Production of extracellular α-amylase by single-stage steady-state continuous cultures of Candida wangnamkhiaoensis in an airlift bioreactor

Griselda Ma. Chávez-Camarillo; Perla Vianey Lopez-Nuñez; Raziel Arturo Jiménez-Nava; Erick Aranda-García; Eliseo Cristiani-Urbina

doi:10.1371/journal.pone.0264734

Abstract

The kinetics of growth and α-amylase production of a novel Candida wangnamkhiaoensis yeast strain were studied in single-stage steady-state continuous cultures. This was performed in a split-cylinder internal-loop airlift bioreactor, using a variety of carbon sources as fermentation substrates. Results showed that the steady-state yields of cell mass from carbohydrates were practically constant for the range of dilution rates assayed, equaling 0.535 ± 0.030, 0.456 ± 0.033, and 0.491 ± 0.035 g biomass/g carbohydrate, when glucose, maltose, and starch, respectively were used as carbon sources. No α-amylase activity was detected when glucose was used as the carbon source in the influent medium, indicating that α-amylase synthesis of C. wangnamkhiaoensis is catabolically repressed by glucose. Contrastingly, maltose and starch induce synthesis of α-amylase in C. wangnamkhiaoensis, with starch being the best α-amylase inducer. The highest α-amylase volumetric and specific activities (58400 ± 800 U/L and 16900 ± 200 U/g biomass, respectively), and productivities (14000 ± 200 U/L·h and 4050 ± 60 U/g biomass·h, respectively) were achieved at a dilution rate of 0.24 h^-1 using starch as the carbon source. In conclusion, single-stage steady-state continuous culture in an airlift bioreactor represents a powerful tool, both for studying the regulatory mechanisms of α-amylase synthesis by C. wangnamkhiaoensis and for α-amylase production. Furthermore, results showed that C. wangnamkhiaoensis represents a potential yeast species for the biotechnological production of α-amylase, which can be used for the saccharification of starch. This offers an attractive renewable resource for the production of biofuels (particularly bioethanol), representing an alternative to fossil fuels with reduced cost of substrates.

Citation: Chávez-Camarillo GM, Lopez-Nuñez PV, Jiménez-Nava RA, Aranda-García E, Cristiani-Urbina E (2022) Production of extracellular α-amylase by single-stage steady-state continuous cultures of Candida wangnamkhiaoensis in an airlift bioreactor. PLoS ONE 17(3): e0264734. https://doi.org/10.1371/journal.pone.0264734

Editor: Shashi Kant Bhatia, Konkuk University, REPUBLIC OF KOREA

Received: September 29, 2021; Accepted: February 15, 2022; Published: March 1, 2022

Copyright: © 2022 Chávez-Camarillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript.

Funding: E.C.-U. - Secretaría de Investigación y Posgrado, IPN. (SIP20201439 and SIP20210590). G.M.C.-C. - Secretaría de Investigación y Posgrado, IPN (SIP20201843 and SIP20210584). The funders of our research had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

Amylases (α, β, and γ) are among the most versatile and important families of enzymes that have numerous biotechnological applications [1,2]. Among them, α-amylases (α-1,4-glucan-4-glucanohydrolase; E.C. 3.2.1.1.) are extensively used in several industries. For instance, food, brewery, syrups, textile, detergent, pulp, paper, pharmaceutical, environmental, biofuel, healthcare, clinical, and analytical chemistry industries, among others [1,3,4]. Nowadays, α-amylases hold the major world market share of enzymes, accounting for approximately 25–30% of the world´s enzyme production [2,3].

α-Amylases catalyze the hydrolysis of starch by cleaving the internal α-1,4-glycosidic linkages to produce limit dextrin, short-chain oligosaccharides, maltotriose, maltose, and glucose [3]. Although α-amylases can be obtained from several sources, such as animals, plants, and microorganisms, most industrial applications use microbial α-amylases, mainly from bacteria and filamentous fungi [5].

