Antonie van Leeuwenhoek (2020) 113:2077–2095

https://doi.org/10.1007/s10482-020-01479-2 (0123456789().,-volV)
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ORIGINAL PAPER

Exploration of yeast communities in fresh coconut, palmyra,

and nipa palm saps and ethanol-fermenting ability
of isolated yeasts
Savitree Limtong . Somjit Am-In . Rungluk Kaewwichian . Chutima Kaewkrajay .
Sasitorn Jindamorakot

Received: 2 July 2020 / Accepted: 2 October 2020 / Published online: 20 October 2020
Ó Springer Nature Switzerland AG 2020

Abstract This study aimed to explore communities and nipa palm saps, whereas Torulaspora delbrueckii
and the ethanol-fermenting ability of yeasts in fresh was found in the palmyra and nipa palm saps. The
coconut, palmyra, and nipa palm saps. From the 90 species with the highest occurrence in the saps of
samples of coconut, palmyra, and nipa palm saps, 204 coconut, palmyra, and nipa palms was S. cerevisiae
yeast isolates were isolated and identified as 15 spe- with 76.67%, 86.70%, and 100% frequency of occur-
cies in the phylum Ascomycota and a species (one rence, respectively. Using principal coordinates anal-
strain) in Basidiomycota. Saccharomyces cerevisiae, ysis for ordination, no marked difference was
Hanseniaspora guilliermondii, and Lachancea ther- observed in the yeast communities from the saps of
motolerans were found in the saps of all three palm the three palm species. A total of 199 isolates were
species. Candida tropicalis and Pichia kudriavzevii found to possess ethanol-fermentation ability when
were obtained from the coconut and palmyra palm cultivated using shake flask in 160 g/L of glucose
saps, Hanseniaspora vineae, Lachancea fermentati, medium at 28°C for 48 h. Lachancea fermentati YSP-
and Pichia manshurica were present in the coconut 383, isolated from nipa palm sap, produced the highest
amount of ethanol (76.74 g/L). Twenty-six isolates of
Candida sanyaensis (1), C. tropicalis (1), H. guillier-
Electronic supplementary material The online version of mondii (7), L. fermentati (8), L. thermotolerans (1),
this article (https://doi.org/10.1007/s10482-020-01479-2) con-
tains supplementary material, which is available to authorized
Pichia kudriavzevii (2), and S. cerevisiae (6) produced
users. high amounts of ethanol ranging from 69.57 to

S. Limtong (&)
Department of Microbiology, Faculty of Science, R. Kaewwichian
Kasetsart University, Bangkok 10900, Thailand Microbiology Program, Department of Science, Faculty of
e-mail: fscistl@ku.ac.th Science and Technology, Bansomdejchaopraya Rajabhat
University, Bangkok 10600, Thailand
S. Limtong
Academy of Science, Royal Society of Thailand, C. Kaewkrajay
Bangkok 10300, Thailand Division of Microbiology, Faculty of Science and
Technology, Phranakhon Si Ayutthaya Rajabhat
S. Am-In
S. Jindamorakot University, Phranakhon Si Ayutthaya 13000, Thailand
National Center for Genetic Engineering and
Biotechnology (BIOTEC), National Science and
Technology Development Agency (NSTDA),
Pathum Thani 12120, Thailand

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76.74 g/L. The result demonstrated that yeasts in the (31.2–57.6 g/L) (Barh and Mazumdar 2008; Misra
palm saps could play roles in the natural fermentation 2016; Minh 2014; Somawiharja et al. 2018; Sudha
of palm saps. et al. 2019). Coconut palm sap is also rich in amino
acids, for example, glutamic acid, threonine, and
Keywords Palm sap
Coconut
Palmyra palm
aspartic acid and vitamins, for example, inositol,
Nipa palm
Yeasts
Ethanol-fermenting yeasts thiamine, nicotinic acid, and pyridoxal (Barh and
Mazumdar 2008; Sudha et al. 2019). Palmyra palm sap
contains 103.6–169.4 g/L total sugars and 8.8–35.6 g/
L reducing sugars which comprise sucrose
Introduction (92.9–174.4 g/L) together with glucose (5.0–18.5 g/
L) and fructose (5.0–18.1 g/L) (Barh and Mazumdar
Coconut (Cocos nucifera L.), palmyra (Borassus 2008; Naknean et al. 2010; Kalaiyarasi et al. 2013). In
flabellifer L.), and nipa (Nypa fruticanspalms W.) addition, the palmyra palm sap has high nutritive value
palms are monocotyledon plants belonging to the as it is rich in vitamins, for example, vitamin C,
family Arecaceae (also known as Palmae). They are vitamin A, and niacin sugars with neutral pH (7.2–7.3)
widely grown in tropical regions. Coconut palm is (Barh and Mazumdar 2008). The raw nipa palm sap
widespread throughout the Western Pacific and Asia, contains total sugars of 126.0–369.7 g/L and reducing
including India, and countries in Southeast Asia, for sugars of 25.9 g/L, with the major sugar components
example, Indonesia, Malaysia, the Philippines, Thai- being sucrose (34.0–188.0 g/L), glucose
land, and Vietnam (Minh 2014; Nguyen et al. 2016; (17.0–39.0 g/L) and fructose (5.0–28.0 g/L), and pH
Sudha et al. 2019). Palmyra palm is widely distributed ranging from 5.25–6.9 (Chongkhong and Puangpee
in tropical countries in Africa, South America, Aus- 2018; Gumienna et al. 2016; Minh 2014; Nguyen et al.
tralia, and South Asia, for example, India, Nepal, Sri 2016; Thorasin 2018). Moreover, 116.0–188.0 mg/
Lanka, Indonesia, Malaysia, the Philippines, Thailand, 100 mL of vitamin C is also reported as being present
and Vietnam (Barh and Mazumdar 2008; Minh 2014; in nipa palm sap samples collected in Thailand
Nguyen et al. 2016; Phaichamnan et al. 2010; Sudha (Thorasin 2018).
et al. 2019). Nipa palm is commonly found in swamp The saps of coconut and palmyra palms are
areas favoring brackish water environments, such as collected from unopened inflorescence (spadix sur-
estuaries. Nipa palm is a mangrove species, which is rounded by spathe), whereas the nipa palm sap is
sometimes called the mangrove palm. This palm is obtained from the inflorescence stalk after cutting off
regarded as an important component of Southeast the fully developed inflorescence, the young inflores-
Asian mangrove vegetation (Hossain and Islam 2015). cence after flowering, or the mature fruits (Nguyen
Nipa palm grows naturally in various countries, for et al. 2016; Minh 2014; Somawiharja et al. 2018;
example, India, Sri Lanka, Bangladesh, Cambodia, Sudha et al. 2019; Thorasin 2018). The sap collection
Indonesia, Malaysia, Myanmar, the Philippines, Thai- from these palms is carried out by non-destructive
land, Vietnam, Australia, Papua New Guinea, the tapping methods, with palms continuing to survive
Solomon Islands, and Nigeria (Mehrotra et al. 2003; after tapping (Nguyen et al. 2016). Before tapping, the
Nguyen et al. 2016; Tsuji et al. 2011). inflorescence or the inflorescence stalk is regularly
Sap is a fluid containing water, sugars, and other beaten to stimulate sap flow; however, the inflores-
compounds. It is produced by plant photosynthesis and cence stalk beating is undertaken for a longer period
transported in xylem or phloem to various plant tissues than the inflorescence beating (Bernal et al. 2010;
for plant growth (Nguyen et al. 2016). The saps of Nguyen et al. 2016). After beating the inflorescence or
coconut, palmyra and nipa palms are fluids that are inflorescence stalk for a certain period, the top end of
sugar-rich, watery, an oyster white color, and translu- the inflorescence or inflorescence stalk is cut and the
cent (Gupta et al. 1980). Coconut palm sap contains sap that flows out is collected daily (Minh 2014;
total sugars ranging from 93.0 to 165.0 g/L with total Thorasin 2018; Nguyen et al. 2016; Somawiharja et al.
reducing sugars from 43.9 to 98.5 g/L and pH from 2018Sudha et al. 2019). Fresh palm saps are used as
6.75 to 7.50. The sugar components are sucrose beverages and as sources of palm sugar, alcoholic
(17.6–57.6 g/L), glucose (22.5–44.6 g/L) and fructose beverages (palm wines), and vinegar production in

