Isolation of haloalkaliphilic fungi from Lake Magadi in Kenya

Abstract

In this study we explored the cultivable fungal diversity in Lake Magadi and their secondary metabolite production. Isolation was done on alkaline media (Potato dextrose agar, Malt extract agar, Oatmeal agar and Sabouraud dextrose agar). A total of 52 unique isolates were recovered from the lake and were characterized using different techniques. Growth was observed at pH, temperature and salinity ranges of between 6 - 10, 25 °C - 40 °C and 0%-20% respectively. Phylogenetically, the isolates were affiliated to 18 different genera with Aspergillus, Penicillium, Cladosporium, Phoma and Acremonium being dominant. A screen for the ability to produce extracellular enzymes showed that different isolates could produce proteases, chitinases, cellulases, amylases, pectinases and lipases. Production of antimicrobial metabolites was noted for isolate 11M affiliated to Penicillium chrysogenum (99%). Cell free extracts and crude extracts from this isolate had inhibitory effects on Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Candida albicans and fungal plant pathogens Schizophyllum commune, Epicoccum sorghinum strain JME-11, Aspergillus fumigatus strain EG11-4, Cladosporium halotolerans CBS 119416, Phoma destructive and Didymella glomerata). In this study we showed that different cultivation strategies can lead to recovery of more phylotypes from the extreme environments. Growth under different physiological characteristics typical of the soda lake environment (elevated temperature, pH and salts) confirmed the haloalkaliphilic nature of the fungal isolates. The use of suitable antimicrobial production media can also lead to discovery of more phylotypes producing diverse biocatalysts and bioactive metabolites.

Full text

Research article
Isolation of haloalkaliphilic fungi from Lake Magadi in Kenya
Philemon Orwa
a
, George Mugambi
b
, Vitalis Wekesa
c
, Romano Mwirichia
a
,
*
a
Biological Sciences, University of Embu, Embu, Kenya
b
School of Pure and Applied Sciences, Meru University of Science and Technology, Meru, Kenya
c
DuduTech Kenya Ltd., Naivasha, Kenya
ARTICLE INFO
Keywords:
Microbiology
Biodiversity
Antibiotics
Mycology
Microbial genomics
Microorganism
Fungi
Extremophiles
Antibiotics
Soda lakes
Kenya
ABSTRACT
In this study we explored the cultivable fungal diversity in Lake Magadi and their secondary metabolite pro-
duction. Isolation was done on alkaline media (Potato dextrose agar, Malt extract agar, Oatmeal agar and Sab-
ouraud dextrose agar). A total of 52 unique isolates were recovered from the lake and were characterized using
different techniques. Growth was observed at pH, temperature and salinity ranges of between 6 - 10, 25 C-40C
and 0%–20% respectively. Phylogenetically, the isolates were affiliated to 18 different genera with Aspergillus,
Penicillium,Cladosporium, Phoma and Acremonium being dominant. A screen for the ability to produce extracellular
enzymes showed that different isolates could produce proteases, chitinases, cellulases, amylases, pectinases and
lipases. Production of antimicrobial metabolites was noted for isolate 11M affiliated to Penicillium chrysogenum
(99%). Cell free extracts and crude extracts from this isolate had inhibitory effects on Bacillus subtilis,Escherichia
coli,Pseudomonas aeruginosa, Salmonella spp., Shigella spp.,Candida albicans and fungal plant pathogens Schizo-
phyllum commune, Epicoccum sorghinum strain JME-11, Aspergillus fumigatus strain EG11-4, Cladosporium hal-
otolerans CBS 119416, Phoma destructive and Didymella glomerata). In this study we showed that different
cultivation strategies can lead to recovery of more phylotypes from the extreme environments. Growth under
different physiological characteristics typical of the soda lake environment (elevated temperature, pH and salts)
confirmed the haloalkaliphilic nature of the fungal isolates. The use of suitable antimicrobial production media
can also lead to discovery of more phylotypes producing diverse biocatalysts and bioactive metabolites.
1. Introduction
Fungi are eukaryotic organisms having either simple unicellular or
multicellular cell structures. Within the environment, fungi are mainly
found in soil and rock surfaces compared to other aquatic habitats. Most
fungal species inhabit soil compared to other environments as they are
organotrophs forming the major group involved in the breakdown of
organic compounds including decaying matter and plant material
(Hasan, 2015). The occurrence of fungi in water is subtle with only
around 3,000 species known to be associated with aquatic habitats,
whereas about 465 species are present in marine waters (Shearer et al.,
2007). Diverse fungal groups have been documented from different
extreme environments such as saline liquids, hot springs, surface of dried
rocks, ocean pits, dry deserts, and very low pH as well as in the polar
environments (Hassan et al., 2016). Filamentous fungi harbour alka-
litolerants lineages similar to those of marine habitats (Grum-grzhimaylo
et al., 2016). Most fungi that inhabit extreme environments are catego-
rized as the imperfect stage of the Ascomycota (Ndwigah et al., 2016).
Different genera, including Cladosporium, Aspergillus, Penicillium, Alter-
naria and Acremonium have been reported to exist as either moderately or
weakly alkali tolerant species in saline environments (Grum-Grzhimaylo
et al., 2013). However, salinity directly affects fungal growth and spor-
ulation. For example, at salinities above 5%, there is increased sporula-
tion and more chlamydospores are formed while conidiogenesis is
inhibited, and there are fewer hyphae (Mulder and El-Hendawy, 1999;
Mandeel, 2006). High ambient salts and pH significantly stress the living
organisms and, therefore, their overall biodiversity may be affected
(Grum-grzhimaylo et al., 2016). To cope with the osmotic stress, fungi in
extreme environments produce extremolytes and extremozymes (Rad-
dadi et al., 2018). In cases of high osmolarity, fungi are able to counteract
loss of water by the accumulation of Kþions into their cells (Plemenita
s
et al., 2014) while others accumulate osmolytes (polysols, sugars and
amino acids) as compatible organic solutes (Roberts, 2005).
