum Ascomycota (such as Penicillium,
Aspergillus, Cladosporium, Pleosporales, and Neodevriesia) and Basidio-
mycota (for instance, Rhodotorula, Leucospridiella, Sporidiobolus, and
Vishniacozyma) were present only at the early stage and disappeared by
Wickerhamomyces the end of fermentation. The abundance of most yeast species increased
anomalus
steadily during the fermentation with Hanseniaspora uvarum being the
Pichia fermentans predominant member, followed by Candida railenensis, and Wick-
erhamomyces anomalus. A low level of Debaryomyces hansenii was also
24 36
read in the first 24 h, but it disappeared at the end of fermentation
(Fig. 5).
3.2. Changes in chemical composition and microbial metabolites during
coffee bean fermentation
The moisture content of the mucilage and endosperm (bean) of the
freshly harvested coffee cherries was 87% and 50%, respectively. The
initial pH of the fermenting mass was 5.40, which decreased to pH 4.95
in 12 h, and further dropped to 4.27 and 3.6 in 24 and 36 h of fer-
mentation, respectively. The pH value of the coffee beans after fer-
mentation was 4.87, while the value for the mechanically de-mucilaged
beans was significantly higher at pH 6.2. Titratable acidity also had a
significant difference between the wet fermented and the mechanically
de-mucilaged beans with the value for the two samples being 1.44 and
1.70 (mg/g), respectively.

5
H. Elhalis, et al.
100%
90%
80%
70%
60%
Rela ve abundance
50%
40%
30%
20%
10%
0%
0 24
Fermenta on me (h)
Fig. 4. Changes in the relative abundance of bacterial operational taxonomic units (OUTs) in coffee beans during wet fermentation.
Fig. 6 shows the changes in sugars, organic acids, and other major
microbial metabolites in the mucilage and endosperm of the coffee
beans during fermentation. The mucilage initially contained three su-
gars, glucose, fructose and sucrose, and the concentration of which all
decreased during fermentation. Fructose was the most abundant sugar
in the mucilage with an initial concentration of 30%, which dropped to
4% at the end of fermentation. Glucose was the second most abundant
sugar and its concentration decreased from 21% to 9% during the fer-
mentation while that of sucrose decreased from 9% to non-detectable
levels (Fig. 6A). Sucrose was the most abundant sugar in the beans with
an initial concentration of 8.2 g/100 g, which gradually declined to
6.7 g/100 g by the end of fermentation. The initial concentrations of
glucose and fructose in the beans were much lower at 1.3 and 0.9 g/
100 g, which dropped slightly to 0.8 and 0.7 g /100 g, respectively,
during fermentation (Fig. 6B).
A low level of mannitol (0.5 g/100 g) was detected in the mucilage
at the start of fermentation, which increased slowly but continuously
during the process to 0.8 g/100 g at the end. Glycerol was not detected
in the mucilage until 24 h with 0.9 g/100 g, which increased slightly to
1.2 g/100 g by the end of fermentation (Fig. 6A). Very low levels of
mannitol and glycerol were also detected in the beans after 16 h and
6
International Journal of Food Microbiology 321 (2020) 108544
Pseudomonas
Acinetobacter
Serra a
Erwinia
Enterobacter
Enterobacteriaceae, other
Gluconobacter
Acetobacter
Methylobacterium
Lactococcus
Leuconostoc
Leuconostocaceae, other
Pediococcus
Lactobacillaceae
Enterococcus
Saccharibacillus
Rhodococcus
36
24 h of fermentation (Fig. 6B).
At the start of fermentation, the mucilage contained four organic
acids: citric, succinic, quinic and malic acids at 0.9 to 2 g/100 g. The
concentration of citric and malic acids decreased slightly in the first
16 h and then increased slightly afterward. The concentration of suc-
cinic acids increased slightly in the first 16 h but remained relatively
stable afterward. The concentration of quinic acid also showed small
variations during fermentation (Fig. 6C). Lactic acid was detected from
16 h and its concentration increased slowly to 0.4 g/100 g.
Citric, succinic, quinic and malic acids were also found in the en-
dosperm, but their levels are much lower than in the mucilage, except
citric acid, which had an initial concentration of 1.4 g/100 g, which
increased continuously during fermentation to 1.8 g/100 at the end.
The levels of the other three organic acids did not change substantially
during fermentation. Similar to the case for the mucilage, lactic acid
was detected in the bean from 16 h, and its concentration increased
continuously during fermentation to 0.3 g/100 g at the end (Fig. 6D).
Table 2 shows the concentration of sugars and organic acids in the
mucilage and the beans of mechanically de-mucilaged coffee beans.
Compared with beans underwent wet fermentation, the mucilage of the
mechanically de-mucilaged samples had significantly higher
H. Elhalis, et al. International Journal of Food Microbiology 321 (2020) 108544

100%
Hannaella
Papiliotrema
90% Vishniacozyma
Cystofilobasidium
Sporobolomyces
80%
Rhodotorula
Sporidiobolus
Leucosporidiella
70%
Colletotrichum
Nectriaceae Gibberella
60% Nectriaceae Fusarium
Relave abundance

