Introduction

Glycerol (glycerin) is a simple sugar alcohol (1,2,3-propanetriol) with various industrial uses in pharmacy, cosmetics, paints, food, paper, dynamite and leather (Zhang et al. 2002; Sivasankaran et al. 2014). Glycerol is also a feedstock for numerous valuable chemicals such as lactic acid, citric acid, 1,3-dihydroxyacetone (DHA), 1,3-propanediol (1,3-PD), dichloro-2- propanol (DCP), acrolein, hydrogen and ethanol (Fan et al. 2010). Glycerol can be synthesized either by chemical or microbial routes. However, the chemical route of glycerol synthesis from petrochemical feedstock or soap manufacture has been declined because of the gradual increase in cost and replacement of soaps by detergents (Wang et al. 2001). In contrast, the fermentative route of glycerol production seems more applicable nowadays due to the simplicity of the method and using low-cost substrates in a short time (Grembecka, 2015). Yeasts have been considered the most widely used microorganisms for the microbial synthesis of glycerol (Yaçlin and Ӧzbas 2005). Production of polyols such as glycerol is an integral part of the normal growth processes of the various yeast strains, which form them according to the requirement of growth (Butinar et al. 2005). Among yeasts, the brewing and baking yeast, Saccharomyces cerevisiae is the most widely glycerol-producing (Taherzadeh et al. 2002). However, technical problems for commercial applications have limited glycerol production by S. cerevisiae, and the other non osmophilic yeasts, these include: (1) a relatively low glycerol yield due to the production of other by-products such as ethanol, acetic acid, acetaldehyde and 2,3-butanediol in significant amounts; (2) using of high amounts of steering agents such as sulfite during the fermentation; and (3) a low productivity rate with a low final concentration in the fermentation medium make the recovery of glycerol expensive and insufficient (Sahoo and Agarwal 2001; Agarwal 1990; Vijaikishore and Karanth 1986). Therefore, the production of glycerol by osmotolerant and marine yeasts is a promising research area for many researchers; this is due to the high potentiality of these yeasts to produce high amounts of glycerol during the growth in its natural habitat to overcome the high external osmotic pressure of marine environment (Connell et al. 2008). The adaptation to an environment with a low water activity (aw), i.e. high osmotic pressure, can vary from a species to another but is generally based on the accumulation of osmolytes as glycerol to recompense the increased external osmotic pressure (Bubnová et al. 2014). Marine yeasts have many advantages for glycerol production compared to conventional yeasts; they do not require anaerobic conditions or steering agents for cell growth and fermentation. Moreover, most marine yeasts can tolerate very high sugar concentrations which necessary for the enhancement of glycerol production yield and rate (Petrovska et al. 1999). Various osmotolerant and marine yeast species were reported as highly glycerol producers including Candida magnolia, Debaryomyces hansenii, Pichia anomala, Candida glycerinogenes and Zygosaccharomyces rouxii (Klein et al. 2017). Obtaining yeast strains with high glycerol fermentation rate considered an extremely challenging task, newly characterized yeasts that are highly efficient in glycerol production can be reached by exploring extreme saline habitats like marine water or by altering important genes in glycerol production pathway through ultraviolet (UV)-mutagenesis (Martinez et al. 2008; Shahsavarani et al. 2012). This study aims to enhance the glycerol production by selected marine yeast W.anomalus HH16 and to investigate the impact of carbon, nitrogen, salinity, and pH of the fermentation medium on glycerol production by this selected mutant.

Materials and methods

Yeast cultures

Seaweed and marine sediment samples were collected from different locations along Suez Canal region in Suez governorate and Suez Gulf area in South Sinai governorate, Egypt. The yeast extract–malt extract (YM) medium was used to isolate potential halotolerant/halophilic yeasts. The YM agar medium consisting of (g l−1 seawater): yeast extract, 3.0; malt extract, 3.0; peptone, 5.0; dextrose, 10.0 and supplemented with 20.0 g agar–agar. The medium pH was adjusted to 5.5 ± 0.2 using 0.1 N HCl and 0.1 N NaOH. Among the isolated yeasts, five isolates were selected for the present study based on their growth rate on YM medium and their strong capability to produce glycerol.

Screening of glycerol production

Preparation of yeast inocula

Yeast inoculum was prepared by inoculating a loopfull of 48 h old culture into 50 ml sterilized YM broth and incubated at 30° C for 48 h on a rotary shaker (150 rpm).