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many countries in the Pacific Islands and Asia, for species found in nipa palm sap comprised Saccha-
example, India, Sri Lanka, Indonesia, Malaysia, the romyces cerevisiae, Torulaspora delbrueckii (Saccha-
Philippines, and Thailand, as well as Cameroon in romyces rosei), Cyberlindnera rhodanensis (Pichia
Africa (Chinnamma et al. 2019; Kapilan et al. 2015; rhodanensis), and Candida parapsilosis (Yamagata
Nguyen et al. 2016; Phaichamnan et al. 2010; et al. 1980). Valder and Nooralabettu (2018) reported
Somawiharja et al. 2018; Stringini et al. 2009). finding Saccharomyces cerevisiae and Lachancea
The sap flow inside the plant is sterile: microor- fermentati, with dominant lactic acid bacteria, namely,
ganisms, including bacteria, filamentous fungi, and Leuconostoc mesenteroides and Fructobacillus fruc-
yeasts, are usually introduced into the sap during tosus in the palmyra palm sap. Ten ethanol-fermenting
tapping and collection (Naknean et al. 2010). Micro- yeast species: S. cerevisiae, L. fermentati, Pichia
bial contamination usually results from non-hygienic kudriavzevii, Shizosaccharomyces pombe, Candida
tapping and/or collection processes. Sap is rich in tropicalis, Zygosaccharomyces rouxii, Saccharomy-
sugar and has some compounds that are nutrients for codes ludwigii, Hanseniaspora guilliermondii, Wick-
microbial growth (Naknean et al. 2010). Conse- erhamomyces anomalus, and P. manshurica were
quently, the contaminating microbial population, isolated from coconut inflorescence sap in Thailand
including yeasts and bacteria, can rapidly increase, (Udomsaksakul et al. 2018b).
resulting in natural fermentation of sugars in the sap. Only a few investigations of yeasts in the saps of
In addition, microbial growth is favored by the high palms have been undertaken: most have focused on
temperatures in tropical regions (Chongkhong and yeasts that play a role in the natural wine fermentation
Puangpee 2018; Naknean et al. 2010). To prevent the of palm saps. Therefore, to increase the knowledge
growth of contaminating microorganisms, some about yeasts in palm saps, the present study was
preservatives are added. In Thailand, kiam wood undertaken with the aim being to investigate yeast
(Cotylelobium lanceolatum) or payom wood (Shorea communities in fresh saps from three palm species,
roxburghii) is traditionally added to the sap during namely coconut, palmyra, and nipa palms growing in
collection to reduce microbial contaminant growth. Thailand, using the culture-dependent approach. In
Kiam wood extract has been found to inhibit the addition, the study determined the ethanol-fermenting
growth of bacteria in the genera Acetobacter, ability of the isolated yeasts.
Flavobacterium, Lactobacillus, Leuconostoc, and Mi-
crococcus, and yeast in the genus Saccharomyces has
been isolated from sap (Chanthachum and Beuchat Materials and methods
1997). In addition, kiam wood extract also inhibited
some pathogenic bacteria viz. Listeria monocytoge- Sample collection and chemical composition
nes, toxigenic Staphylococcus aureus, and enterotox- analysis
igenic Bacillus cereus. In Indonesia, limestone at a
concentration of 3 g/L is used for the preservation of Thirty samples each of coconut palm (Cocos nucifera
coconut palm sap (Somawiharja et al. 2018). L.), palmyra palm (Borassus flabellifer L.), and nipa
The large variations of chemical composition and palm (Nypa fruticanspalms W.) were collected. They
microbial load of palm saps depend on various factors were collected, respectively, from one province in
including geographic locality, variety, age, number of central Thailand (Samut Songkhram), three provinces
fruits, tapping and collecting methods, and the season (two of which were in central Thailand [Phetchaburi
when tapping (Chandrasekhar et al. 2012; Manel et al. and Chai Nat] and one in eastern Thailand [Chacho-
2011; Naknean et al. 2010). engsao]), and two provinces in southern Thailand
The microorganisms that are present and responsi- (Nakhon Si Thammarat and Trang) (Fig. 1). Each sap
ble for the fermentation of palm sap are mainly yeasts sample was collected from a different tree in the same
in the genus Saccharomyces and lactic acid bacteria or different garden. In the same garden, each tree was
(Uzochukwu et al. 1999). High densities of yeasts and far from the others by 100–150 m, and each garden
bacteria result in turbidity of the sap, with protein and was far from the others at least 5 km. The saps were
polyphenol compounds also causing sap turbidity obtained after tapping inflorescences of coconut and
(Lasekan et al. 2007; Naknean et al. 2010). The yeast palmyra palms, and after tapping stalks of the nipa