Fungi from extreme environments such as the soda lakes have
adapted to elevated temperatures and alkaline saline conditions, which
may lead to evolution and modifications of various fungal pathways
* Corresponding author.
E-mail address: mwirichia.romano@embuni.ac.ke (R. Mwirichia).
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyon
https://doi.org/10.1016/j.heliyon.2019.e02823
Received 1 July 2019; Received in revised form 13 October 2019; Accepted 4 November 2019
2405-8440/©2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Heliyon 6 (2020) e02823

(Brakhage and Schroeckh, 2011). A lot of secondary metabolites
including antimicrobial agents (antibiotics), pigments and toxins are
produced from the modified fungal and actinomycetes pathways
(Satyanarayana et al., 2005;Brakhage and Schroeckh, 2011;Liao et al.,
2015). Fungal genera that are known to produce secondary metabolites
include Fusarium, Aspergillus, Acremonium and Penicillium (Wilson and
Brimble, 2009). Studies on the physiology and genetics of fungi isolated
from unusual habitats are important to foster the understanding of both
Table 1. Growth characteristics of different isolates under varied physiological conditions (temperature, pH and NaCl).
Isolate Salt tolerance Temperature tolerance pH tolerance
0% 5% 10% 20% 25 C30
C35
C40
C pH6 pH7 pH8 pH9 pH10
1M þþ þþþ þ þ þ þþ þþ þþ þ þ þ þ þ
2M þþ þ --þþ þþþ þ þ -þþ þþ þ þþ
5M þþ þþþ þþþ -þþ þþ þþþ þþþ þþ þþ þþþ þ þþ
9M þþþ þþ þþ -þþ þþþ þ þ -þ þþþ þ þþþ
10M þ þþþ þþ -þþþþ-þ þ þ þþþ
11M þþ þþþ þþ -þþ þþþ þþþ þþ þ þþ þþþ þ þþ
13M þþ þ þ -þþ þþ þþ þþ þ þ þþ þ þ
14M þþ þþ --þ þþþ þþ þþþ -þþþþ-
15M þ þþþ þ -þþ þþ þþþ þ þ þ þþþ þþ þ
16M þþ þþ þ -þþ þþþ þþ þ þþ þ þþ þ þþ
18M þþ þþ þ -þ þ þþþ þ -þ þþ þþþ þ
22M þþ þþ þ -þ þþ þþ þ þþ þþ þ þþ þþ
24M þþ þþ þþ þ þ þþ þ þ þþ þþ þþ þþ þþ
25M þþþþ-þþ þþ þþ þ þþ þ þþþ þþ þ
29M þþ þ --þ þþ þ þþ þ þ þþ þ þ
30M þþþ þþþ þþþ -þþ þþþ þþ þþ þþ þ þþþ þþ þþ
31M þþþþ-þþ þþ þþ þþ þ þ þ þ þ
32M þþþþþ-þþþþ-þþ þþ þþþ þ þ
36M þþþþþ-þ þþ þþ þ þþ þ þþ þ þ
38M þþ--- þþ ----þþþ
39M þþþ þþþ þþþ þ þ þ þ þ þþ þþ þþ þþ þþþ
40M þ þþ þþ þ þ þþ þþþ þþ -þþ þþþ þ þ
53M þþ þþ þ þ þ þþ þþ þþ þ þ þþþ þ þþ
56M þþ þ --þþþþþþþþþþþþþ
57M þ þþþ þ þ þ þþ þ þþþ þþþ þ
58M þþþþ-þþþþþþ--þþ þþ þ
59M þþ þþþ þþþ -þþ þþþ þþ þþ -þþ þþ þ þþ
60M þþþ--þþ þþ þþ þ þ þþ þþ þ þ
64M þ þþþ þþ þ þ þþ þ þ þ þþ þþ þ þþ
65M þ þþ þ þ þþ þþþ þþþ þ -þþþþþ
67M þ þþþ þ -þþ þþ þþ þþ -þþþþþ
68M þþ þþ þ -þþþþþþþ-þ þþþ þþþ þ
69M þþ þ --þþ þþþ þ þ -þþ þþ þ þþ
70M þþ þþ þ -þþ þ --þþþ þ þþþ -þþþ
71M þþþ-þ þþþ þþ þþ þþ þ þþ þ þ
72M þþþþ-þþ þþ þ þþ þþ þ þþ þ þþ
73M þ þþþ þ -þþ þþþ þþ þþ -þþ þþ þþ þþ
80M þþ þ þ -þþþþþþþ-þþþþþ
82M þþþþþ-þ þþ þþ þ þþ þ þþ þ þ
87M þþ þþþ þ þ þ þþ þ þ -þþþþþþ
89M þþ þþþ þþþ -þ þþ þ þ þþþ þþþ þþþ þþ þþþ
90M þþþþ-þ þþ þþþ þ -þ þ þþþ þ
94M þþþþþþþþ þ --þþ-þ
95M þþ þ þ -þ þþþ þþ þ -þþþþþ
100M þ þþþ þþ þ þ þþþ þþþ þ --þþ þ þþþ
108M þþþ þþþ þþþ -þ þþ þ þ þþ þ þþþ þþ þþþ
111M þ þþþ þ -þ þþ þ þ þ þ þþþ þþþ þ
113M þþþþ-þ þþþ þ þ þþ þ þþ þ þ
114M þþþþ-þ þþ þþ þ þ þ þþ þ þþ
120M þ þþþþþ þ þþ þþþþ þþþþþþþþþþ
122M þþ þþ þþ þ þ þþ þ þþþ -þ þ þþþ þþ
123M þþþ þþ þ -þþþþþþ þþþþþþþþ
KEY: - (no growth), þ(0 –2 mm, slight growth), þþ (2.1 –4mm, moderate growth), þþþ (>5 mm, abundant growth).