Fermentation process

The used glycerol fermentation medium is composed of (g l−1 distilled water): glucose, 180; yeast extract, 1; peptone, 1; KH2PO4, 1; MgSO4, 0.5 and NaCl, 58.44 (1 M). The medium pH was adjusted to 6 ± 0.2 using 0.1 N HCl and 0.1 N NaOH. The fermentation process was conducted in glass bottles of 100 ml volume each containing 50 ml medium inoculated with 20% of the selected yeasts (HH1, HH16, HH56, HH60, HH91) inocula (106 cells ml−1) and incubated at aerobic conditions for 96 h on a rotary shaker (150 rpm) at 30° C.

Glycerol extraction

The concentrations of both intracellular and extracellular glycerol were measured to determine the total glycerol produced and accumulated by the used yeast strains. Yeast cultures were centrifuged at 4000g for 10 min. The cell pellets were used for the intracellular glycerol measurement while the supernatant was used to determine the extracellular glycerol (Aslankoohi et al. 2015). The cell pellets were washed twice with PBS (Phosphate Buffered Saline); 1× contains 0.137 M NaCl, 0.0027 M KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4. After washing, the cell pellets were added into 3 ml 0.1 mol l−1 TRIS/HCl buffer (pH 7.7) containing 2 mmol l−1 EDTA for and boiled for 5 min. After centrifugation at 11,000g for 10 min to remove the cell debris, the glycerol concentration was measured in the supernatant using sodium periodate method.

Glycerol determination

Glycerol was quantitatively determined using sodium periodate method according to Kuhn et al. (2015). http://www.sciencedirect.com/science/article/pii/S0717345815000226—af0005. For each experiment, the following parameters were calculated according to the following equations: intracellular glycerol concentrations (Pin), extracellular glycerol concentrations (Pex), total glycerol concentrations (Pm), biomass (Xm), glycerol coefficient yield (YP/S), biomass coefficient yield (YX/S), volumetric glycerol productivity (Qp) and average substrate uptake (Qs). Glycerol and biomass coefficient yields were based on initial sugar concentration (SGo) and expressed in g g−1:

$$ Y_{\text{p/s}} \left[ {{\text{g g}}^{ - 1} } \right]\; = \;X_{\text{m}} /S_{\text{G0}} $$
(1)
$$ Y_{\text{x/s}} \left[ {{\text{g g}}^{ - 1} } \right]\; = \;P_{\text{m}} /S_{\text{G0}} . $$
(2)

Volumetric glycerol productivity (Qp) was calculated as grams of glycerol formed per liter per hour (g l−1 h−1):

$$ Q_{\text{p}} \; = \;dP/dt. $$
(3)

Whereas average substrate uptake (Qs) was calculated as grams of substrate consumed per liter per hour (g l−1 h1):

$$ Q_{\text{s}} \; = \; - \;dS/dt. $$
(4)

To determine the microbial kinetic growth, the specific glycerol production rates (vg) and the specific growth rate (µx) values were calculated. Specific glycerol production rates (vg) were expressed as g g−1 h−1 and calculated from the following relationship by using the changes in glycerol and dry biomass concentrations with time:

$$ v_{\text{g}} = dP/Xdt. $$
(5)

The specific growth rate values were calculated from the logarithmic plots of the dry weight data with the fermentation time.

$$ \mu_{x} = dX/dt . $$
(6)

Furthermore, the maximum values of specific growth rates (µmax), maximum specific glycerol production rates (vmax), the maximum dry weight (Xmax) and the maximum glycerol concentrations (pmax) were also calculated.

Total residual sugars (TRS) determination

The residual sugars were determined by dinitrosalicylic acid (DNS) method as described by Miller (1959). Three ml of DNS reagent was added to3 ml of diluted fermentation medium and the mixture was boiled for 5 min. After cooling down to room temperature, 1 ml of Rochell salt (40% potassium sodium tartarate) was added and absorbance was measured at 510 nm by UV-9200 VIS spectrophotometer. DNS reagent was prepared by dissolving 10.0 g of sodium hydroxide, 10 g of 3, 5-dinitrosalicylic acid, 2.0 g of phenol and 50 mg of sodium sulfite in one liter distilled water.

Biomass determination

The biomass was determined as dry weight (g l−1) by drying the collected cells from fermentation medium in an oven at 85° C for 24 h and then was weighted (Zhang et al. 2018).