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palm after cutting off the mature fruits (Fig. 1). Before Corporation, Milford, MA, USA) using a Sugar-pak
tapping, the inflorescences of coconut and palmyra column (6.5 9 300 mm) (Waters Corporation, Mil-
palms were beaten and bent every day (10–15 min) for ford, MA, USA). The deionized water was used as a
4–5 days and 7 days, respectively, whereas the stalks mobile phase, running with a flow rate of 0.4 mL min-
1
of the nipa palm were beaten (30 min) every day for under a column temperature of 90 °C. The pH was
9–14 days. The inflorescences and stalks were cut in determined by a pH meter (TOA, HM-35 V, Tokyo,
the evening and left overnight to allow the sap to flow Japan).
from the cut into containers, to which strips of
Cylylelobium lanceolatum wood were added to inhibit Yeast isolation
bacterial growth. In the morning, the sap samples were
collected, stored in an icebox, and transported to the Yeasts were isolated from the individual sap samples
laboratory for yeast isolation. using the dilution plate technique. A tenfold serial
The sugar content in the fresh saps was determined dilution was prepared and 0.1 ml of a diluted sample
as a Brix value (°Bx) using a portable refractometer was spread on yeast extract-malt extract (YM) (0.3%
(HI 96,801, Trans, Singapore). The concentrations of yeast extract, 0.3% malt extract, 0.5% peptone, and
sucrose, glucose, fructose, and ethanol were deter- 1% glucose) agar supplemented with 0.02% chloram-
mined by high performance liquid chromatography phenicol and 0.025% sodium propionate and incu-
(HPLC) (2690) (Waters Corporation, Milford, MA, bated at 25°C for three days. From the individual sap
USA) with a refractive index detector (RI 410, Waters samples, yeast colonies of different morphologies

B1
D1
B2

C1 D3 D2
C2

Fig. 1 Map of Thailand showing location of provinces where palmyra palm sap (C1) and palmyra palm sap samples (C2);
saps were collected (provinces with the darker colour in the and nipa palm fruit (D1), nipa palm sap (D2) and nipa palm sap
map) (A); tapping and collecting of coconut palm sap (B1) and samples (D3)
coconut palm sap samples (B2); tapping and collecting of

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were picked and purified by repeated cross streaking Biodiversity Analysis

on YM agar. Purified yeast isolates were suspended in
YM broth supplemented with 10% (v/v) glycerol and The similarity of yeast communities associated with
maintained in a deep freezer at -80°C (MDF- the sap samples from these three palm species was
DU702VX TwinGuard ULT, PHC Cooperation, calculated using the classical Jaccard similarity coef-
Gunma, Japan). ficient. The calculation was performed using PAST
software version 3.25 (Hammer et al. 2001). The
DNA sequencing and yeast identification. principal coordinates analysis (PCoA) for the ordina-
tion of yeast communities in the 90 sap samples from
The nucleotide sequences of the D1/D2 domains of the the three palm species was based on Jaccard similarity
large subunit (LSU) rRNA gene and the internal indices through employing PAST software version
transcribed spacer (ITS) regions were determined 3.25. The frequency of occurrence (FO) (in %) was
from polymerase chain reaction (PCR) products calculated as the number of sap samples in which a
amplified from genomic DNA extracted from cells, particular species was observed as a proportion of the
using method of Lachance et al. (1999). The ampli- total number of sap samples.
fication of the D1/D2 domains of the LSU rRNA gene
was carried out by PCR with the forward primer NL1 Ethanol production
(59-GCATATCAATAAGCGGA GGAAAAG-39)
and the reverse primer NL4 (59-GGTCCGTGTT To prepare the yeast inoculum, yeast was grown in
TCAAGACGG-39) (Kurtzman and Robnett, 1998) yeast extract peptone dextrose (YPD) broth (1% yeast
using a T100TM thermo cycler (Bio-Rad, Hercules, extract, 2% peptone, 2% glucose) and incubated on a
CA, USA). The ITS regions were amplified with the rotary shaker at 28 °C for 24 h and at cell concentra-
forward primer ITS5 (59-GGAAGTAAAAGTCG tion as optical density (OD) was determined by a
TAACAAGG-39) and the reverse primer ITS4 (59- spectrophotometer (Cintra 404, GBC, Australia) at
TCCTCCGCTTATT GATATGC-39) (White et al., 600 nm. The inoculum was inoculated into 50 mL
1990). The PCR products were purified by GenepH- YPD broth containing 16% glucose in a 250 mL
lowTM gel/PCRKit (Geneaid Biotech Ltd., Taiwan) in Erlenmeyer flask to give an initial cell concentration
accordance with the manufacturer’s instructions. determined as OD at 600 nm of 0.5. The incubation
Sequencing was performed by Macrogen (Seoul, was performed on a rotary shaker (Innova 4330
South Korea) using Sanger sequencing with the PCR refrigerated incubator shaker, New Brunswick, NJ,
primers. USA) at 150 rpm at 28 °C for 48 h. Ethanol and cell
Yeasts were identified by analysis of the sequence concentrations were measured at 0 h, 24 h, and 48 h.
similarity of the D1/D2 domains of the LSU rRNA The ethanol concentration was analyzed by gas
gene in the GenBank database using the nucleotide chromatography (GC) (Shimadzu GC-2010 Plus,
BLAST search program (Altschul et al. 1997). In this Tokyo, Japan) with a flame ionization detector using
study, ascomycetous yeast isolates which differed by an Agilent DB-225 capillary column (0.25 mm inner
0–3 nucleotide differences were considered to be the diameter and 30 m in length) (Agilent, Santa Clara,
same species, and isolates that showed greater than 1% CA, USA) packed with 10% polyethylene glycol
nucleotide substitutions (six nucleotide substitution in 20 M. Operating conditions were as follows: helium
the ca. 600 nucleotides of the D1/D2 region) compared was used as the carrier gas at a flow rate of 0.44 mL/
with the type strains of species represented a distinct min, and the column, injection, and detector temper-
species (Kurtzman and Robnett 1998). For basid- atures were 90 °C, 200 °C, and 250 °C, respectively.
iomycetous yeasts, isolates that differed by two or Propane-1-ol served as an internal standard. Reducing
more nucleotides in the D1/D2 region from their type sugar (glucose) concentration was analyzed by using
strains were taken to be different taxa (Fell et al. dinitrosalicylic acid (DNS) method (Miller, 1959).
2000), however, the similarity of ITS sequences had to Cell concentration was determined as OD by a
be co-considered. After identification, the yeast spectrophotometer (Cintra 404, GBC, Braeside, Vic-
species were counted as presence or absence in each toria, Australia) at 660 nm. Ethanol concentration was
sample. reported as gram ethanol per liter of the fermentation