P. Orwa et al. Heliyon 6 (2020) e02823
2

the ecological roles and potential industrial applications (Prakash and
Sharma, 2016). Due to the few numbers of fungal isolates described from
the soda lake ecosystems, their diversity, function and potential to pro-
duce secondary metabolites is still not well studied. In this study, we used
different media and cultivation conditions in an effort to recover novel
phylotypes with the potential to produce bioactive metabolites.
2. Results
2.1. Characterization of the isolates
A total of 52 isolates were obtained with most of them being from
sampling site S3 (wet sediments). The other sites (S3 Dry sediments, S2
wet sediments, S5 wet sediments, microbial mats, grassland soil, and
brine sediments) had a few isolates. After plating, growth of the isolates
on the respective media was monitored and recorded after a period of
between 7-21 days of incubation at 30 C. Spore formation was observed
after 14 days of growth for the spore-farmers. Malt extract agar, Sabo-
urauds dextrose agar, potato dextrose agar and oatmeal agar supported
growth of most of the fungi both in high and low nutrient composition.
The isolates showed varied macroscopic and microscopic characteristics
in terms of colony pigmentation, surface morphology and the hyphae
under the microscope. The hyphae were either septate (hyaline) or
aseptate in others.
2.2. Physiological characteristics
Moderate growth was observed in all the isolates at NaCl concen-
tration of between 0% and 5%. When the salt concentration was
increased to 10%, most of the isolates showed slow growth with isolate
9M,10M, 11M, 24M, 32M, 36M, 40M, 64M, 82M and 100M growing the
same as in the lower concentration. However, isolate 5M, 30M, 39M,
59M and 108M showed optimum growth at 10% while isolate 2M, 14M,
29M, 38M, 56M, 60M, 69M did not show any visible colonies on plates
media (no growth). The highest salt concentration tested was 20% and
only 13 isolates showed growth albeit slow while the rest did not grow
(Table 1). Notable is that isolates 1M, 24M, 39M, 64M, 65M, 100M,
120M and 122M were able to grow across all the tested salt concentra-
tions (Table 1). Different isolates showed varied growth patterns under
different pH ranges. Except for pH 6 where quite a number of the isolates
did not grow completely, most of the fungal isolates grew in pH 7, 8, 9
and 10. Optimum growth for majority of the fungal isolates was recorded
at pH 8–10 (Table 1).
It was observed that the optimum growth temperature for most of the
isolates was 30 C. At 25 C, slow growth was observed in all isolates
except isolate 38M which did not grow at all. When the temperature was
raised to 40 C, isolates M5, M14 and M122 exhibited fastest growth
while four isolates (32M, 38M, 70M and 94M) did not grow at all at this
temperature.
Table 2. Sumary of enzymatic activity of some of the fungal isolates. Only isolates positive for at least one substrate are shown.
Isolate Site Sta. CMC Cas. Pec. Xan. Lig. T20 Gt. Chit. Cel.
1M mats - - - - - - - - þþ -
2M mats þþ -þþ ---þþ -
5M S3S þ---- ---- -
13M S3S - - - - - - - - þ-
14M S3S - - - - - - - - þ-
29M S3S - - þ-- -þ-- þ
31M mgs - - þ-- -þ-þþ -
32M S3S - - þ-- -þ-- -
38M mgs - - - - - - - - þþþ -
39M S3S - - - - - - - - þþ -
40M S3S - - þ-- -þ-þ-
57M S3S - - - - - - - - þ-
59M mgs þþ -þþ -- -- þ
64M S3S - - - - - - - - þ-
68M S3S - - - - - - - - þ-
69M mats þþ þ þ þ -þ-- þþ
70M S3S - - - - - - - - þ-
71M S3S - - - - - - - - þ-
73M S3S - - - - - - - - þþ -
87M S5S þþþ-þþ -- -þþ
90M S3S þþþ-þþ þþþ ---- þþþ
94M S3S - - - - - - - - þþ -
95M S3S - þ--- ---þ-
100M br2 þ-þþþ -þ-þþ þþ
108M S3S - - - - - - - - þ-
111M S3S þþ þ þ þ ---- þ
113M S3dry þþ þ þ þ ---- þ
120M S3S - - - - - - - - þþ
122M S3S - - - - - - - - þ-
123M S3S - - - - - - - - þþ -
KEY: - (no activity), þ(0–3 mm), þþ (3.1–6 mm), þþþ (>6 mm) Iso-isolate, Sta-starch CMC- carboxymethylcellulose, Cas-casein, Pec-pectin, Xan-xanthan, Lig-lignin,
T20-tween 20, Gt- Glyceryl Tributyrate, Chit-chitin, Cel-cellulose.