Nectriaceae; Other
Hanseniaspora
50%
Wickerhamomyces
Debaryomyces

40% Candida
Torulaspora
Meyerozyma
30% Penicillium
Byssochlamys
Aspergillus
20%
Ascomycota; Other
Neodevriesia
10% Cladosporium
Cladosporiaceae; Other

0%
1 2 3
Fermentaon me (h)

Fig. 5. Changes in the relative abundance of fungal operational taxonomic units (OUTs) in coffee beans during wet fermentation.

concentrations of reducing sugars, but the levels of citric, succinic,
malic and quinic acids were similar between the two types of mucilage.
The concentration of sucrose inside the mechanically de-mucilaged
beans was 8.2 g/100 g, which was significantly higher than that in the
fermented beans (6.0 g/100 g), but the levels of fructose and glucose
were similar between the two types of beans. As expected, the con-
centrations of lactic acid, glycerol, and mannitol in the mucilage of the
mechanically de-mucilaged samples, which were mostly produced by
microbial metabolism during fermentation, were much lower than
those in the wet fermented samples, and they were not detected in the
mechanically de-mucilaged beans.
4. Discussion
Results obtained by the culture-dependent methods demonstrated
that yeasts, LAB, AAB, and Enterobacteriaceae grew significantly (about
10 times) during the wet fermentation of Australian coffee beans con-
ducted at a laboratory bench scale. Yeast counts remained high
throughout the fermentation with Hanseniaspora uvarum and Pichia
kudriavzevii being the predominate species. Hanseniaspora uvarum has
also been identified in coffee fermentation elsewhere (Masoud et al.,
2004; Pereira et al., 2014); however, Pichia kudriavzevii was isolated for
the first time in the present study. Pichia kudriavzevii was detected in a
wide range of fermented products such as alcoholic beverages, cheeses,
cereal-based products, and cocoa beans. In wine production, Pichia
kudriavzevii potentially used to deacidify wine and improve final pro-
duct sensory quality (del Monaco et al., 2014). In cheese, it was iden-
tified during the cheese maturation process and reported with high
protease activities which can potentially affect the final product texture
and flavour (Zheng et al., 2018). In bread, it was isolated during the
sourdough fermentation and was responsible for the leavening and
texture improvement (Verheyen et al., 2015). During cocoa fermenta-
tion, Pichia kudriavzevii was detected and identified with its superior
aroma profile (Pereira et al., 2017). Overall, Pichia kudriavzevii was
detected during the fermentation of several fermented products in-
cluding coffee. In addition, its potential enzymatic activities and cap-
ability to produce flavour compounds were reported in previous works.
These indicate its possible important roles in coffee bean fermentation
and provide light to future studies to be evaluated and used as a starter
culture.

7
H. Elhalis, et al. International Journal of Food Microbiology 321 (2020) 108544

Sucrose Glucose B Sucrose Glucose Fructose

A
Fructose Mannitol Mannitol Glycerol
Glycerol
40 10

Concentra on g/100 g

Concentra on g/100g
30 8
6
20
4
10
2
0 0
0 10 20 30 40 0 10 20 30 40
Time (h) Time (h)

C Citric acid Succinic acid D Citric acid Succinic acid

Malic acid Lac c acid Malic acid Lac c acid
Quinic acid Quinic acid
2.5

Concentera on g/100g
2
Concentra on g/100 g

2
1.5
1.5
1
1
0.5 0.5
0 0
0 10 20 30 40 0 10 20 30 40
Time (h)
Time (h)

Fig. 6. Changes in the concentration of sucrose, glucose, fructose, mannitol and glycerol (A, B) and organic acids (C, D) in the mucilage (A, C) and beans (B, D) during
the fermentation of coffee beans.

Table 2 regarding their contribution to coffee flavour (Evangelista et al., 2014a;