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medium. Ethanol productivity, defined as gram of As previously mentioned, the sugar content and pH
ethanol per liter per hour, was calculated from the final of the sap of each palm species depended on various
ethanol concentration. Ethanol yield was calculated as factors; therefore, the geographic locality and variety
the ratio of ethanol produced to sugar consumed. of each palm species were included (Chandrasekhar
et al. 2012; Manel et al. 2011; Naknean et al. 2010).
Therefore, the study compared the sugar compositions
Results and discussion and the pH of the saps of each palm species examined
in this study with those of the same palm species, as
Chemical compositions of palms saps previously reported in Thailand. The palmyra palm
sap results in the present study were compared with
The fresh saps of coconut, palmyra, and nipa palms those in a report on a previous study conducted in
ranged from oyster-white to yellow-white in color Songkhla province, in southern Thailand (Naknean
with slight turbidity. Their chemical compositions et al. 2010). The results revealed that the ranges of
were analyzed and the results were shown in Table 1. sucrose concentration and pH levels obtained from
The sugar content, the concentration of sugars (su- both studies were similar but, in the present study, the
crose, glucose, and fructose), and ethanol varied highest values of glucose, fructose, and ethanol
between the sap samples from each palm. The average concentrations were observed to be higher than those
sugar and fructose concentrations of saps of all three detected in the previous study. The nipa palm sap
palm species were not significantly different results were compared with results of a prior study that
(p \ 0.05). The average sucrose concentration of the analyzed nipa palm sap collected in the same province,
nipa palm sap was significantly higher than in the Nakhon Si Thammarat, as the present study (Thorasin
coconut and palmyra palm saps, which had no 2018). The pH ranges of both studies were not much
significantly different concentrations. Whereas the different, except that the highest values of sucrose,
glucose concentration in the coconut palm and glucose, fructose, and ethanol detected in the present
palmyra palm saps were the same but significantly study were higher than those obtained in the previous
higher than in the nipa palm sap. All samples study.
contained low ethanol concentrations, which the
concentration in the coconut and nipa palm saps were Yeast isolation from palm saps and their
the same but significantly higher than in the palmyr- identification
a palm saps. The average pH levels of the saps were
slightly acid, and in coconut palm sap was signifi- Yeasts could be isolated from all 90 sap samples of the
cantly higher than in the palmyra palm and nipa palm three palm species. Seventy-six and 69 yeast isolates
and saps (Table 1). were obtained from the inflorescence sap samples of

Table 1 Chemical compositions and pHs of coconut, palmyra and nipa palm saps
Compositions Coconut palm sap Palmyra palm sap Nipa palm sap
Range Average Range Average Range Average

Sugar content 15.8–18.0 16.9 ± 0.7a 12.8–19.0 15.9 ± 1.9a 11.4–22.8 17.50 ± 3.0a
(% Brix)
Sucrose (g/L) 22.71–189.90 73.14 ± 47.1a 3.24–139.29 82.73 ± 43.1a 0.48–211.01 112.45 ± 64.7b
a a
Glucose (g/L) 2.94–57.19 30.64 ± 16.8 1.85–66.71 30.23 ± 19.7 1.19–57.00 18.21 ± 17.5b
a a
Fructose (g/L) 3.84–57.82 33.56 ± 17.6 4.16–70.79 24.84 ± 18.2 0.74–76.68 23.23 ± 22.9a
a b
Ethanol (g/L) 15.55–46.94 26.54 ± 9.4 7.53–45.52 19.8 ± 8.4 6.03–62.79 26.76 ± 16.9a
a b
pH 4.6–5.8 5.2 ± 0.7 3.8–5.5 4.5 ± 0.5 3.8–5.1 4.76 ± 0.6b
The values with different superscript letters in a row are significantly different (p \ 0.05)

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the coconut palms and palmyra palms, respectively, present study investigated the fresh sap, after the
whereas 59 yeast isolates were derived from sap palms were tapped and the sap was collected using
samples from the nipa palm inflorescence stalks common local procedures that involved an overnight
(Tables 2, 3 and 4). In this study, fresh sap samples collecting period (evening tapping and morning col-
that were stimulated by tapping, then collected and lection of the sap for further use), yeasts were able to
carried using sterile equipment were examined for be isolated in all samples. These results implied that
yeasts, with no yeasts found (data not shown). This yeasts were introduced after the sap left the palms and
result agreed with a previous study that found the sap that ethanol fermentation had taken place as ethanol
inside the plant to be sterile and that it was during the was detected in the sap. These results were in
tapping and collecting processes that microorganisms agreement with results from prior investigations
from the environment were introduced to the sap (Chinnamma 2019; Stringini et al. 2009; Thorasin
(Chinnamma 2019; Naknean et al. 2010). When the 2018).