P. Orwa et al. Heliyon 6 (2020) e02823
3

2.3. Screening for enzymatic activity
All the isolates were screened for their ability to produce extracellular
proteases, celullases, pectinases, chitinases, amylases, xylanases, lipases
and esterases by growing them on a medium with the respective sub-
strate. Production of cellulases was observed by positive enzymatic ac-
tivity on cellulose and carboxymethylcellulose in 9 and 8 isolates
respectively (Table 2). Utlization of the substrate was scored as positive if
there was a halo around the colony after flooding the plate with iodine
solution. Eight isolates were positive for amylase production. Positive
protease activity was observed in 8 of the isolates by way of a clearance
zone around colony on casein agar. Pectinase and chitinase production
was observed after degradation of substrates pectin and chitin by 8 and
22 isolates respectively. However, 22 isolates did not show any enzy-
matic activity on any of the substrates. None of the isolates showed
peroxidase/laccase activity. Some isolates were able to produce more
than one enzyme with a few producing up to four enzymes. Interesting
isolates in terms of polyenzymatic activity were 69M, 87M, 90M, 69M,
59M and 2M. Chitinase activity was observed in 22 of the isolates. This
enzyme is important as a virulence factor in entomopathogenic fungi. It
was observed that most fungi recovered in this study showed low enzy-
matic activity as indicated by the diameter of clearing zones around the
colony. Those with a clearance zone measuring up to 3mm were scored as
(þ). Moderate enzymatic activity was observed in isolates 1M, 2M, 39M,
69M, 87M, 94M, 100M, 123M whereby they had clearing zones of be-
tween 3.1-6mm and was recorded as (þþ). A few isolates 38M and 90M
showed high enzymatic activity for chitinase and cellulase respectively,
they recorded diameter of halo zones above 6mm which was scored as
(þþþ)(Table 2).
2.4. Phylogenetic analysis
BLAST analysis was used to evaluate the phylogenetic affiliation or
relatedness of the individual isolates to nearest neighbors and the results
expressed as percentage similarity (Table 3). All the isolates clustered
with the phylum Ascomycota and were distributed in 18 genera. Among
the isolates, 15 were affiliated to the genus Aspergillus with most of them
exhibiting between 99%-100% similarities. The other genera were also
represented in small numbers and had close affiliation with known
genera in the phylum Ascomycota. They included: Penicillium (9 isolates),
Acremonium (3 isolates), Phoma (4 isolates), Cladosporium (3), Septoriella
(1), Talaromyces (2), Zasmidium (1), Chaetomium (1), Aniptodera (1),
Pyrenochaeta (1), Septoria (1), Juncaceicola (1), Paradendryphiella (1)
Sarocladium (2) Phaeosphaeria (1) and Juncaceicola (1), Biatriospora (2).
The constructed phylogenetic tree indicated the phylogenetic position of
the isolates (Figure 1). The bootstrap values showed that different strains
of isolates within a given genus were closely related (Figure 2). Based on
bootstrap values, Zasmidium and Aspergillus are sister groups in the same
clade and are therefore closely related. The genera Juncaceicola, Septor-
iella and Phaeosphaeria fall in the same clade and therefore have common
evolutionary history. Chaetomium, Aniptodera, Sarocladium fall in the
same clade with Acremonium (commonly known fungal genera). Inter-
estingly, isolate 69M, 87M and 90M had sequence similarity of 96 %,
97% and 95% respectively therefore they could represent novel species.
However, isolates 72M, 80M, 95M, 108M, 113M and 100M had sequence
similarity values below 94% which means they could potentially repre-
sent new genera (Table 3).
2.5. Antimicrobial screening
In primary screening, one isolate (11M) had positive antimicrobial
activity against human enteric pathogens Bacillus subtilis,Escherichia coli,
Pseudomonas aeruginosa, Salmonella spp., Shigella spp. and fungal human
pathogen Candida albicans. The isolate also inhibited fungal plant path-
ogens Alternaria tenuissima and Didymella glomerata. Positive antimicro-
bial activity was qualitatively indicated by presence of inhibition zones.
Both crude filtrates and cell free extracts from isolate 11M showed
antimicrobial effects against several test pathogens as compared to the
results observed in primary screening. Cell free extracts gave larger in-
hibition zones as compared to zones observed from inhibition by crude
extracts. Extracts from production media (PM3) showed positive results
for inhibition against 13 different test pathogens. Production media
Table 3. BLAST analysis results of the fungal isolates from Lake Magadi and their
close relative.
Isolate Code Closest Relative (BLAST) Identity
1M Aspergillus sp. strain 3Y 100%
2M Phoma herbarum strain BZYB-1 99%
5M Aspergillus fumigatus strain EG11-4 98%
9M Cladosporium cladosporioides 98%
10M Penicillium oxalicum strain TGQM01 100%
11M Penicillium chrysogenum CBS 306.48 99%
13M Cladosporium halotolerans CBS 119416 99%
14M Phaeosphaeria luctuosa strain CBS 577.8 99%
15M Aspergillus fumigatus strain EG11-4 99%
16M Aspergillus versicolor isolate F 100%
18M Penicillium commune CBS 343.51 100%
22M Penicillium limosum CBS 339.97 100%
24M Pyrenochaeta nobilis CBS 407.76 96%
25M Acremonium roseolum strain CBS 289.62 99%
29M Aspergillus versicolor isolate CWJ2 100%
30M Uncultured eukaryote clone NYS002060 98%
31M Aspergillus versicolor strain MPE9 99%
32M Sarocladium sp. strain CG-MB01 100%
36M Zasmidium cellare isolate F-14 100%
38M Chaetomium globosum strain F0909 99%
39M Paradendryphiella arenariae isolate F-15 99%
40M Acremonium sclerotigenum CBS 124.42 99%
53M Sarocladium kiliense CBS 122.29 99%
56M Aspergillus keveii strain CBS 209.92 28S 99%
57M Aspergillus glaucus JCM 1575 98%
58M Penicillium citrinum strain IITG_KP1 99%
59M Aspergillus flavipes NRRL 302 98%
60M Phoma destructiva 99%
64M Aniptodera chesapeakensis 99%
65M Phoma sp. LF617 99%
67M Juncaceicola alpina CBS 456.84 99%
68M Septoriella leuchtmannii CBS 459.84 99%
69M Cladosporium velox 96%
70M Aspergillus versicolor strain MF557 99%
71M Aspergillus sp. strain DX4H 99%
72M Penicillium viridicatum CBS 390.48 81%
73M Acremonium roseolum strain CBS 289.62 99%
80M Uncultured fungus clone 42_Wound2L 80%
82M Biatriospora carollii 99%
87M Aspergillus keveii strain CBS 209.92 28S 97%
89M Talaromyces marneffei strain Tm-HIV 92%
90M Uncultured eukaryote clone BSLe1 95%
94M Phoma destructiva isolate: MUCC0064 99%
95M Aspergillus terreus strain AZM03 84%
100M Penicillium oxalicum strain TGQM01 93%
108M Aspergillus flavipes NRRL 302 83%
111M Aspergillus sp. strain AON1 98%
113M Talaromyces marneffei strain Tm-HIV 92%
114M Penicillium polonicum CBS 222.28 98%
120M Septoria senecionis CBS 102366 98%
122M Biatriospora carollii CCF4484 99%
123M Penicillium citrinum strain IITG_KP1 98%
P. Orwa et al. Heliyon 6 (2020) e02823
4

YESD and YPSS however did not give good inhibition results as their
extracts showed positive results against few pathogens (Cladosporium.