Comparison of chemical composition and microbial metabolites in the mucilage Evangelista et al., 2014b; Pereira et al., 2014; Pereira et al., 2015;
and endosperm of wet fermented and mechanically de-mucilaged coffee beans. Ribeiro et al., 2017), and further studies are needed to fill this gap of
Compound Mucilage (g/100 g)a Bean (g/100 g) knowledge. Moreover, yeasts were reported predominate in several
fermented plant and animal origin foods, such as alcoholic beverages,
bread, cheese, cocoa beans, and soy sauce. For instance, Pichia and
b
Fermented Mechanically de- Fermentedb Mechanically de-
mucilaged mucilaged
Candida spp. were detected in wine with a potential role in improving
Sucrose ND 5.0 ± 0.3 6.0 ± 0.1 8.2 ± 0.4 the sensory quality of the final product by producing fruity flavour
Glucose 9.0 ± 0.5 15.0 ± 0.2 0.8 ± 0.0 0.9 ± 0.1 compounds such as isoamyl acetate (Romi et al., 2014). Pichia ku-
Fructose 4.0 ± 0.2 18.0 ± 0.7 0.8 ± 0.0 1.4 ± 1.2 driavzevii was isolated from a wide range of fermented products with
Mannitol 0.8 ± 0.0 ND 0.1 ± 0.0 ND potential roles as discussed above. Hanseniaspora uvarum was also de-
Glycerol 1.2 ± 0.0 ND 0.1 ± 0.1 ND
Citric acid 0.9 ± 0.0 0.8 ± 0.0 1.8 ± 0.1 1.6 ± 0.0
tected in wine and dairy products and identified with high enzymatic
Succinic acid 1.8 ± 0.0 1.6 ± 0.1 0.2 ± 0.1 0.2 ± 0.0 activities such as protease and b-glucosidase which catalysed the pro-
Malic acid 1.8 ± 0.1 1.7 ± 0.2 0.2 ± 0.1 0.3 ± 0.0 duct internal contents releasing aromatic compounds and improving
Lactic acid 0.4 ± 0.0 ND 0.3 ± 0.0 ND the final product quality (Charoenchai et al., 1997; Ferreira and
Quinic acid 1.7 ± 0.0 1.4 ± 0.1 0.5 ± 0.1 0.5 ± 0.0
Viljoen, 2003). In cocoa bean fermentation, yeasts were predominate,
a
Results are means ± standard deviation in triplicates. especially in the early stage of the fermentation, where Hanseniaspora,
b
Results at the end of fermentation. Pichia, and Candida genera are among the most frequently isolated
yeasts with a potential contribution to cocoa beans sensory quality (Ho
Other Pichia species reported for coffee fermentation elsewhere in- et al., 2014; Nielsen et al., 2005). Such findings in this wide range of
clude P. fermentans, P. guilliermondii, P. caribbica, P. kluyveri, and P. fermented products may indicate the possible roles of yeasts in coffee
anomala (Masoud et al., 2004;Pereira et al., 2014;Wouters et al., 2012). bean fermentation and final product quality.
Saccharomyces spp. and Schizosaccharomyces spp. were not detected in Leuconostoc mesenteroides and Lactococcus lactis were the pre-
this study, but they are common isolates with a high population in other dominant isolates of LAB. These two LAB have been isolated from coffee
studies (Agate and Bhat, 1966; Pereira et al., 2014). These findings fermentation in different locations around the world (Avallone et al.,
demonstrate that yeasts isolated from wet fermentation of coffee beans 2001; Evangelista et al., 2015; Ribeiro et al., 2017). High levels of LAB
in different regions have both common and region-specific species. have been reported for most wet fermentation of coffee beans; however,
Most of these yeast isolates have been reported with high pectinolytic their contribution to mucilage degradation and flavour development of
enzyme activities, which are expected to contribute to the mucilage coffee is poorly understood. The most predominant AAB was Acet-
degradation during wet fermentation of coffee beans (Agate and Bhat, obacter persici, the population of which more grew by more than
1966; Frank et al., 1965; Pereira et al., 2014; Silva et al., 2013; Vaughn 2 log CFU/g by the end of fermentation. A few studies have reported the
et al., 1958). Yeasts are well known for their production of secondary occurrence of AAB such as Gluconobacter, Acetobacter in wet coffee
flavour metabolites such as esters, ketones, higher alcohols, aldehydes fermentation (De Bruyn et al., 2017; Zhang et al., 2019), but their role
and organic acids (Hammond, 1993; Janssens et al., 1992; Jayaram in the fermentation is not clear. The growth of yeasts and LAB produce
et al., 2013; Torner et al., 1992). However, there is scant information organic acids, ethanol, and mannitol, which would favor the growth of
AAB at later stages of the fermentation as they can oxidize these