Table 2 Sample location, sample codes and numbers, and yeast isolate numbers of coconut, palmyra and nipa palm saps
Sampling location and date Sap sample Code No No. of yeast isolates

Coconut palm saps

Bang Krabue, Bang Khonthi, Samut Songkhram province C1–C5 5 10
11 July 2017
Jompluak Bang Khonthi, Samut Songkhram province C6–C11 6 15
11 July 2017
Khlong Khoen, Mueang Samut Songkhram, C12–C21 10 24
Samut Songkhram province
11 July 2017
Tha Kha, Amphawa, C22–C30 9 27
Samut Songkhram province
12 July 2017
Total number (coconut palm saps) 30 76
Palmyra palm saps
Saphan Krai, Ban Lat, Phetchburi province T1–T5 5 10
24 April 2018
Nong Kapu, Ban Lat, Phetchburi province T6–T10 5 13
24 April 2018
Tham Rong, Ban Lat, Phetchburi province T1–T15 5 12
25 April 2018
Huai Krot, Sankhaburi, Chai Nat province T16 – T25 10 21
9 May 2018
Pak Nam, Bang Khla, Chachoengsao province T26–T30 5 13
10 May 2018
Total number (palmyra palm saps) 30 69
Nipa palm saps
Khanab Nak, Pak Phanang, Nakhon Si Thammarat province@17 August 2017 N1–N7 7 12
Khanab Nak, Pak Phanang, Nakhon Si Thammarat province@18 August 2017 N8–N13 6 11
Khanab Nak, Pak Phanang, Nakhon Si Thammarat province@3 March 2018 N14–N23 10 18
Ban To Mueang, Bang Ma, Kantang, Trang province@3 March 2018 N24–N30 7 18
Total number (nipa palm saps) 30 59

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Table 3 Yeasts species (204 isolates) isolated from the sap of coconut (30 samples), palmyra (30 samples), and nipa palm (30
samples) and their frequency of occurrences
Species Coconut palm Palmyra palm Nipa palm
No. of isolates FO (%)* No. of isolates FO (%)* No. of isolates FO (%)*

Ascomycetous yeast
Candida sanyaensis – – 1 3.3 – –
Candida tropicalis 11 36.7 4 13.3 – –
Hanseniaspora guilliermondii 19 63.3 26 86.7 4 13.3
Hanseniaspora valbyensis – – – – 2 67
Hanseniaspora vineae 7 23.3 – – 1 3.3
Lachancea fermentati 6 20.0 – – 13 43.3
Lachancea thermotolerans 1 3.3 5 16.7 3 10.0
Metschnikowia sp. – – 1 3.3 – –
Meyerozyma guilliermondii – – – – 1 3.3
Pichia kudriavzevii 3 1.0 5 16.7 - -
Pichia manshurica 6 20.0 – – 1 3.3
Saccharomyces cerevisiae 23 76.7 25 83.3 30 100
Torulaspora delbrueckii – – 1 3.3 1 3.3
Torulaspora nypae** – – – – 2 6.7
Wickerhamomyces anomalus – – – – 1 3.3
Basidiomycetous yeast
Moniliella sp. – – 1 3.3 – –
Total 76 69 59
*Frequency of occurrence (%) = number of samples, where a particular species was observed, as a proportion of the total number of
samples
**New species (Kaewwichian et al. 2020)

From 204 yeast isolates from the sap samples of the genera (Table 3). S. cerevisiae was the species with the
three palm species, they were identified by analysis of highest occurrence, followed by H. guilliermondii,
the D1/D2 domain of the LSU rRNA gene sequence with these two species present in 26 and 25 sap
similarity, 203 isolates were identified as 15 species in samples, equivalent to FOs of 86.7% and 83.33%,
the phylum Ascomycota and one isolate was identified respectively. The other seven species were detected in
as a species in the phylum Basidiomycota (Fig. 2 and 1–5 sap samples. In the nipa palm sap samples, 11
Supplementary Table S1). Seventy-six yeast isolates yeast species from seven genera were identified
from the coconut palm sap samples were identified as (Table 3). S. cerevisiae were found in all samples of
being from eight species in five genera (Table 3). nipa palm sap, equivalent to an FO of 100%. The
Saccharomyces cerevisiae was the species with the species with the second highest occurrence was
highest occurrence, as it was found in 23 of the 30 sap L. fermentati, which was present in 13 samples,
samples, representing a frequency of occurrence (FO) equivalent to an FO of 43.33%. The other species
of 76.67%. The species with the second highest were only found in 1–4 samples. Yeast species found
occurrence, H. guilliermondii, was found in 19 sap in the saps of all three palm species comprised
samples (FO = 63.33%). The remaining species were S. cerevisiae, H. guilliermondii, and Lachancea ther-
present in 1–11 sap samples. Identification of 69 yeast motolerans (Fig. 3). The other 13 yeast species were
isolates derived from the palmyra palm sap samples present in the saps of one or two palm species.
showed that they belonged to nine species in eight

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 Table 4 Yeasts species isolated from sap of coconut (30 samples), palmyra (30 samples), and nipa palm (30 samples), and their frequency of occurrences
Sample Coconut palm
no sap
Candida Hanseniaspora Hanseniaspora Lachancea Lachancea Pichia Pichia Saccharomyces
tropicalis guilliermondii vineae fermentati thermotolerans kudriavzevii manshurica cerevisiae

1 1 1
2 1
3 1
4 1 1 1 1 1
5 1
6 1 1 1
7 1 1 1
8 1 1
Antonie van Leeuwenhoek (2020) 113:2077–2095

9 1 1
10 1 1
11 1 1 1
12 1 1
13 1 1
14 1 1 1
15 1 1
16 1 1
17 1 1 1
18 1 1 1
19 1 1 1
20 1
21 1 1 1
22 1 1 1
23 1 1 1 1
24 1 1 1
25 1 1 1
26 1 1
27 1 1 1
28 1 1 1 1 1
29 1 1
2085

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 Table 4 continued
2086

Sample Coconut palm

no sap

123
Candida Hanseniaspora Hanseniaspora Lachancea Lachancea Pichia Pichia Saccharomyces
tropicalis guilliermondii vineae fermentati thermotolerans kudriavzevii manshurica cerevisiae

30 1 1
Total 11 19 7 6 1 3 6 23
isolate
FO (%)* 36. 7 63.3 23.3 20.0 3.3 10.0 20.0 76. 7
Palmyra
palm sap
Sample no Candida Candida Hanseniaspora Lachancea Metschnikowia Pichia Torulaspora Saccharomyces Moniliella
sanyaensis tropicalis guilliermondii thermotolerans sp. kudriavzevii delbrueckii cerevisiae sp.

1 1
2 1 1
3 1 1
4 1 1
5 1 1 1
6 1 1 1
7 1 1
8 1 1 1
9 1 1 1
10 1 1
11 1 1 1
12 1 1
13 1 1 1
14 1 1
15 1 1
16 1 1
17 1 1 1 1
18 1 1
19 1 1 1
20 1 1
21 1 1
22 1 1
23 1
Antonie van Leeuwenhoek (2020) 113:2077–2095
 Table 4 continued
Palmyra
palm sap
Sample no Candida Candida Hanseniaspora Lachancea Metschnikowia Pichia Torulaspora Saccharomyces Moniliella
sanyaensis tropicalis guilliermondii thermotolerans sp. kudriavzevii delbrueckii cerevisiae sp.