Yeasts offer several advantages for the commercial production of amylases, including: their easier cultivation, higher specific growth rate and shorter generation time than filamentous fungi ensure more productivity. Because of their GRAS (generally regarded as safe) status, their products are safe for human consumption, and yeasts are commonly deemed superior to bacteria in terms of their extracellular products. Yeasts are also amenable to easy genetic modification. Enzymes-derived from yeast tend to be more economically relevant than enzymes derived from other sources [6–10].

α-Amylase production is not widespread among yeast species but is found principally among species of the genera Candida, Cryptococcus, Lipomyces, Pichia, Saccharomycopsis, and Schwanniomyces [4,6,7]. Amylolytic yeasts have garnered much attention in recent years because of their great potential to produce single-cell oils, single-cell proteins, ethanol, and alcoholic beverages from starchy substrates [6,8,11,12]. Furthermore, yeast amylases have properties that differ from those produced by other microorganisms, which make them potentially useful in several industries, including food, biofuels, clinical, pharmaceutical, analytical chemistry, and those involving environmental remediation [3,6,10].

Recently, Hernández-Montañez et al. [11] reported that only 1 of 385 yeast strains isolated from soil and foods was able to degrade starch. This yeast strain was genetically identified by sequencing the D1/D2 domain of the 26S rDNA gene as Wickerhamia sp. X-Fep. It is now designated as Candida wangnamkhiaoensis [13]. This yeast species was recently reported in the literature but has scarcely been studied. Therefore, its physiological and metabolic capacities, as well as its potential for use in biotechnological processes, are unknown. Our research group previously demonstrated that C. wangnamkhiaoensis can produce α-amylase and reported some biochemical and molecular properties of the enzyme [11]. We also demonstrated that C. wangnamkhiaoensis α-amylase is capable of extensive starch hydrolysis and exhibits an elevated capacity for starch saccharification. In the light of this information, further research to determine the potential of C. wangnamkhiaoensis as an α-amylase producer will be of great value.

To optimize the α-amylase production process of C. wangnamkhiaoensis, it is essential to investigate the mechanisms underlying yeast growth and enzyme synthesis.

The continuous and uncontrollable changes in the environmental conditions during microbial culture growth lead to continuous alteration of the culture phenotype. This makes the results of batch studies on regulatory mechanisms of enzyme synthesis difficult to interpret. Furthermore, it is generally not possible to alter the growth environmental composition in batch cultivation without simultaneously changing the culture growth rate and consequently the culture physiology [14,15].

In contrast, the chemostat is a powerful tool for studying the physiology of microbial cultures because cell growth occurs under steady-state conditions. Here, the environmental growth conditions and consequently the culture phenotype, remain invariant throughout the cultivation period. Additionally, a continuous chemostat culture permits the alteration of environmental growth conditions independent of the specific growth rate of the microbial culture [14–16].

Continuous chemostat cultures have potential advantages for production purposes against batch cultures because the specific rate of microbial growth can be controlled. Additionally, long-term, higher volumetric and specific productivities, and higher yields of products can be achieved by manipulating the feed flow rate. This allows maintenance of low metabolic product concentrations in the chemostat to reduce growth inhibition [17,18]. Furthermore, compared to batch cultures, continuous chemostat cultures reduce time-consuming activities such as cleaning, filling, and sterilization of bioreactors, allow the use of smaller bioreactors, reduce capital investments, labor, production costs, and are easier to control at steady state [18,19].

Most studies on the production of yeast α-amylases have been performed in batch systems and the few studies available in the literature on amylolytic yeasts in continuous systems have focused on the production of biomass (unicellular protein) [20–23]. Furthermore, studies on amylolytic yeasts have been carried out in flasks or mechanically shaken bioreactors. To our knowledge, no studies have been reported using amylolytic yeasts grown in pneumatic bioreactors, such as in airlift bioreactors or bubble columns; these exhibit several economic and operational advantages over mechanically stirred bioreactors.

This study reports the production kinetics of α-amylase produced by C. wangnamkhiaoensis in single-stage steady-state continuous cultures in an airlift bioreactor, using different carbon and energy sources as fermentation substrates.