8
H. Elhalis, et al.
compounds into acetic acid and acetoin (De Angelis and Gobbetti,
2004; Papalexandratou et al., 2011; Van der Meulen et al., 2007;
Wouters et al., 2012). In cocoa bean fermentation, the diffusion of
acetic acid into the beans contributes to the death of the embryo with
consequent disruption of the cellular organization and contact between
various degradative enzymes (e.g., protease and invertase), which plays
a key role in producing flavour precursors (Ho et al., 2014). Similar
phenomena may also occur in coffee bean fermentation.
The bacterial species detected in this study were also common iso-
lates in other fermented products. LAB such as L. mesenteroides and L.
lactis were detected in rice wine, acid-leavened bread, vegetable, meat,
and fish products and were responsible for the flavour and texture of
the final products (Coppola et al., 2006; Quigley et al., 2011; Rhee
et al., 2011). Gluconobacter and Acetobacter were also isolated in cocoa
beans fermentation and contributed to the acidity and metabolism of
the beans (Ho et al., 2014; Schwan, 1998). Enterobacteriaceae was in-
volved in the fermentation of meat and cassava products and contribute
to the product ripening and texture (Cocolin et al., 2011; Lücke, 2015;
Marty et al., 2012). These can illustrate the possible contributions of
these isolates in the coffee bean fermentation process and the final
product quality.
Results obtained by culture-independent methods showed that there
was considerable diversity in the microbial community in wet fer-
mentation of coffee beans. Lactococcus, Leuconostoc, Gluconobacter,
Enterobacter, and Serratia were the dominant bacterial genera while
dominant yeast was Hanseniaspora uvarum. These results were broadly
consistent with those obtained by the culture-dependent methods.
Surprisingly, the culture-independent methods did not find any mem-
bers of Pichia, the second most predominant yeasts detected by the
culture-dependent methods. One possible reason for this might be the
limitation of the databases for yeasts, which are not yet as complete as
those for bacteria. The culture-independent analysis revealed a rela-
tively high abundance of filamentous fungi at the start, which dis-
appeared with the progress of fermentation. Surprisingly, unidentified
members of Nectriaceae had a high initial level and grew to form 97% of
the fungal population in the first 24 h. Nectriaceae were isolated in other
fermented foods such as fermented dairy and beverages (Bokulich et al.,
2014; De Bruyn et al., 2017; Ratomahenina et al., 1995) however, their
impact on the products was not clear. The capability of Nectriaceae to
degrade polysaccharides by its pectinase and cellulase enzymes has
recently been reported (Zhang et al., 2018), and it is possible they may
contribute to the de-mucilage of coffee beans during fermentation, but
further studies are needed to confirm this possibility.
The mucilage layers of the Australian coffee beans were found to be
rich in sugars, which gradually decreased to low levels, as they were
utilized as a carbon source for microbial growth during fermentation. In
contrast, only minor changes to sugars occurred inside the beans.
Sucrose in the beans decreased from the initial level of about 9% to the
final concentration of about 6%, which is most likely due to the con-
version of the sugar to glucose and fructose by the endogenous in-
vertase activity (Hansen et al., 1998). Surprisingly, the levels of glucose
and fructose inside the bean did not show a corresponding increase but
stayed relatively constant throughout fermentation. This might be due
to the sugars leaching out from the inside of the beans to the sur-
rounding environment (Wootton, 1974). Another reason could be the
switch of the metabolism of the beans post-harvest from aerobic to
anaerobic pathways. More specifically, during the wet fermentation,
microbial activities consumed most of the available oxygen in the
surrounding water, leading to the metabolic activity of the bean to
switch from aerobic respiration to lactic/alcoholic fermentation. The
latter pathway is less efficient than the former and requires more glu-
cose and fructose to produce the same amount of ATP molecules
(Roberts et al., 1984; Summers et al., 2000). As a comparison, the
mechanically de-mucilaged beans had a higher level of all the sugars
than the wet fermented beans in both the mucilage and the beans. The
difference in the concentration of sugars, especially the reducing sugars
9
International Journal of Food Microbiology 321 (2020) 108544
inside the beans, could have a significant impact on the development of
coffee color, aroma and flavour after roasting, as the sugars are key
participants of Maillard reaction and caramelization (Fischer et al.,
2001;Knopp et al., 2006; Rohan and Stewart, 1967).