24 1
25 1 1
26 1 1
27 1 1 1
28 1 1
29 1 1
30 1 1 1 1
Total 1 4 26 5 1 5 1 25 1
isolate
Antonie van Leeuwenhoek (2020) 113:2077–2095

FO (%)* 3.3 13.3 86.7 16.7 3.3 16.7 3.3 83.3 3.3

Nipa palm sap

Sample Hanseniaspora Hanseni- Hanseni- Lachancea Lachancea Meyerozyma Pichia Saccha- Torulaspora Torulaspora Wicker-
no guilliermondii aspora aspora fermentati thermo- guillier- manshurica romyces delbrueckii nypae ** hamomyces
valbyensis vineae tolerans mondii cerevisiae anomalus

1 1
2 1 1
3 1
4 1 1
5 1 1
6 1 1 1
7 1
8 1 1
9 1 1
10 1
11 1 1
12 1
13 1 1 1
14 1
15 1
16 1 1
2087

123
 Table 4 continued
2088

Nipa palm sap

Sample Hanseniaspora Hanseni- Hanseni- Lachancea Lachancea Meyerozyma Pichia Saccha- Torulaspora Torulaspora Wicker-

123
no guilliermondii aspora aspora fermentati thermo- guillier- manshurica romyces delbrueckii nypae ** hamomyces
valbyensis vineae tolerans mondii cerevisiae anomalus

17 1 1
18 1 1 1
19 1 1
20 1 1
21 1 1
22 1 1
23 1
24 1 1 1
25 1 1 1
26 1 1
27 1 1 1
28 1 1 1
29 1 1
30 1 1
Total isolate 4 2 1 13 3 1 1 30 1 2 1
FO (%)* 13.3 6.7 3.3 43.3 10.0 3.3 3.3 100 3.3 6.7 3.3
*Frequency of occurrence (%) = number of samples, where a particular species was observed, as a proportion of the total number of samples
** New species (Kaewwichian et al., 2020)
Antonie van Leeuwenhoek (2020) 113:2077–2095
 Table 5 Ethanol production by yeasts isolated from palm saps cultivated in yeast extract peptone glucose broth containing 160 g/L glucose, on a rotary shaker at 150 rpm at
28 °C for 48 h
Yeast species Source of sap No. of No. of isolates Produced B 49 g/L Produced 50–59 g/L Produced 60–69 g/L Produced C 70 g/L
isolates with no ethanol ethanol ethanol ethanol
production
Ethanol (g/ No. of Ethanol (g/ No. of Ethanol(g/L) No. of ethanol No. of
L) isolates L) isolates isolates Ethanol (g/L) isolates

Candida sanyaensis Palmyra palm 1 73.97 1

Candida tropicalis Coconut palm 11 58.87 1 61.67–66.73 10
Palmyra palm 4 63.03–67.74 3 71.03 1
Hanseniaspora guilliermondii Coconut palm 19 56.08–58.53 2 61.79–67.37 17
Palmyra palm 26 63.65–69.65 21 71.19–73.52 5
Nipa palm 4 60.05–69.11 4
Hanseniaspora valbyensis Nipa palm 2 57.83 1 65.37 1
Antonie van Leeuwenhoek (2020) 113:2077–2095

Hanseniaspora vineae Coconut palm 7 62.05–68. 01 7

Nipa palm 1 68.77 1
Lachancea fermentati Coconut palm 6 66.94–68.16 4 70.11,72.85 2
Nipa palm 13 59.54 1 63.07–66.28 6 71.69–76.74 6
Lachancea thermotolerans Coconut palm 1 62.99 1
Palmyra palm 5 59.28 1 64.33–66.28 3 73.68 1
Nipa palm 3 59.96–65.74 3
Metschnikowia sp. Palmyra palm 1 24.96 1
Meyerozyma guilliermondii Nipa palm 1 53.29 1
Moniliella sp. Palmyra palm 1 55.36 1
Pichia kudriavzevii Coconut palm 3 67.99–68.70 2 71.04 1
Palmyra palm 5 65.24–68.87 4 69.59 1
Pichia manshurica Coconut palm 6 4 25.68,29.41 2
Nipa palm 1 1 0
Sacchromyces cerevisiae Coconut palm 23 61.07–66.58 23
Palmyra palm 25 62.71–68.73 23 70.20,73.16 2
Nipa palm 30 59.00 1 60.20–69.38 25 70.48–75.95 4
Torulaspora delbrueckii Palmyra palm 1 65.90 1
Nipa palm 1 63.01 1
Torulaspora nypae Nipa palm 2 68.36,69.52 2
Wickerhamomyces anomalus Nipa palm 1 66.83 1
Total 204 5 3 9 163 24
2089

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2090 Antonie van Leeuwenhoek (2020) 113:2077–2095

Fig. 2 Yeast species and the number of coconut, palmyra and nipa palms sap samples which these yeasts were detected. (Color
figure online)

It is not surprising that S. cerevisiae was found to be

the dominant species in the saps of all palm species
investigated in this study. S. cerevisiae was previously
reported as being detected in saps of various palm
species, including coconut palm (Udomsaksakul et al.
2018a, 2018b), palmyra palm (Valder and Noorala-
bettu 2018), nipa palm (Yamagata et al. 1980) and oil
palm (Stringini et al. 2009). The second most preva-
lent yeast species in coconut and palmyra palm saps,
and present in four samples of nipa palm sap in this
study, H. guilliermondii, was previously reported as
being one of the ethanol-fermenting yeast species
obtained from coconut palm sap in Thailand (Udom-
saksakul et al. 2018b). Lachancea fermentati, found in
the present study to be one of the two species that
occurred most often in the nipa palm sap samples and
also detected in coconut palm sap (at FO = 20%), was Fig. 3 Principle coordinates analysis (PCoA) plots of yeast
previously reported to be found in coconut palm sap in communities in 90 sap samples of coconut, palmyra, and nipa
palms using Jaccard similarity coefficient. (Color figure online)
Thailand (Udomsaksakul et al. 2018b) and in palmyra
palm sap in India (Valder and Nooralabettu 2018).