halotolerans, Phoma destructiva, Dickeya dianthicola,Phoma destructiva,
Schizophyllum commune isolate ScGD28) pathogens. Crude extracts from
PM3 media showed inhibition zones ranging between 11.33 
0.03–14.33 0.03 and were active against Shigella sp, Pseudomonas
aeruginosa, Escherichia coli and Staphylococcus aureus as the only human
pathogens. However, five plant pathogenic fungi were inhibited by cell
free extracts from PM3. Crude extracts from YESD media showed activity
against Aspergillus fumigatus strain EG11 (422.33 0.03), Cladosporium
Figure 1. Unrooted Phylogenetic tree created using Neighbor-joining method based on a comparison of the 18S ribosomal DNA sequences of Lake Magadi isolates and
their closest phylogenetic relatives. Percentages of bootstrap sampling derived from 1000 replications are indicated by the numbers on the tree.
P. Orwa et al. Heliyon 6 (2020) e02823
5

halotolerans CBS 119416 (22.33 0.09), Phoma destructiva (20.67 
0.03) while cell free extracts had inhibitory effect against Schizophyllum
commune isolate ScGD28 (9.67 þ0.03). Finally, crude extracts from YPSS
media and cell free extracts showed inhibitory effects against Cladospo-
rium halotolerans CBS 119416 (21.67 0.03) and Schizophyllum commune
isolate ScGD28 (9.33 0.03) respectively. Positive controls using broad
spectrum antimicrobial drugs chloramphenicol and nystatin showed
inhibition zones ranging closer to 13.00 0.06 and 15.00 0.06
respectively (see Figures 3and 4).
3. Discussion
The present study focused on the isolation of fungi from Lake Magadi,
and new fungal diversity was determined using various identification
Figure 2. A representative sample of the 52 isolates based on colony and cell characteristics (Plate A1: colony on plates) and (Plate A2: under a compound microscope
magnification x40).
Figure 3. Antimicrobial activity (inhibition zones) of crude extract from isolate 11M screened against test organisms using agar well diffusion method on plates. A1-
Didymella glomerata; A2- Schizophyllum commune isolate ScGD28; A3- Epicoccum sorghinum strain JME-11; A4- Phoma destructive; A5- Candida albicans; A6- Pseudomonas
aeruginosa; A7- Bacillus subtilis; A8- Shigella spp.; A9- Salmonella typhi; A10- Escherichia coli; C1- Positive control with Nystatin; C2- Positive control with
Chloramphenicol.
P. Orwa et al. Heliyon 6 (2020) e02823
6

techniques. Modification of the culture dependent techniques in combi-
nation with molecular analysis using 18S rRNA gene aided in the search
for new diversity in the soda lake. Fungal isolation using commercial
media prepared both in high and low nutrient composition and diluted
with soda lake water is a modification specific to this study. High and low
nutrient media prepared from Malt extract agar, Oatmeal agar, Sabour-
aud dextrose agar and Potato dextrose agar showed varied results in
terms of the number of isolates obtained. For example, more isolates (14)
were obtained using MEA-L than MEA-H whereby only five isolates were
recovered. The dominant genera found in this study were present and
includes: Penicillium, Acremonium,Phoma, and Cladosporium. The same
scenario was observed for PDA and oat meal agar. It is therefore clear that
nutrient fluctuations within different sites in the lake may have little
contribution to the diversity of fungal communities.
Unique morphological characteristics specific to haloalkaliphilic
fungi identified in this study was useful in the description of potential
new species and genera isolated from Lake Magadi and other soda lakes.
Morphologically based characterization was an important parameter in
determining various cell features that are specific to the entire fungi
kingdom. Specific cell structures for example the nature of hyphae/
mycelium justified the occurrence of the isolates in group Ascomycota.
Septate hyphae and oval spores (ascospores) is mainly attributed to
phylum Ascomycota (Raja and Shearer, 2007). However, unique features
including the thick mycelium observed in Phoma herbarum strain BZYB-1
–isolate 2M are important in stress tolerance. Thick mycelium has been
studied in Wallemia ichthyophaga, the most halotolerant fungi known and
it is known to have 3-fold cell wall thickening as a specific feature to
withstand high NaCl concentrations (Gostin
car et al., 2009;Palkov
a and
V
achov
a, 2006). The pigmented fungi isolated for example Zasmidium
cellare isolate F-14 (isolate 36M) and others that are dark pigmented
(Aspergillus keveii-56M, Cladosporium velox-69M) is a feature that enable
such yeasts to thrive in the harsh environments, such as the surface of
stone monuments (Liu et al., 2018). The hyphae produces melanin which
brings about the black coloration in the fungi, an important feature to
stress survival (Plemenita
s et al., 2008). The black coloration was also
observed on most stone monuments, like Liu et al. (2018) reported.