The reduction of the sugars during fermentation was accompanied
by the accumulation of lactic acid, glycerol, and mannitol in the fer-
menting beans and none of these compounds was detected in the me-
chanically de-mucilaged beans, demonstrating that they were products
of microbial metabolism. Similar findings have been reported elsewhere
(De Angelis and Gobbetti, 2004; De Bruyn et al., 2017; Van der Meulen
et al., 2007; Wouters et al., 2012). These compounds are well known
microbial metabolites with a favorable impact on coffee quality. For
example, mannitol has a favorable sweet and cool taste (Soetaert et al.,
1999), which is mainly produced by heterofermentative LAB (e.g.,
Leuconostoc mesenteroides) through metabolizing fructose (De Vuyst
et al., 2010; Saha and Racine, 2011), while glycerol has a sweet taste
and gives a smooth mouth-feel, and is mainly produced from the me-
tabolism of sugars by yeasts (Swiegers et al., 2005).
The production of lactic acid was accompanied by a corresponding
decline of pH from 5.4 to 3.6 in the fermentation mass while four other
organic acids, such as citric and malic acids, were also detected but
their levels did not change significantly during fermentation. Lactic
acid was reported to be produced by wide range of bacteria and fungi
(Komesu et al., 2017). The acidification process could have an impact
on the sensory quality of coffee, as the fermented beans have a lower
pH (4.7) compared with the mechanically de-mucilage beans (6.2).
Moreover, acidification has also been reported to facilitate the de-
gradation of the mucilage layer by swelling and loosening the poly-
saccharide network of the mucilage and facilitating their washing out
(Avallone et al., 2001; dAuzac, 1996).
5. Conclusion
In conclusion, the present study has shown that there is a large and
diverse microbial community consisting of yeasts, bacteria and fila-
mentous fungi in the wet fermentation of Australian coffee beans. Most
of the microbial species in the community have been reported for wet
fermentation of coffee beans conducted in other parts of the world, but
the yeast Pichia kudriavzevii was isolated for the first time in the wet
fermentation. The bacterial community was dominated by aerobic
mesophilic bacteria (AMB) with Citrobacter being the predominant
genus. Hanseniaspora uvarum and Pichia kudriavzevii were the pre-
dominant yeasts while Leuconostoc mesenteroides and Lactococcus lactis
were the predominant LAB. The yeasts and bacteria grew significantly
during fermentation, utilizing sugars in the mucilage and produced
mannitol, glycerol, and lactic acid, leading to a significant decrease in
pH. Future studies should examine the impact of microbial growth and
metabolism on coffee quality, especially flavour.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
References
Agate, A.D., Bhat, J.V., 1966. Role of pectinolytic yeasts in the degradation mucilage
layer of Coffea Robusta cherries. Appl. Environ. Microbiol. 14, 256–260.
Agrifutures Australia, 2017. Agrifutures Australia Coffee. viewed on 30 October 2019.
https://www.agrifutures.com.au/farm-diversity/coffee/.
Angelucci, E., Arima, H.K., Mantovani, D.M.B., Figueiredo, I.B., 1982. Analise qumica de
café. Instituto de Tecnologica de Alimentos, Campinas Sao Paulo.
Australian Subtropical Coffee Association, 2020. Australian Subtropical Coffee
Association. viewed 7 January 2020. http://www.astca.org.
Avallone, S., Guyot, B., Brillouet, J.M., Olguin, E., Guiraud, J.P., 2001. Microbiological
and biochemical study of coffee fermentation. Curr. Microbiol. 42, 252–256.
Bokulich, N., Ohta, M., Lee, M., Mills, D., 2014. Indigenous bacteria and fungi drive
H. Elhalis, et al.
traditional kimoto sake fermentations. Appl. Environ. Microbiol. 80 (17), 5522–5529.
Borém, F.M., Isquierdo, E.P., Fernandes, S.M., Fernandes, M., 2008. Armazenamento do
café. In: Borem, F.B. (Ed.), Pós-colheita do café, Lavras, Brazil. Editora UFLA.
Brando, C.H.J., 1999. Cereja Descascado, desmucilado, fermentado, despolpado ou la-
vado. In: Anais 25th Congresso Brasileiro de Pesquisas Cafeeiras, Franca, Brazil, pp.
342–346.
Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K.,
Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., 2010. QIIME allows
analysis of high-throughput community sequencing data. Nat. Methods 7 (5), 335.
Charoenchai, C., Fleet, G.H., Henschke, P.A., Todd, B.E.N.T., 1997. Screening of non-
Saccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes.
Aust. J. Grape Wine Res. 3 (1), 2–8.
Cocolin, L., Rantsiou, K., Iacumin, L., Cantoni, C., Comi, G., 2002. Direct identification in
food samples of Listeria spp. and Listeria monocytogenes by molecular methods. Appl.
Environ. Microbiol. 68, 6273–6282.
Chin, S.T., Eyres, G.T., Marriott, P.J., 2015. Application of integrated comprehensive/