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Antonie van Leeuwenhoek (2020) 113:2077–2095 2091

Even though C. tropicalis found in this study has been agaves NRRL Y-17915 T by 1.6% nucleotide substi-
reported from various habitats in nature, such as tutions (9 nucleotide substitutions in 555 nucleotides)
plants, forest soil, agricultural soil, and peat in peat in the D1/D2 region of the LSU rRNA gene. Whereas,
swamp forests, especially in tropical climate where isolate YSP-478 differed from the type stain of closest
temperature and humidity enhance its adaptability species, Moniliella megachiliensis CBS190.92 T by
(Amprayna et al. 2012; Boonmak et al. 2020; Into et al. 0.5% nucleotide substitutions (3 nucleotide substitu-
2020; Limtong and Koowadjanakul 2012; Limtong tions in 551 nucleotides) in the D1/D2 region and 4.9%
et al. 2014; Srisuk et al. 2019Yang et al. 2012), it was nucleotide substitutions (22 nucleotide substitutions of
also known as clinical yeasts that was reported to be 464 nucleotides) in the ITS region. However, addi-
one of the important opportunistic pathogenic yeasts tional isolates are needed to officially propose these as
(Colombo et al. 2017; Guinea 2014; Wang et al. 2016; two new species.
Zuza-Alves et al. 2017).
In comparing yeasts associated with coconut palm Biodiversity Analysis
sap samples collected in Samut Songkhram province,
Thailand and detected in the present investigation with The classical Jaccard similarity coefficient was used to
those found in the previous study reported by Udom- compare the similarity of the yeast communities from
saksakul et al. (2018b), it was revealed that six species, the saps of each of these three palm species. The
C. tropicalis, H. guilliermondii, L. fermentati, P. similarity coefficient values were in the range of
kudriavzevii, P. manshurica, and S. cerevisiae were 0.25–0.46 (Supplementary Table S3). The average
found in both investigations. However, two species value was 0.38, which meant that the sap of each palm
viz. H. vineae and L. thermotolerans obtained in the species shared 38% of the yeast species. A compar-
present study were not observed in the previous atively higher similarity was observed between the
investigation. On the other hand, four other species saps of the coconut palm and the nipa palm. In
(Shizosaccharomyces pombe, Zygosaccharomyces contrast, the similarity comparison of the saps of the
rouxii, Saccharomycodes ludwigii, and Wicker- palmyra palm and the nipa palm showed the lowest
hamomyces anomalus) reported in the previous study similarity index values. However, the yeast commu-
were not detected in the present investigation. The nities of the 90 sap samples collected from the three
reason could be that the two investigations used palm species showed no marked differences in sim-
different methods for yeast isolation. Enrichment ilarity using a PCoA plot base on Jaccard similarity
cultivation in glucose peptone yeast extract broth indices (Fig. 3).
containing 30 ml/L ethanol was used in the previous
study. Stringini et al. (2009) investigated yeast in palm Ethanol production
wine during the first five days of tapping oil palms
(Elaeis guineensis) in Cameroon and reported that The 204 yeast isolates isolated from the sap samples of
Sac. ludwigii was the dominant yeast species at day the three palm species were tested for their ethanol-
zero, followed by Saccharomyces cerevisiae and fermenting ability by cultivation in YPD broth con-
Z. bailii. After three days, S. cerevisiae became the taining 16% glucose in a rotary shaker at 28 °C for
dominant yeast species, with Sac. ludwigii occasion- 48 h. The results showed that only five isolates of
ally detected in the fermented sap. In addition, Pichia manshurica were incapable of ethanol fermen-
Hanseniaspora uvarum, Candida parapsilopsis, Can- tation, whereas the other 199 isolates (97.5%) were
dida fermentati, and Pichia fermentans were detected. capable of fermenting ethanol (Table 5; Supplemen-
In the present study, one new yeast species isolated tary Table S2). Of the ethanol-fermenting isolates,
from nipa palm sap was explored and proposed as most isolates (196 isolates) produced ethanol in the
Torulaspora nypae (Kaewwichian et al. 2020). In range of 53.29–76.74 g/L and only two P. manshurica
addition, two potential new species in the genera isolates and one Metschnikowia sp. isolates produced
Metschnikowia (isolate YSP-519) and Moniliella ethanol at a level lower than 30 g/L. Of the 196
(isolate YSP-478) were observed (Supplementary ethanol-fermenting isolates, which produced more
Table S1). The isolate YSP-519 differed from the than 50 g/L ethanol, L. fermentati YSP-383 from nipa
type strain of its closest species, Metschnikowia palm sap produced the highest ethanol concentration