Phenotypic characterization

Based on the glycerol production capability, W. anomalus HH16 was found to be the yeast isolate that showed the highest glycerol yield. W. anomalus HH16 was selected to characterize phenotypically and genotypically as well. W. anomalus HH16 isolate was morphologically characterized on four different types of culture media; the colony characteristics including color, texture, appearance, elevation, and margin are examined in cultures streaked on YM agar medium. The cell morphology, budding, formation of pellicle and ascospores were examined on 5% malt broth medium (Ali and Khan 2014) and sodium acetate agar medium (Sulieman et al. 2015). The formation of pseudohyphae or true hyphae was studied on cornmeal agar using the coverslip method (Lodder and Kreger-van Rij 1952). Traditional biochemical and physiological tests were used for isolate identification. The isolate was tested for the production of enzymes; amylase, cellulase, and urease. Other biochemical criteria were tested as diazonium Blue B (DBB) test, citrate, indole, methyl red (MR), voges-proskauer (VP), growth in vitamin free and osmotic medium, assimilation and fermentation of various carbon compounds. Furthermore, the temperature and halo-tolerance profiles of the isolate were performed.

Genotypic identification

DNA extraction and PCR amplification

Genomic DNA (gDNA) was extracted according to Kumar et al. (2010) using chloroform-extraction and ethanol-precipitation method. The primers ITS1:5′-TCCGTAGGTGAACCTGCGG-3′and ITS4 5′ TCCTCCGCTTATTGATATGC-3′ (White et al. 1990) were used to amplify ~ 750 bp from the internal transcribed spacer (ITS) Region. PCR reactions were performed in a final volume of 100 µl with the following reagent concentrations: Taq buffer (1×); dNTP mixture (200 µM each); Forward and reverse primers (0.2 µM each); Taq DNA polymerase (2.5 U/100 µl); ~ 50:100 ng of gDNA template and the total volume of the PCR reaction adjusted to 100 µl with nuclease-free H2O. The complete reaction mixture was incubated at an automated MJ research thermal cycler (USA). PCR amplification included an initial denaturation step at 95° C for 3 min, followed by 35 cycles: 95° C for 30 s, annealing at 55° C for 30 s, extension 72° C for 60 s, and a final extension at 72° C for 5 min. All PCR amplicons were analyzed by gel electrophoresis through 1% agarose gels in 1×Tris-acetate-EDTA (TAE) buffer (40 mMTris, 20mM Acetate and 1 mM EDTA, pH 8.0). The sizes of the PCR amplicons were estimated against a DNA ladder (Applied Biotechnology Co, Egypt) and visualized under UV light after staining with ethidium bromide (5 µg ml−1).

Nucleotide sequence analysis

The amplified PCR product was sent to Solgent Co Ltd (South Korea) for sequencing. The resulted sequences were trimmed and assembled in Geneious software (Biomatters). Consequently, the trimmed sequences were identified by search in basic local alignment tool (BLAST) in GenBank. The full-length sequences obtained were matched with previously published sequences available in NCBI using BLAST at NCBI website: http://www.ncbi.nlm.nih.gov/BLAST/ to assess the degree of DNA similarity.

UV-mutagenesis of the selected yeast isolate

Wickerhamomyces anomalus HH16 culture was inoculated in 10 ml of YM medium and incubated overnight at 30 °C until cell density reached 2 × 108 cell ml−1. The cells were diluted to obtain a final density of 10–100 cell ml−1. A 100 μl of the diluted suspension was taken and inoculated on YM agar plate. Exposure was conducted at UV irradiation intensity (wavelength 254 nm) of 200 mW cm−2 for 5, 10, 15 and 20 min. To stop photoreactions, plates were kept in dark for 24 h then incubated for 3 days at 30 °C. The surviving mutant colonies were picked and streaked on YM medium.

Optimizations of glycerol production by the selected mutant isolate W. anomalus HH16 MU5

Parameters such as carbon, nitrogen sources, salinity, and pH were investigated to obtain the maximum yield of glycerol by the UV mutant isolate. The fermentation medium was supplemented individually with organic different carbon sources (glucose, fructose, sucrose, maltose, and lactose) at a concentration of 18% of each carbon source separately. Furthermore, determination of growth and glycerol production kinetics at different glucose concentrations was achieved. Effect of individual addition of various organic and inorganic nitrogen substances (peptone, yeast extract, beef extract, malt extract, urea, ammonium phosphate and ammonium sulfate) at 0.3% level on glycerol production was examined. Also, the influence of the combination of different nitrogen sources (yeast extract- peptone- urea) at different ratios were investigated. The effect of different salinity concentrations (0.5 M, 1 M, 2 M, 3 M, 4 M, and seawater) on glycerol production was studied. To determine optimum pH for glycerol production, the pH of fermentation was controlled using 2.5 N Na2CO3. The effect of pH was tested at uncontrolled pH, pH 4.0, pH 5.0, pH 6.0, pH 7.0 and pH 8.0.