Cladosporium species for example is known to be a halophilic species
dominant in black yeasts (Nazareth, 2014).
Slight differences of the sampling sites in terms of biotic and abiotic
factors (pH, salts, moisture, vegetation cover and trace elements) indi-
cated little difference in the fungal communities present. However, the
highest level of fungal diversity was found in communities from S3
sediments, the common genera being Penicillium, Acremonium,Phoma,
Cladosporium,Zasmidium,Aniptodera,Pyrenochaeta,Septoria,Paraden-
dryphiella and Biatriospora. This may be attributed to the composition of
various physiochemical parameters especially the pH, Na
þ
,K
þ
ions and
trace elements which differed from the other sampling sites. S3 sedi-
ments recorded the highest amounts of Kþand Na
þ
therefore the mi-
croorganisms present probably adopted the salt in strategy to survive the
salt stress. The salt-tolerant yeast D. hansenii for example is known to
maintain a relatively high internal sodium concentration to cope with
salt stress (Prista et al., 1997,2005). In addition, sediments in parts of the
lake are considered to have high nutrient levels because of the contin-
uous erosion of the surroundings which eventually are deposited as silts
(Blomqvist et al., 2004).
The complex effects of physiological parameters (pH, salt concen-
tration and temperature) are also vital in shaping the composition of
fungal species within Lake Magadi (Table 1). It was revealed in this study
that the majority of fungi isolated from Lake Magadi are alkaliphiles
since the pH of the lake had a range of pH 9–10. It is however interesting
that there are moderately halophilic fungal species which grow well in
salt concentrations of up to 5%. In addition, halotolerant strains which
grew well in up to 20% salts were also reported from the lake. The spe-
cific species that were recovered from 20% salts included Aniptodera
chesapeakensis, Phoma sp. and Aspergillus sp. Their ability to tolerate salt
stress is a unique characteristic for such species especially in biotech-
nology. There was no specific temperature requirement for most of the
fungi recovered from Lake Magadi because good growth was observed to
range between 25 - 45 C. Most halophilic microorganism are known to
grow optimally between 25 –45 C(Oren, 2016). It is evident from this
study that some of the fungal isolates recovered are polyextremophiles
because they are able to colonize environments having more than one
extreme condition (Dhakar and Pandey, 2016). In these recent studies,
such polyextremophiles have attracted great attention because of their
possible application to biotechnology and also in aspects relevant to
ecology (Dhakar and Pandey, 2016). Enzymatic activity of specific fungal
isolates indicated their ability to produce at least more than one enzyme
type. Interesting isolates for industrial enzymes included: 2M, 31M, 40M,
59M, 69M, 87M, 90M, 100M and 111M (Table 2). Their ability to pro-
duce proteases, amylase, lipases, esterase cellulases and chitinases makes
them important extremophiles for industrial applications (Sheridan,
2004).
Lake Magadi has phylogenetic diversity composed of fungal com-
munities with various dominant taxa in addition to species exhibiting
spatial and temporal variations at low frequencies. Sequence analysis
indicates a total of 18 different genera that were recovered in this study
and all belong to the phylum Ascomycota except for 3 isolates which were
grouped as unclassified fungi. These findings are similar to the results
from Salano et al. (2017) who found the phylum Ascomycota as the
dominant fungal group from Lake Magadi. Members of the same phylum
Figure 4. Antimicrobial activity (inhibition zones) of cell free extract from isolate 11M screened against test organisms using agar well diffusion method on plates. B1-
Epicoccum sorghinum; B2- Schizophyllum commune isolate ScGD28; B3- Didymella glomerata; B4- Cladosporium halotolerans CBS 119416; B5- Aspergillus fumigatus strain
EG11-4; B6- Shigella spp.; B7- Pseudomonas aeruginosa; B8- Escherichia coli; B9- Staphylococcus aureus; C1- Positive control with Nystatin; C2- Positive control with
Chloramphenicol.
P. Orwa et al. Heliyon 6 (2020) e02823
7

were also found in large proportions in Tundra soils after sequencing 125
cloned fungi (Schadt et al., 2003). Fungal communities from other hy-
persaline environments studied by Santini et al. (2015) indicated that
phylum Ascomycota was dominant with a score of 73% of the total OTUs.
In contrary, reports from other hypersaline environments indicated that
phylum Basidiomycota was the dominant fungal group from deep-sea
environments (Singh et al. 2011;Bass et al., 2007).
The different fungal genera that were dominant in this study were
Aspergillus, Penicillium, Cladosporium, Phoma, and Acremonium. Most of
these genera have been recovered from saline habitats, Aspergillus for
example was found to be the dominant species in the sediments of Lake
Magadi as well as species of Phaeosphaeria (Kambura, 2016). Hypersaline
waters of salterns have also been previously studied and Penicillium and
Aspergillus species were present in diverse levels (Gunde-Cimerman et al.,
2005). Several strains of genus Cladosporium have also been isolated from
Caspian Sea waters (Sadati et al., 2015). Moreover, a similar study by
Salano (2011) indicated the presence of genera Aspergillus, Penicillium,
Cladosporium, Talaromyces, and Acremonium from Lake Magadi. The
presence of dominant species from different fungal genera is a suggestion
that they are highly adaptable to the extreme conditions of the soda lake.
The above genera commonly found together in saline environments share
ecological preferences to extreme conditions.