multidimensional gas chromatography with mass spectrometry and olfactometry for
aroma analysis in wine and coffee. Food Chem. 185, 355–361.
Cocolin, L., Dolci, P., Rantsiou, K., 2011. Biodiversity and dynamics of meat fermenta-
tions: the contribution of molecular methods for a better comprehension of a complex
ecosystem. Meat Sci. 89 (3), 296–302 (icrobiol. 68, 6273-6282).
Coppola, S., Fusco, V., Andolfi, R., Aponte, M., Blaiotta, G., Ercolini, D., Moschetti, G.,
2006. Evaluation of microbial diversity during the manufacture of Fior di Latte di
Agerola, a traditional raw milk pasta-filata cheese of the Naples area. J. Dairy Res. 73
(3), 264–272.
dAuzac, J., 1996. Toxic oxygen: protection against pathogens. Plantations, Research
Development 3, 153–170.
De Angelis, M., Gobbetti, M., 2004. Environmental stress responses in Lactobacillus: a
review. Proteomics 4, 106–122.
De Bruyn, F., Zhang, S.J., Pothakos, V., Torres, J., Lambot, C., Moroni, A.V., Callanan, M.,
Sybesma, W., Weckx, S., De Vuyst, L., 2017. Exploring the impacts of postharvest
processing on the microbiota and metabolite profiles during green coffee bean pro-
duction. Appl. Environ. Microbiol. 83, e02398–16.
De Vuyst, L., Lefeber, T., Papalexandratou, Z., Camu, N., 2010. The functional role of
lactic acid bacteria in cocoa bean fermentation. In: Biotechnology of Lactic Acid
Bacteria: Novel Applications. 301. pp. 325.
del Monaco, S.M., Barda, N.B., Rubio, N.C., Caballero, A.C., 2014. Selection and char-
acterization of a Patagonian Pichia kudriavzevii for wine deacidification. J. Appl.
Microbiol. 117 (2), 451–464.
DeSantis, T.Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E.L., Keller, K., Huber, T.,
Dalevi, D., Hu, P., Andersen, G.L., 2006. Greengenes, a chimera-checked 16S rRNA
gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72
(7), 5069–5072.
Dick, R.H., 1990. Coffee and tea. In: Helrich, K. (Ed.), Official Method of the Association
of Official Analytical Chemists. AOAC, Virginia, USA, pp. 2.
Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST.
Bioinformatics 26 (19), 2460–2461.
Edgar, R.C., 2013. UPARSE: highly accurate OTU sequences from microbial amplicon
reads. Nat. Methods 10 (10), 996.
Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves
sensitivity and speed of chimera detection. Bioinformatics 27 (16), 2194–2200.
Esteve-Zarzoso, B., Belloch, C., Uruburu, F., Querol, A., 1999. Identification of yeasts by
RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed
spacers. Int. J. Syst. Bacteriol. 49, 329–337.
Evangelista, S.R., Silva, C.F., Miguel, M.G.P.C., Cordeiro, C.S., Pinheiro, A.C.M., Duarte,
W.F., Schwan, R.F., 2014a. Improvement of coffee beverage quality by using selected
yeast strains during the fermentation in dry process. Food Res. Int. 61, 183–195.
Evangelista, S.R., Miguel, M.G.P.C., Cordeiro, C.S., Silva, C.F., Pinheiro, A.C.M., Schwan,
R.F., 2014b. Inoculation of starter cultures in a semi-dry coffee (Coffea arabica)
fermentation process. Food Microbiol. 44, 87–95.
Evangelista, S.R., Miguel, M.G.P.C., Silva, C.F., Pinheiro, A.C.M., Schwan, R.F., 2015.
Microbiological diversity associated with the spontaneous wet method of coffee
fermentation. Int. J. Food Microbiol. 210, 102–112.
Ferreira, A.D., Viljoen, B.C., 2003. Yeasts as adjunct starters in matured Cheddar cheese.
Int. J. Food Microbiol. 86 (1–2), 131–140.
Fischer, M., Reimann, S., Trovato, V., Redgwell, R.J., 2001. Polysaccharides of green
Arabica and Robusta coffee beans. Carbohydr. Res. 330 (1), 93–101.
Frank, H.A., Lum, N.A., Dela Cruz, A.S., 1965. Bacteria responsible for mucilage-layer
decomposition in Kona coffee cherries. Appl. Microbiol. 13, 201–207.
Hamdouche, Y., Meile, J.C., Nganou, D.N., Durand, N., Teyssier, C., Montet, D., 2016.
Discrimination of post-harvest coffee processing methods by microbial ecology ana-
lyses. Food Control 65, 112–120.
Hammond, J.R.M., 1993. In: Rose, A.H., Harrison, J.S. (Eds.), The Yeat Technology, 2nd
eds. vol 5. Academic Press, London, pp. 7–67.
Hansen, C.E., del Olmo, M., Burri, C., 1998. Enzyme activities in cocoa beans during
fermentation. J. Sci. Food Agric. 77, 273–281.
Ho, V., Zhao, J., Fleet, G., 2014. Yeasts are essential for cocoa bean fermentation. Int. J.
Food Microbiol. 174, 72–87.
International Coffee Organization, 2019. International Coffee Organization, production.
viewed on 8 January 2020. http://www.ico.org/prices/po-production.pdf.
Janssens, L., De Pooter, H.L., Schamp, N.M., Vandamme, E.J., 1992. Process Biochem. 27,
195–215.
Jayaram, V.B., Cuyvers, S., Lagrain, B., Verstrepen, K.J., Delcour, J.A., Courtin, C.M.,
2013. Food Chem. 136, 301–308.
Knopp, S., Bytof, G., Selmar, D., 2006. Influence of processing on the content of sugars in
green Arabica coffee beans. Eur. Food Res. Technol. 223, 195–201.
10