Results and discussion

Screening of glycerol and biomass production by the tested yeast isolates

Results in Table 1 showed that all the tested marine yeast isolates (HH1, HH6, HH56, HH60 and HH91) could produce and accumulate glycerol at variable concentrations. The highest amount of total glycerol (66.55 g l−1) was produced by the isolate HH16 with volumetric glycerol productivity 0.92 g l−1h−1.On the other hand, the isolate HH60 produced the lowest amount of total glycerol (52.30 g l−1), with volumetric glycerol productivity 0.73 g l−1 h−1. The potentiality of marine yeasts to survive the external high osmotic pressure with a low water activity (aw) can vary from species to a species but in general based on the amount of osmolytes that accumulated in the cells (Hohmann 2002; Thome 2007). Passoth et al. (2006) reported that the ascomycetous yeast can adapt to surrounding osmotic stress by the production of excess glycerol as an intracellularly osmolyte. The intracellular glycerol can be adjusted by exporting some of the synthesized glycerol extracellular to balance the osmotic pressure across plasma membrane.

Table 1 Glycerol and biomass production by five yeast isolates
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Characterization and identification of the isolate W. anomalus HH16

The morphological characteristics of the isolate HH16 were shown in Table 2. The colony on YM agar was tannish-white colored butryous, smooth, glistening and umbonate. Cells’ morphological characters were measured on 5% Malt extract broth and sodium acetate agar media; it was obvious that the cell shape was sub-globose to ovoidal with monoploar, bipolar and multilateral budding. Diploid budding cells are transformed into asci containing 2–8 hat-shaped ascospores. Pseudohyphae formation was studied on cornmeal agar using the coverslip method which indicated that pseudohyphae are formed and branching out bearing clusters of ovoidal blastoconidia. Pseudohyphae also have ramified chains of cells bearing dense groups of ovoidal blastoconidia in verticils, the cells are transformed into asci with ascospores (Fig. 1). The morphological characters obtained in this study were as described by Kurtzman and Fell (1998) who characterized the yeast W. anomalus as heterothallic, ascomycetous yeast, forming one to four hat-shaped ascospores.

Table 2 Morphological characteristics of the isolate HH16
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Fig. 1
figure 1

Morphological characteristics of the yeast isolate HH16. a Conjugation of yeast cells on sodium acetate agar (100×), b Cells are subglobose, asci containing two to eight hat shaped ascospores on 5% malt broth(100×), c Scanning electron microscope (SEM) of subglobose budding yeast cells on 5% malt broth (3.500×), d Pseudohyphae formation on cornmeal agar (10x), e, f Long and branched pseudohyphae with numerous ovoid blastospores occur in clusters on cornmeal agar (40×, 100×), g SEM of pseudohyphae formation on cornmeal agar (3.500×)

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The biochemical and physiological characteristics (Table 3) revealed that the isolate HH16 is ascomycetous yeast relying on its negative reaction to diazonium blue B test (DBB). This test is employed to determine whether asexual yeast belongs to a basidiomycetous (+ve reaction) or an ascomycetous genus (−ve reaction) (Ghindea et al. 2009; Kurtzman and Fell 1998). The isolate was positive to voges-proskauer (VP) and negative to both of methyl red (MR) and indole tests. This isolate showed the ability to utilize citrate, glucose, sucrose, maltose, lactose, galactose, l- arabinose, erythritol, cellobiose, cellulose, starch and, urea, but not d-xylose. The results also indicate that W.anomalusHH16 is unable to ferment lactose and galactose but can assimilate these two sugars. Results showed that this non-conventional isolate could grow under extreme environmental stress conditions such as high osmotic pressure (50% glucose) and vitamin free medium. It could tolerate sodium chloride concentrations up to 4.0 M and grow well at temperature range 8–42 °C.