This study reports the presence of new diversity of fungi thriving in
the sediments and soils of Lake Magadi which has not been previously
reported. New fungal genera and different species have been recovered in
this study including: Septoriella leuchtmannii,Phoma sp., Zasmidium cel-
lare,Chaetomium globosum,Aniptodera chesapeakensis,Pyrenochaeta
nobilis,Septoria senecionis,Paradendryphiella arenariae, Sarocladium
kiliense, Juncaceicola alpina and Biatriospora carollii. which has been iso-
lated from other different saline environments (Georgieva et al., 2012;
Bonugli-santos et al., 2015). This is the first report on the occurrence of
this new genera isolated from Lake Magadi. Studies on Dead Sea, saline
habitats of Wadi El-Natrun, Egypt however was able to isolate Chaeto-
mium globosum (Perl et al., 2018). Ndwigah (2017) isolated a fungal
strain from Kenyan saline Lake Sonachi that had 100% alignment with
Sarocladium kiliense (HQ232198). Grum-grzhimaylo et al. (2016) also
recovered the same isolate in soda soil. There are limited reports on the
recovery of Biatriospora sp. from saline environments however it is known
to be a potential producer of potent antibiotics and a diverse set of me-
tabolites (Kola
rík et al., 2017). The marine fungus Paradendryphiella
arenariae is a marine fungus and has been isolated from Thailand; in-
vestigations have indicated that it produces bioactive secondary metab-
olites (Yoiprommarat et al., 2015). Septoriella leuchtmannii,Zasmidium
cellare,Aniptodera chesapeakensis,Pyrenochaeta nobilis,Septoria senecionis,
and Juncaceicola alpine are common causes of human and plant diseases.
The diverse groups of fungi present in the lake are terrestrial fungi
and may have originated from the surrounding agricultural/vegetation
soils. They are carried by surface run offs and deposited into various
sediments in the lake either in the form of spores or fungal hyphae. Such
fungi develop effective strategies to cope with the extreme conditions of
salts, alkaline pH and temperatures. Further studies on their metabolite
production potential will be of interest because of their ability to adapt to
extreme saline environments. The discovery of fungi that are common
causes of plant and animal diseases from the soda lake will ignite more
research on their genome structures so as to determine their toxin
pathways. Appropriate strategies to control such pathogens can thus be
developed.
Antimicrobial metabolite production noted for isolate 11M affiliated
to Penicillium chrysogenum CBS 306.48 (99%) is of interest because it
produces active agents against both human pathogenic bacteria and plant
pathogenic fungi. The use of modified production medium, in this case
PM3, is an important consideration in the search for antimicrobial me-
tabolites from haloalkaliphilic fungi. Since the discovery of Penicillium
chrysogenum as the source for the first antibiotic penicillin, various
studies on antimicrobial production have been done on various species of
Penicillium. Halophilic and halophilic species of Penicillium are known to
be producers of various polyketides including penicillic acids, antibiotics
(penicillins) and amino acid derived extrolites (Frisvad, 2005). In
particular, different strains of Penicillium chrysogenum have been isolated
from saline environments (Nayak et al., 2012;Gunde-cimerman and
Zalar, 2014;Cantrell et al., 2006). Antimicrobial agents (β-lactam anti-
biotics) from Penicillium chrysogenum and other Penicillium species are
known to be active against most of the Gram positive pathogenic bacteria
(Salo, 2016). Penicillium chrysogenum CBS 306 strain recovered in this
study proved to be active against Gram negative, Gram positive, Candida
albicans and also inhibited various plant pathogenic fungi (Schizophyllum
commune, Epicoccum sorghinum strain JME-11, Aspergillus fumigatus strain
EG11-4, Cladosporium halotolerans CBS 119416, Phoma destructive and
Didymella glomerata). Similar findings on the same strain of Penicillium
has not been reported in previous studies. Further purification and
identification of the specific metabolites produced by Penicillium chrys-
ogenum CBS 306 strain will be useful in agricultural and pharmaceutical
systems especially when the active agents are formulated into products.
Exploration of extreme environments therefore enhances the isolation of
new strains of producer fungi mainly with the modification of growth
and production medium. This will ignite industrial improvement pro-
grams that will enhance activities to develop high metabolite producing
strains.
4. Materials and methods
4.1. Isolation of fungal strains
Soil samples used in this study were collected from the hypersaline
Lake Magadi located in the East African Rift valley (2 S and 36 E). The
lake lies about 660 m above sea level, depth ranging between 1-5 m and
covers an area estimated to be 90 km
2
(Behr and R€
ohricht, 2000). Serially
diluted samples (0.1 g in 1 ml of sterile lake water) were plated onto
Potato dextrose agar (PDA), Malt extract agar (MEA), Oatmeal agar and
Sabouraud dextrose agar (SDA) with chloramphenicol (100 mg/L) as an
antibiotic to inhibit bacterial growth. Onto each medium, 100
μ
l from
each dilution was spread plated and the plates incubated at 28 C until
visible colonies appeared. The colonies were picked and transferred
several times onto fresh medium until axenic cultures were obtained.
4.2. Physiological and biochemical characterization
Morphological features (color, pigmentation and colony surface
morphology) together with cellular features (hyphae type) were used to
select unique isolates from the different media. Physiological character-
ization was done to test the ability of the selected isolates to grow at
elevated pH ranges (6, 7, 8, 9, and 10), different NaCl concentrations
{(w/v) 0%, 5%, 10% and 20%} and varied temperature ranges (25 C, 30
C, 35 C and 40 C). Growth was scored in terms of the size of the colony
after 48 h of growth. Growth was scored depending on the colony size.
Ability of the different isolates to produce exoenzymes was tested on a
basal medium (Tryptone 10g, Sodium chloride 10g, and Yeast Extract 5g)
supplemented with the respective substrate as summarized in Table 4.