International Journal of Food Microbiology 321 (2020) 108544
Komesu, A., de Oliveira, J.A.R., da Silva Martins, L.H., Maciel, M.R.W., Maciel Filho, R.,
2017. Lactic acid production to purification: a review. BioResources 12 (2),
4364–4383.
Leong, K.H., Chen, Y.S., Pan, S.F., Chen, J.J., Wu, H.C., Chang, Y.C., Yanagida, F., 2014.
Diversity of lactic acid bacteria associated with fresh coffee cherries in Taiwan. Curr.
Microbiol. 68, 440–447.
Lücke, F.K., 2015. Quality improvement and fermentation control in meat products. In:
Advances in Fermented Foods and Beverages. Woodhead Publishing, pp. 357–376.
Marty, E., Buchs, J., Eugster-Meier, E., Lacroix, C., Meile, L., 2012. Identification of
Staphylococciand dominant lactic acid bacteria in spontaneously fermented Swiss
meat products using PCR–RFLP. Food microbiol 29 (2), 157–166.
Masoud, W., Cesar, L.B., Jespersen, L., Jakobsen, M., 2004. Yeast involved in fermenta-
tion of Coffea arabica in East Africa determined by genotyping and by direct dena-
turating gradient gel electrophoresis. Yeast 21, 549–556.
Nielsen, D.S., Hønholt, S., Tano-Debrah, K., Jespersen, L., 2005. Yeast populations asso-
ciated with Ghanaian cocoa fermentations analysed using denaturing gradient gel
electrophoresis (DGGE). Yeast 22 (4), 271–284.
Ovreas, L., Forney, L., Daae, F.L., Torsvik, V., 1997. Distribution of bacterioplankton in
meromictic Lake Saelenvannet, as determined by denaturing gradient gel electro-
phoresis of PCR-amplified gene fragments coding for 16S rRNA. Appl. Environ.
Microbiol. 63, 3367–3373.
Papalexandratou, Z., Falony, G., Romanens, E., Jimenez, J.C., Amores, F., Daniel, H.M.,
De Vuyst, L., 2011. Species diversity, community dynamics, and metabolite kinetics
of the microbiota associated with traditional Ecuadorian spontaneous cocoa bean
fermentations. Appl. Environ. Microbiol. 77, 7698–7714.
Pereira, G.V.M., Soccol, V.T., Pandey, A., Medeiros, A.B.P., Andrade, J.M.R.L., Gollo, A.L.,
Soccol, C.R., 2014. Isolation, selection and evaluation of yeasts for use in fermenta-
tion of coffee beans by the wet process. Int. J. Food Microbiol. 188, 60–66.
Pereira, G.V.M., Neto, E., Soccol, V.T., Medeiros, A.B.P., Woiciechowski, A.L., Soccol,
C.R., 2015. Conducting starter culture-controlled fermentations of coffee beans
during on-farm wet processing: growth, metabolic analyses, and sensorial effects.
Food Res. Int. 75, 348–356.
Pereira, G.V.M., Neto, D.P.C., Medeiros, A.B.P., Soccol, V.T., Neto, E., Woiciechowski,
A.L., Soccol, C.R., 2016. Potential of lactic acid bacteria to improve the fermentation
and quality of coffee during on-farm processing. Int. J. Food Sci. Technol. 51,
1689–1695.
Pereira, G.V., Alvarez, J.P., Neto, D.P.D.C., Soccol, V.T., Tanobe, V.O., Rogez, H., Góes-
Neto, A., Soccol, C.R., 2017. Great intraspecies diversity of Pichia kudriavzevii in
cocoa fermentation highlights the importance of yeast strain selection for flavor
modulation of cocoa beans. LWT 84, 290–297.
Pereira, G.V.M., Soccol, V.T., Pandey, A., Medeiros, A.B.P., Andrade, J.M.R.L., Gollo, A.L.,
Soccol, C.R., 2014. Isolation, selection and evaluation of yeasts for use in fermenta-
tion of coffee beans by the wet process. Int. J. Food Microbiol. 188, 60–66.
Quigley, L., O’Sullivan, O., Beresford, T.P., Ross, R.P., Fitzgerald, G.F., Cotter, P.D., 2011.
Molecular approaches to analysing the microbial composition of raw milk and raw
milk cheese. Int. J. Food Microbiol. 150 (2–3), 81–94.
Ratomahenina, R., Van den Booms, S., Galzy, P., Dieu, B., 1995. Study of growth para-
meters of Cylindrocarpon sp., a mould isolated from saint nectar cheese. Chem.
Microbiol. Techno. Lebens. 17, 169–171.
Ribeiro, L.S., Miguel, M.G.C.P., Evangelista, S.R., Martins, P.M.M., Mullem, J., Belizario,
M.H., Schwan, R.F., 2017. Behavior of yeast inoculated during semi-dry coffee fer-
mentation and the effect on chemical and sensorial properties of the final beverage.
Food Res. Int. 92, 26–32.
Roberts, J., Callis, J., Wemmer, D., Walbot, V., Jardetzky, O., 1984. Mechanisms of cy-
toplasmic pH regulation in hypoxic maize root tips and its role in survival under
hypoxia. Proc. Natl. Acad. Sci. 81 (11), 3379–3383.
Rohan, T.A., Stewart, T., 1967. The precursors of chocolate aroma: production of free
amino acids during fermentation of cocoa beans. J. Food Sci. 32 (4), 395–398.
Romi, W., Keisam, S., Ahmed, G., Jeyaram, K., 2014. Reliable differentiation of
Meyerozyma guilliermondii from Meyerozyma caribbica by internal transcribed
spacer restriction fingerprinting. BMC Microbiol. 14 (1), 52.
Saha, B.C., Racine, F.M., 2011. Biotechnological production of mannitol and its appli-
cations. Appl. Microbiol. Biotechnol. 89 (4), 879–891.
Schillinger, U., Boehringer, B., Wallbaum, S., Caroline, L., Gonfa, A., Huch, M., Holzapfel,
W.H., Franz, C.M.P., 2008. A genus-specific PCR method for differentiation between