Table 3 Biochemical and physiological characteristics of the isolate HH16
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The ITS region sequence was used for genotyping and confirm the identity of some selected yeast isolates during the current study. ITS sequences were deposited into GenBank and assigned the following accession numbers: MK156297–MK156309, MK182823, and MK182824. Using BLASTN search tool (Altschul et al. 1990), the ITS sequence of W.anomalus HH16 (GenBank accession MK182824) matched the type sequence of W. anomalus 1033L1 strain 1033L1 (Accession KP638741) with identities = 562/562 (100%) and gaps = 0/546 (0%). Wickerhamomyces anomalus, formerly known as Pichia anomala and Hansenula anomala was recently assigned to the genus Wickerhamomyces based on phylogenetic analysis of a dataset of D1/D2 LSU rRNA gene sequences, which has caused great changes in the yeasts classification analysis (Kurtzman et al. 2011). W. anomalus is a biotechnologically important yeast species isolated from diverse natural habitats including marine environments. This organism exhibits unique and potentially exploitable physiological and metabolic characteristics (Padilla et al. 2018); this is due to the efficient capability to tolerate extreme environmental conditions like oxidative, salt, and osmotic stresses (Walker 2011).

UV-mutagenesis of W. anomalus HH16

The influence of (UV) irradiation on the marine isolate W. anomalus HH16 was studied to enhance its glycerol production. The results revealed that there was a dramatic reduction in the percentage of the surviving yeast colonies number as the UV exposure time increased (Fig. 2). Data showed that only 12% of the yeast colonies survive after 5 min UV irradiation, 5% surviving after 10 min and only one yeast colony survive after 15 min, whereas at UV exposure time (20 min), no colonies were reported. Two surviving mutants were selected for glycerol production, the selection based on the resistance to exogenous ethanol in contrast to the wild isolate at medium containing 10–30% (v/v) ethanol. The alcohol resistance of the selected irradiated strains, possibly due to yeast mutation during UV irradiation (Revin et al. 2018). UV irradiation can induce mutagenic and cytotoxic DNA lesions such as cyclobutane–pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4 PPs); however, yeast cells have developed a wide range of DNA-damage repair mechanisms to counteract the UV effect. Photoreactivation with photolyase enzyme considered one of the most effective repair mechanisms developed by yeasts (Sancar and Smith 1989). Zhang et al. (2015a, b) found that several genes were highly expressed only in the ethanol-tolerant mutant but not in the parent strain; the ethanol-tolerant mutant also exhibited resistance to other stresses including high osmosis (Zhao and Bai 2009). Random mutagenesis in yeast by mutagens such as mutagenic agents and UV light is a promising strategy for improving lipid production (Tapia et al. 2012), ethanol production (Watanabe et al. 2011) and sugar alcohols production (Zhang et al. 2015a, b; Savergave et al. 2013). UV exposure causes various types of DNA damage, the intensity and time exposure of UV irradiation can highly affect both the activation of yeast (as a protective mechanism against oxidative stress) and mutation that can occur (Kumari and Pramanik 2012; Ikehata and Tetsuya 2011). At high UV doses, most recombination events reflect the repair of two sister chromatids broken at the same position whereas, at lower UV doses, most events involve the repair of a single broken chromatid which could cause mutagenesis in different regions (Yin and Petes 2013), this might explain why the mutant HH16 MU5 which exposed to UV irradiation for 5 min gives high glycerol production (80.15 g l−1) compared to the mutant HH16 MU15 which irradiated for 15 min (Table 4).

Fig. 2
figure 2

The percentage of surviving yeast colonies at different exposure time to UV

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Table 4 Glycerol and biomass production by the wild and UV-mutant isolates
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Optimizations of glycerol production by the selected mutant isolate W. anomalus HH16MU5

Glycerol yield by yeasts can be affected by various growth and environmental factors; substrate type, initial substrate concentration, nitrogen source, pH and salinity are among these factors (Zhao et al. 2015).