4.3. Molecular characterization
A sterile loop was used to aseptically scrape the mycelia/spores into
100
μ
l sterilized resuspension buffer (50mM Tris pH 8.5, 50mM EDTA pH
8.0 and 25 % sucrose solution) in an Eppendorf tube. To it was added
400
μ
l of the lysis buffer (10mM Tris pH 8.5, 5mM EDTA pH 8.0 and 1 %
SDS), 10
μ
l of Proteinase K (20 mg/l) followed by incubation at 65 C for
60 min. Phase separation was achieved by addition of an equal volume of
chloroform and centrifugation at 13,200 rpm for 10 min at 4 C. The
supernatant was carefully transferred to a new tube and recovered using
the standard sodium acetate/isopropanol/ethanol method. An aliquot
(3
μ
l) of the recovered DNA was run on 1% agarose to confirm the pres-
ence and quality.
P. Orwa et al. Heliyon 6 (2020) e02823
8

The 18S rDNA gene was amplified using the primer pair Fungi 683f
(50-GCTCGTAGTTGAACCTTTGG-30) and Fungi1394r (50-
TCTGGACCTGGTGAGTTTC-30) on a Surecycler 8800 machine (Agilent
Technologies). The PCR mix consisted of 0.6
μ
l dNTP's, 6
μ
l of PCR buffer
(10), 1.5
μ
l (5pmol) of FF390r reverse primer, 1.5
μ
l (5pmol) of Fung5f
forward primer, 0.5
μ
l of template DNA, 0.1
μ
lTaq polymerase and 18.3
μ
l
of water in a final volume of 30
μ
l. Cycling was done as follows:36 cycles.
Initial denaturation at 95 C for 3 min followed by 30 cycles of dena-
turation at 95 C for 1 min; Primer annealing at 58 C for 45 s and
extension at 72 C for 1 min. A final extension step at 72 C for 5 min was
included. An aliquot of 5
μ
l of the PCR products was run on a 1 % agarose
gel stained with Cyber green in 1TAE buffer and visualized under ul-
traviolet. The PCR products were purified using ExoSAP-IT™(Applied
Biosystems™) as described by the manufacturer. The cleaned amplicons
were sent for Sequencing to Inqaba Biotech, South Africa.
4.4. Screening for antimicrobial activity
Fungal isolates were grown for 14 days in broth at 30 C in a shaker
incubator at 100rpm. The crude extracts were tested for antimicrobial
activity against Gam positive bacteria Staphylococcus aureus and Bacillus
subtilis, Gram negative bacteria Escherichia coli,Pseudomonas aeruginosa,
Salmonella spp., Shigella spp. and fungal human pathogen Candida albi-
cans. to allow for a qualitative selection of bioactive isolates (Arora et al.,
2016). The antimicrobial assay was done using the agar well diffusion
method (Rajpal et al., 2016) on Muller Hinton agar (pH 8 and 5% NaCl).
The fungal isolates were also tested for antagonistic effect against com-
mon fungal plant pathogens to determine their potential as biocontrol
agents using agar well diffusion method. The test fungal pathogens used
were Epicoccum sorghinum strain JME-11, Alternaria tenuissima, Didymella
glomerata,Schizophyllum commune isolate ScGD28, Phoma destructive,
Cladosporium halotolerans CBS 119416, Aspergillus fumigatus EG11-4 iso-
lated from the lake and plant bacterial pathogen Dickeya dianthicola ob-
tained from infected vegetable. Isolates that showed positive
antimicrobial activity in primary screening were qualitatively selected
for secondary screening using agar well diffusion method on Muller
Hinton agar. However, for this test, the producer strain was grown on
three production media (YESD, PM3 and YPSS, pH 9 and 5% NaCl) in a
shaker incubator at 30 C at 100rpm. Cell free extracts obtained after
centrifugation of crude extracts at 10000 rpm for 10 min and crude ex-
tracts were used. Inhibition zones were measured as mean diameter of
the wells 0.6cm plus the clearing zone in triplicates for every test or-
ganism used. Broad-spectrum antibiotic and antifungal (chloramphenicol
20
μ
g/ml/nystatin 5
μ
g/ml) were used as positive controls. The media
used are:
Media A (YESD)-g/L Tryptone soya broth 30.0, yeast extract 5.0, tap
water (VanderMolen et al., 2013).
Media B (PM3)-g/L glucose 0.5, glycerol- 2.5ml, oat meal 5.0, soy
bean 5.0, casamino acids 2.0, yeast extract 0.5, 1ml from solutions of
calcium chloride (156mg/10ml), magnesium chloride (190mg/10ml)
and manganese chloride (12.58 mg/10ml) and tap water (Jose and
Jebakumar, 2013).
Media C (YPSS)-g/L Yeast extract 4.0, starch 14.0, dibasic K2HPO4
1.0, MgSO4.7H2O 0.5, tap water (VanderMolen et al., 2013).
Declarations
Author contribution statement
Philemon Orwa, Romano Mwirichia: Conceived and designed the
experiments; Performed the experiments; Analyzed and interpreted the
data; Contributed reagents, materials, analysis tools or data; Wrote the
paper.
George Mugambi, Vitalis Wekesa: Analyzed and interpreted the data;
Contributed reagents, materials, analysis tools or data; Wrote the paper.
Funding statement
The work was supported by the Alexander von Humboldt Stiftung
Equipment Grant, DAAD Material Resources Programme, Equipment
donation by Seeding Labs, USA, The National Research Fund, Kenya and
The World Academy of Sciences. Philemon Orwa was supported by the
University of Embu postgraduate scholarship.
Competing interest statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
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