Leuconostoc and Weissella and its application in identification of heterofermentative
lactic acid bacteria from coffee fermentation. FEMS Microbiol. Lett. 286, 222–226.
Schwan, R.F., 1998. Cocoa fermentations conducted with a defined microbial cocktail
inoculum. Appl. Environ. Microbiol. 64 (4), 1477–1483.
Sfredo, M.A., Finzer, J.R.D., Limaverde, J.R., 2005. Heat and mass transfer in coffee fruits
drying. J. Food Eng. Essex 70, 15–25.
Silva, C.F., Schwan, R.F., Dias, E.S., Wheals, A.E., 2000. Microbial diversity during ma-
turation and natural processing of coffee cherries of Coffea arabica in Brazil. Int. J.
Food Microbiol. 60, 251–260.
Silva, C.F., 2014. Microbial activity during coffee fermentation. In: Schwan, R.F., Fleet, G.
(Eds.), Cocoa and Coffee Fermentation. CRC Press, Boca Raton, FL.
Silva, C.F., Batista, L.R., Abreu, L.M., Dias, E.S., Schwan, R.F., 2008. Succession of bac-
terial and fungal communities during natural coffee (Coffea arabica) fermentation.
Food Microbiol. 25, 951–957.
Silva, C.F., Vilela, D.M., Souza, C.C., Duarte, W.F., Dias, D.R., Schwan, R.F., 2013.
Evaluation of a potential starter culture for enhance quality of coffee fermentation.
World J. Microbiol. Biotechnol. 29, 235–247.
Soetaert, W., Vanhooren, P.T., Vandamme, E.J., 1999. The production of mannitol by
fermentation. Carbohydrate biotechnology protocols. Humana Press, pp. 261–275.
Summers, J., Ratcliffe, R., Jackson, M., 2000. Anoxia tolerance in the aquatic monocot
Potamogeton pectinatus: absence of oxygen stimulates elongation in association with
H. Elhalis, et al.
an unusually large Pasteur effect. J. Exp. Bot. 51 (349), 1413–1422.
Swiegers, J.H., Bartowsky, E.J., Henschke, P.A., Pretorius, I., 2005. Yeast and bacterial
modulation of wine aroma and flavour. Aust. J. Grape Wine Res. 11 (2), 139–173.
Torner, M., Martínez-Anaya, M., Antuña, B., Benedito de Barber, C., 1992. Headspace
flavour compounds produced by yeasts and lactobacilli during fermentation of pre-
ferments and bread doughs. Int. J. Food Microbiol. 15 (1–2), 145–152.
Van der Meulen, R., Scheirlinck, I., Van Schoor, A., Huys, G., Vancanneyt, M., Vandamme,
P., De Vuyst, L., 2007. Population dynamics and metabolite target analysis of lactic
acid bacteria during laboratory fermentations of wheat and spelt sourdoughs. Appl.
Environ. Microbiol. 73, 4741–4750.
Vaughn, R.H., Camargo, R.D.E., Fallange, H., Mello, A.G., Sergedello, A., 1958.
Observations on the microbiology of the coffee fermentation in Brazil. Food Technol.
12, 57–60.
Velmourougane, K., 2013. Impact of natural fermentation on physicochemical, micro-
biological and cup quality characteristics of Arabica and Robusta coffee. Proc. Natl.
Acad. Sci. 8, 233–239.
Verheyen, C., Albrecht, A., Elgeti, D., Jekle, M., Becker, T., 2015. Impact of gas formation
kinetics on dough development and bread quality. Food Res. Int. 76, 860–866.
Vilela, D.M., Pereira, G.V.M., Silva, C.F., Batista, L.R., Schwan, R.F., 2010. Molecular
ecology and polyphasic characterization of the microbiota associated with semi-dry
processed coffee (Coffea arabica L.). Food Microbiol. 27, 1128–1135.
Wootton, A.E., 1974. The Dry Matter Loss From Parchment Coffee During Field
11
International Journal of Food Microbiology 321 (2020) 108544
Processing.
Wouters, D., Grosu-Tudor, S., Zamfir, M., De Vuyst, L., 2012. Bacterial community dy-
namics, lactic acid bacteria species diversity and metabolite kinetics of traditional
Romanian vegetable fermentations. J. Sci. Food Agric. 93, 749–760.
Zhang, J., Kobert, K., Flouri, T., Stamatakis, A., 2013. PEAR: a fast and accurate Illumina
Paired-End reAd mergeR. Bioinformatics 30 (5), 614–620.
Zhang, W., Zhang, X., Li, K., Wang, C., Cai, L., Zhuang, W., Xiang, M., Liu, X., 2018.
Introgression and gene family contraction drives the evolution of lifestyle and host
shifts of hypocrealean fungi. Mycology 9 (3), 176–188.
Zhang, S.J., De Bruyn, F., Pothakos, V., Contreras, G.F., Cai, Z., Moccand, C., Weckx, S.,
De Vuyst, L., 2019. Influence of various processing parameters on the microbial
community dynamics, metabolomic profiles, and cup quality during wet coffee pro-
cessing. Front. Microbiol. 10, 2621.
Zheng, X., Li, K., Shi, X., Ni, Y., Li, B., Zhuge, B., 2018. Potential characterization of yeasts
isolated from Kazak artisanal cheese to produce flavoring compounds.
Microbioliologyopen 7 (1), e00533.
Wouters, D., Grosu-Tudor, S., Zamfir, M., De Vuyst, L., 2012. Bacterial community dy-
namics, lactic acid bacteria species diversity and metabolite kinetics of traditional
Romanian vegetable fermentations. J. Sci. Food Agr. 93, 749–760. https://doi.org/
10.1002/jsfa.5788.
Rhee, S.J., Lee, J.E., Lee, C.H., 2011. Importance of lactic acid bacteria in Asian fermented
foods. Microb. Cell Fact. (Vol. 10, No. 1, p. S5). BioMed Central.