Effect of different carbon sources

Data in Table 5 show that the wild and mutant isolates could assimilate different sugars and produce glycerol at variable concentrations. The yeast osmotic stress and glycerol production is carbon source dependent (Babazadeh et al. 2017). According to Leandro et al. (2011), marine yeasts could utilize a variety of substrates, the various sugars and carbon sources used in fermentation might exhibit osmotic pressure of different degrees on the yeast cell. Moreover, our results indicated that the highest glycerol yields were achieved by both the wild and mutant isolate W. anomalus HH16MU5 using a growth medium supplemented with monosaccharides such as glucose or fructose. The maximum glycerol yields were recorded as 0.3697 and 0.4452 for the wild and the mutant isolates, respectively, using glucose as a carbon source. Fructose was the second-best substrate with glycerol and biomass yields recorded as 0.4268, 0.0881 g g−1 respectively, and average fructose uptake of 2.1289 g l−1 h−1 for the mutant isolate. The minimum glycerol production was achieved using medium supplemented with lactose and maltose with glycerol yields 0.2766, 0.3077 g g−1 for the mutant and 0.3083, 0.3005 for the wild isolate, respectively. These results were consistent with Piddocke et al. (2009) who revealed that glycerol production was higher in the fermentation of glucose-rich medium than in medium supplemented with maltose. The reason could be that at high monosaccharide medium more DHAP and NADH were generated (Orlic et al. 2010).

Table 5 Effect of different carbon sources on glycerol and biomass yields by the selected mutant isolate
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Effect of different initial glucose concentrations

The integration of experimental studies with mathematical modeling makes it is possible to obtain quantitative and meaningful interpretation of the experimental results and provide new aspects of microbial physiology (Snoep et al. 2009). In shake-flask experiments, the effects of different initial concentrations of glucose (50–250 g l−1) at specific time intervals were examined. For each time interval, the changes in glycerol production concentration and dry biomass were determined. Variations in glycerol concentration and biomass of the isolate W. anomalus HH16 MU5during fermentation time were shown in Fig. 3. The initial glucose concentration of the medium was chosen as independent variable for the specific growth rate and glycerol production rate of the culture. The growth data was represented by the following Monod model:

$$ \mu \; = \;\frac{{\mu_{\text{max} } \; + \;S_{^\circ } }}{{ks\; + \;S_{^\circ } }}, $$
(7)

where µmax is the maximum specific growth rate, So is initial glucose concentration, Ks is substrate utilization constant of the Monod model.

Fig. 3
figure 3

Variation in a biomass and b glycerol production of W. anomalus HH16MU5 on glucose during different fermentation periods

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It was noted that the maximum specific growth rate obtained at an initial glucose concentration of 175 g l−1 whereas the maximum specific glycerol production rate was obtained at high initial concentration of glucose (200 g l−1). High glucose concentrations in the growth medium increase the extracellular osmotic pressure, and as a result, osmotolerant yeasts retain more glycerol in the fermentation medium (Petrovska et al. 1999). The role of high sugar is to create a high osmotic pressure thus force the organism to produce the compatible solutes as glycerol to overcome the surrounding environment. According to Nissen et al. (2000), a high concentration of sugar is necessary to obtain maximum yield of glycerol, where respiration is repressed, reducing the capability of the organism to reoxidize surplus amounts of NADH formed in biosynthetic reactions. Furthermore, under intracellular conditions, an increase towards glycerol 3-phosphate dehydrogenase was reported and glycerol accumulated inside the cell during growth under osmotic stress as an efficient osmolyte protecting the cell against lysis. However, our data revealed that both the specific growth and glycerol production rate were declined above the optimum glucose concentration; this may be due to the inhibitory effect of glucose. The maximum specific growth and glycerol production rates for the mutant isolate were correlated by using the non-linear regression method as follows:

$$ \mu \; = \;\frac{{0.21\; + \;S_{\text{o}} }}{{43.15\; + \;S_{\text{o}} }},\quad R^{2} = 0.860, $$
$$ v\; = \;\frac{{0.103\; + \;S_{\text{o}} }}{{89.03\; + \;S_{\text{o}} }} ,\quad R^{2} = \; 0.840. $$

It was estimated that the maximum specific growth rate (μmax) and maximum specific glycerol production rates (vmax) were calculated as 0.21 h−1 and 0.103 g gly g−1 yeast cells (Fig. 4). These results were inconsistent with Yaçlin and Ӧzbas (2005) who studied the growth and glycerol kinetics of S.cerevisiae Kalecik 1 at different sugar concentrations and estimated that an inhibition effect was noted at initial sugar concentrations were above the optimum substrate concentrations necessary for yeast growth.