123
2092 Antonie van Leeuwenhoek (2020) 113:2077–2095

of 76.74 ± 2.09 g/L with high productivity and yield 2019; Lombardi et al. 2018; Martin et al. 2018; Xu
at 1.60 ± 0.04 g/L/h and 0.49 ± 0.01 g/g, respec- et al. 2006). They play a significant role at the
tively. (Table 5; Supplementary Table S2). Whereas beginning of fermentation, producing ethanol and
26 isolates of Candida sanyaensis, C. tropicalis, H. aroma compounds, such as ethyl ester, ethyl acetate,
guilliermondii, L. fermentati, L. thermotolerans, and phenyl acetate, and isoamyl alcohol. Yeasts from the
Sacchromyces cerevisiae produced high levels of genus Lachancea possess the characteristics for
ethanol concentration (69.57–75.95 g/L). Most iso- ethanol production and for the accumulation of lactic
lates (160 isolates) produced ethanol in the range of acid (Bellut et al., 2019; Vilela, 2018). Therefore, L.
59.96–69.37 g/L (Table 5; Supplementary Table S2). fermentati and L. thermotolerans are of interest in the
In another study, it was reported that 26 isolates of production of sour beers (Bellut et al. 2019; Domizio
S. cerevisiae, isolated from coconut palm sap in et al. 2016; Porter et al. 2019). Pichia manshurica,
Thailand, produced ethanol at levels greater than 50 g/ with its ability to produce both ethanol and esters, has
L by standing cultivation at 30°C for 48 h in a medium been used in some traditional beverages (Toyotome
containing 180 g/L glucose (Udomsaksakul et al. et al. 2019; Zhang et al. 2017). However, this species
2018a). Udomsaksakul et al. (2018b) reported that a was also reported as a spoilage yeast in wine as it
high ethanol concentration of 66.92 g/L was produced produced phenols at a level of volatility that was too
by S. cerevisiae NC027, which was isolated from high (Perpetuini et al., 2020; Saez et al., 2011).
coconut palm sap in Thailand after 48 h of standing Torulaspora spp. are fermentative yeasts, now widely
cultivation in a medium containing 180 g/L glucose. used in wine making due to various advantages when
In the same investigation, the other yeast species compared to S. cerevisiae; for example, they produce
obtained from coconut palm sap viz. C. tropicalis, lower acetic acid, produce higher glycerol, and
H. guilliermondii, L. fermentati, P. kudriavzevii, P. produce higher aroma compounds, as well as having
manshurica, Sac. ludwigii, Shi. pombe, W. anomalus, higher malolactic acid fermentation ability (Nardi
and Z. rouxii also showed the ability to ferment et al. 2019; Ramı́rez and Velázquez, 2018; Zhang et al.
ethanol. 2018). Torulaspora delbrueckii has been reported to
Most yeast species obtained from palm saps in the improve the aroma profile by enhancing the produc-
present study, except for C. sanyaensis, P. kudriavze- tion of higher alcohols (3-methyl-1-propanol and
vii, W. anomalus, and the novel species Torulaspora phenylethyl alcohol) and ethyl esters (ethyl decanoate
nypae, have been reported to perform ethanol fermen- and ethyl butanoate) in wine when employed with S
tation and to contribute the sensory characteristics of cerevisiae, without having delays in ethanol and
alcoholic beverages (Jespersen, 2003; Lleixà et al. malolactic acid fermentation (Zhang et al. 2018). On
2019; Martin et al. 2018; Walker and Stewart 2016; the other hand, Pichia kudriavzevii and C. tropicalis
Xu et al. 2006). Saccharomyces cerevisiae has the revealed the ability to produce high levels of ethanol at
ability not only to ferment ethanol but also to produce the evaluated temperatures from both glucose and
aroma compounds (Walker and Stewart, 2016). It is xylose (Chamnipa et al. 2018; Jamai et al. 2001;
the predominant yeast species in various indigenous Pongsanat and Miyuki 2018; Yuangsaard et al. 2013).
fermented foods and alcoholic beverages: in addition, Therefore, the yeast species discovered in the saps
it has traditionally been the primary fermenting yeast of the three palm species in the present study may have
for the production of alcoholic beverages by the roles in natural fermentation to produce palm wines.
fermentation of sugars in various raw materials
(Walker and Stewart, 2016). For example, rum forms
from sugarcane juice and molasses, and wine and Conclusion
brandy forms from grapes. Co-cultures of S. cerevisiae
and C. tropicalis have been noted in the production of In this study, we confirmed that the sap inside plants is
traditional sorghum beers (Alloue-Boraud et al. 2015; sterile and that microorganisms from the environment
N’Guessan et al. 2010). The three Hanseniaspora are introduced during the tapping and collecting
species, H. guilliermondii, H. valbyensis, and H. processes. The exploration of yeast communities in
vineae, detected in the present study have been shown the fresh saps of coconut, palmyra, and nipa palms
to have oenological impact on wines (Lleixà et al. using common tapping and collecting procedures

123
Antonie van Leeuwenhoek (2020) 113:2077–2095 2093

revealed that all but one yeast isolates obtained were in fermentati strain KBI 12.1 for low alcohol beer brewing.
the phylum Ascomycota. No marked difference was J Am Soc Brew Chem 77:157–169
Chamnipa N, Thanonkeo S, Klanrit P, Thanonkeo P (2018) The
observed in the yeast communities in the saps from the potential of the newly isolated thermotolerant yeast Pichia
three palm species. Saccharomyces cerevisiae, Hanse- kudriavzevii RZ8-1 for high-temperature ethanol produc-
niaspora guilliermondii, and Lachancea thermotoler- tion. Braz J Microbiol 49:378–391
ans were common in the saps of all three palm species. Chandrasekhar K, Sreevani S, Seshapani P, Pramodhakumari J
(2012) A review on palm wine. Int J Res Biol Sci 2:33–38
Most (97.5%) of the identified yeast isolates possessed Chanthachum S, Beuchat LR (1997) Inhibitory effect of kiam
ethanol-fermentation ability; therefore, they could (Cotylelobium lanceotatumcraih.) wood extract on gram-
play roles in natural fermentation to produce palm positive food-borne pathogens and spoilage micro-organ-
wines. isms. Food Microbiol 14:603–608
Chinnamma M, Bhasker S, Hari BM, Sreekumar D, Madhav H
(2019) Coconut neera-a vital health beverage from coconut
palms: harvesting, processing and quality analysis. Bev-
Author contributions SL: Designed study, data discussion and erages 5:22. https://doi.org/10.3390/beverages5010022
writing the manuscript; SA: Performed experiments and data Chongkhong S, Puangpee S (2018) Alternative energy under the
analysis; PK: Data analysis.; CK: Data analysis; SJ: Performed royal initiative of his majesty the king: ethanol from nipa
experiments and data analysis. sap with yeast isolated. Songklanakarin J Sci Technol
40:648–658
Funding This work was supported by the Thailand Research Colombo AL, Ju nior JNA, Guinea J, (2017) Emerging mul-
Fund through a TRF Research Team Promotion grant tidrug-resistant Candida species. Curr Opin Infect Dis
(RTA6080004) that is providing to Savitree Limtong. 30:528–538
Domizio P, House JF, Joseph CML, Bisson LF, Bamforth CW
Compliance with ethical standards (2016) Lachancea thermotolerans as an alternative yeast
for the production of beer. J Inst Brew 122:599–604
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conflict of interest. A (2000) Biodiversity and systematics of basidiomycetous
yeasts as determined by large-subunit rDNA D1/D2
Ethical approval This article does not contain any studies domain sequence analysis. Int J Syst Evol Microbiol
with human participants or animals performed by any of the 50:1351–1371
authors. Felsenstein J (1985) Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39:783–791
Guinea J (2014) Global trends in the distribution of Candida
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Publisher’s Note Springer Nature remains neutral with
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regard to jurisdictional claims in published maps and
the production of fermented beverages. Beverages 2(4):30.
institutional affiliations.
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