Fig. 4
figure 4

The experimental and model predicted specific growth and glycerol production rates by W. anomalus HH16MU5 at different glucose concentrations

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Effect of different nitrogen sources on glycerol production

The type of nitrogen in the fermentation medium greatly affects the glycerol and biomass yields. Effect of different organic (peptone, yeast extract, beef extract and, malt extract) and inorganic (urea, ammonium phosphate and ammonium sulfate) nitrogen sources on glycerol production was investigated (Table 6).The maximum glycerol production rate was achieved with peptone as the sole nitrogen source in the fermentation medium with recorded glycerol and biomass yields 0.3725 g g−1 and 0.0481 respectively. Junior et al. (2008) found that peptone supplementation enhanced the fermentation performance of the yeast. Both yeast and beef extracts had no significant difference for glycerol production, these extracts provide convenient growth factors for yeast growth (Nancib et al. 2001). Ammonium sulphate is a cheap and efficient nitrogen source for yeast growth; it does not produce a toxic effect towards the microbial enzymes (Bamforth 2005). However, the minimum glycerol yields were recorded from the addition of ammonium sulphates and ammonium phosphate individually with no significant difference, this may be due to that these inorganic nitrogen sources probably contain only the nutrients that satisfy no more than the minimum requirement for growth (Costa et al. 2002). Nitrogen deficiency dramatically declines the yeast growth and possibly the metabolic reactions, this decline results from the inhibition of the synthesis of protein transporting sugars through the cell membrane to the interior of the cells (Varela et al. 2004). The combination of different organic nitrogen sources has a marked effect on the metabolic pathways of the microorganism which regulates the production of numerous metabolites (Bhasin and Modi 2012). Therefore, four different fermentation media supplemented with different combinations of the three organic nitrogen sources (yeast extract, peptone, and urea) were investigated (Fig. 5). The maximum glycerol productivity 1.146 g l−1 h−1was obtained with medium supplemented with peptone and yeast extract at the ratio of 1:3% (w/v) with glycerol and biomass yields recorded as 0.4120 and 0.0886 g g−1 respectively. This increase in glycerol productivity might be due to the availability of different amino acids, peptides, water-soluble vitamins and carbohydrates in both of peptone and yeast extract (Costa et al. 2002). However, our results indicated that the supplementation of urea to other nitrogen combinations has no significant impact on glycerol production, which is consistent with Zhuge et al. (2001) who observed that the addition of commercial urea to other nitrogen sources has a little effect on glycerol production by C. glycerinogenes.

Table 6 Effect of different nitrogen sources on glycerol and biomass yields by the selected mutant isolate
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Fig. 5
figure 5

Effect of combination of different nitrogen sources: medium A, peptone + yeast extract (1:1); medium B, peptone + yeast extract (1:3); medium C, peptone + yeast extract (3:1); medium D, peptone + yeast extract + urea (1:1:3) on glycerol and biomass production parameters

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Effect of salinity stress on glycerol production

The effect of different salt concentrations on glycerol production was investigated. Data in Fig. 6 revealed that intracellular glycerol accumulation was enhanced by increasing sodium chloride concentrations, whereas extracellular glycerol production showed a marked decline. Abdel Nasser and El-Moghaz (2010) studied the salt tolerance of Pichia pastoris and reported that salt stress was accompanied by an increase in the intracellular level of glycerol to decrease the intracellular water potential, restore water influx and consequently restore cell volume and turgor pressure. Shen et al. (1999) revealed that yeast cells grown under salt stress could accumulate much higher amounts of glycerol than cells grown without sodium chloride, suggesting that the metabolic flux may be changed to favor glycerol biosynthesis and the membrane permeability to glycerol may be reduced under salt-stress conditions. Our data explained that the accumulation of glycerol as a compatible solute is a strategy for the marine yeasts at high salt concentrations and plays a central role in the osmotic adaptation of the yeast cells (Silva-Graça and Lucas 2003). Moreover, our results revealed that both the wild and mutant isolates could tolerate high salt concentrations up to 4 M; however, the total glycerol and biomass yields showed a marked decrease at very high salt concentrations. This reduction of growth might be due to the disproportional deviation of carbon and energy into production of the cellular osmolytes (Fernanda et al. 1999). Seawater was a good substitute for distilled water supplemented with NaCl; our mutant achieved glycerol production equal to 73.33 g l−1 and volumetric glycerol productivity 1.0185 g l−1 h−1. In contrast, the wild isolate could achieve glycerol production equal to 67.334 g l−1 using seawater based medium. This may be due to the wide spectrum of minerals content of seawater that may avoid the addition of essential nutrients currently required for commercial fermentation medium (Lin and Tanka 2006). Thus, using marine yeasts in industrial fermentations could potentially enhance the overall economics of the process.

Fig. 6
figure 6

Effect of seawater and different salinity concentrations (0.5 M, 1 M, 2 M, 3 M and 4 M) on glycerol and biomass production parameters

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Effect of hydrogen ion concentration

The effect of different hydrogen ion concentrations (pH) on glycerol and biomass production was investigated. The resulted data in Fig. 7 revealed that glycerol production rate gradually increased along with the increase in pH values with a sharp increase in the glycerol yield of at the pH values 7 and 8 for both the two tested isolates. The maximum glycerol production 86.55 g l−1 was achieved by the mutant isolate at slightly alkaline pH 8.00 with glycerol productivity 1.2021 g l−1 h−1 and the substrate uptake rate achieved 2.5833 g l−1 h−1.The positive effect of increasing pH on glycerol production may explained in relation with the activity of the enzyme aldehyde dehydrogenase which increased at high pH values and cause the fixation of acetaldehyde, eliminating the ethanol formation and diverting the excess NADH towards glycerol formation by reduction of dihydroxyacetone phosphate to glycerol-3-phosphate in order to maintain the redox balance of the cell (Wang et al. 2001). Thus, commercially industrial glycerol may be enhanced by alkali-stressed (Taherzadeh et al. 2002). Patil (2013) also indicated that pH 8.0 is optimum for glycerol production by thermotolerant yeast H. anomala. This was in agreement with Yaçlin and Ӧzbas (2008) who studied the effect of pH on growth and glycerol production kinetics of two indigenous wine yeast strains S. Cerevisiae Kalecik 1 and Narince 3. The maximum biomass yield 0.0886 g g−1 was achieved by the mutant isolate at uncontrolled pH with glycerol production 82.49 g l−1 and glucose uptake rate 2.5194 g l−1 h−1. Walker (1998) stated that yeast grow very well between pH 4.5–6.5, but nearly all species can be able to grow in more acidic or alkaline media that low or high pH values may cause chemical stress on yeast cell, which was in consistence with the results presented in the current study.

Fig. 7
figure 7

Effect of different pH values (uncontrolled pH, pH 4.0, pH 5.0, pH 6.0, pH 7.0 and pH 8.0) on glycerol and biomass production parameters

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Comparison of glycerol production in this study with previously reported studies

Data in Table 7 showed a comparison of the glycerol production in the current study with previously reported studies. Overproduction of glycerol by S. cerevisiae can be achieved by forming a complex of acetaldehyde with the bisulfite ion (known as the steering agent) that limits ethanol production and promotes reoxidation of glycolytically formed NADH by glycerol synthesis (Wang et al. 2001). Several other efforts such as mutagenesis, strain breeding or overexpression of the genes associated with glycerol formation have been attempted to improve glycerol synthesis (Remize et al. 2001; Overkamp et al. 2002). Glycerol production via osmotolerant yeasts is an attractive approach that can produce significant amounts without a need for a steering agent (Zhang et al. 2002; Zhuge et al. 2001). However, the selection of yeast strains, optimization of fermentation conditions and yeast genetic modifications are critical parameters for glycerol overproduction (Mirończuk et al. 2016; Ehsani et al. 2009). Based on the comparison table, it was revealed that our mutant isolate has promising features for glycerol production especially using seawater based medium.

Table 7 Comparison of glycerol production in this study with previously reported studies
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Conclusions

Marine yeasts are an interesting group with great potentials for large scale production of worthy industrial chemicals such as glycerol. UV-mutagenesis is a significant tool to improve the glycerol productivity. It was found that the substrate type, initial concentration and fermentation periods significantly affects the yeast growth and glycerol production. Glucose is the best substrate for glycerol production by W. anomalus HH16 MU5. The maximum specific growth rate was obtained at an initial glucose concentration of 175 g l−1, whereas the maximum specific glycerol production rate was achieved at a high initial concentration of glucose (200 g l−1). Other fermentation parameters as the nitrogen source, salinity and pH are critical factors influence glycerol accumulation and production. It was revealed that the combination of different nitrogen sources (peptone + yeast extract) could enhance glycerol productivity. Although the isolate W. anomalus HH16 MU5 could tolerate up to 4 M, the highest total glycerol production was achieved at 1 M. Furthermore, the results indicated that low or high pH values may cause chemical stress on yeast cell and the highest glycerol production was achieved at pH 8.