2. Materials and methods

2.1. Microorganism

A novel anamorphic yeast strain that was isolated from lemon and genetically identified as Candida wangnamkhiaoensis was used in this study. This yeast strain was acquired from the Microbial Culture Collection of the Industrial Microbiology Laboratory, Microbiology Department of the Biological Sciences National School, National Polytechnic Institute, Mexico City, Mexico. The C. wangnamkhiaoensis yeast strain was preserved on YPG (1% yeast extract, 1% casein peptone, and 2% glucose) agar slants and agar plates at 4°C.

2.2. Culture media composition

The culture medium used for the single-stage steady-state continuous culture experiments was made from Castañeda medium (0.375 g/L KH₂PO₄, 0.625 g/L dibasic ammonium citrate, 1 g/L yeast extract, 0.275 g/L MgSO₄·7H₂O, 0.25 g/L NaCl, and 0.375 g/L Na₂CO₃) [24] supplemented with either 20 g/L of glucose, maltose, or soluble starch. The culture media were sterilized in an autoclave at 121°C for 15 min. The pH of the culture medium was 6.0 ± 0.1. Analytical grade reagents provided by BD Bioxon (Mexico) and JT Baker (Mexico) were employed for the preparation of preservation and culture media.

2.3. Inoculum preparation

The inocula of C. wangnamkhiaoensis were grown in 200 mL of Castañeda’s culture medium supplemented with 20 g/L glucose, maltose, or soluble starch in 1000-mL Erlenmeyer flasks in a water bath shaker (Cole-Parmer, Inc., Vernon Hills, IL, USA) at 28 ± 2°C and 150 rpm for 15 h. The resulting yeast cell suspension was used as inoculum in subsequent continuous culture experiments performed in a pneumatic bioreactor.

2.4. Split-cylinder internal-loop airlift bioreactor

Candida wangnamkhiaoensis is an anamorphic yeast [13], highly susceptible to shear stress occurring in mechanically agitated bioreactors. This causes damage to pseudo-mycelial yeast cells and reduces α-amylase production. Airlift bioreactors are known to be suitable for shear stress-sensitive microorganisms, prone to physical damage by mechanical agitation or fluid turbulence. In addition, airlift bioreactors consume lower amounts of energy than mechanically stirred reactors, and are considered one of the most economic designs for aerobic industrial production systems [25,26]. Therefore, to minimize damage to C. wangnamkhiaoensis yeast cells, increase α-amylase production, and reduce production costs, an airlift bioreactor was used in the present study.

After performing a comprehensive literature search, no information was found on α-amylase production by yeasts in airlift bioreactors, so this is the first study on this subject matter.

Single-stage steady-state continuous culturing was performed in a split-cylinder internal-loop airlift bioreactor with a liquid operating volume of 1.8 L. Fig 1 displays a schematic diagram of the airlift bioreactor. The cylindrical column and base of the bioreactor were made of borosilicate glass. The column bioreactor had a height of 50 cm and an inner diameter of 8 cm. Inside the column, there was a centered stainless-steel baffle, to divide the bioreactor column into two equal vertical parts. One part is the riser and the other is the downcomer. Therefore, the cross-sectional area ratio of the riser-to-downcomer is 1.0. The bottom and top clearances were 2 and 4 cm, respectively. The column body and the baffle side edges are so tightly joined that even liquids and air cannot pass along their juncture. The cylindrical column also had a port through which the culture samples were collected.

Download:

Fig 1. Schematic representation of the split-cylinder internal-loop airlift bioreactor.

a) air pump; b) outflow effluent duct; c) silicone stopper; d) air-in filter; e) acid/alkali solution reservoir; f) culture medium reservoir; g) and i) peristaltic pumps; h) antifoam solution reservoir; j) gas diffuser; k) pH electrode; l) pH controller; m) baffle; n) effluent reservoir.

https://doi.org/10.1371/journal.pone.0264734.g001

The upper part of the column has a silicone stopper with ports for the addition of sterile air, alkali, acid, inoculant, and to introduce pH and oxygen electrodes. Sterile air was sparged into the bottom of the bioreactor through a porous diffuser attached to the air duct outlet. The airflow rate was controlled using a pressure-regulating valve and measured using a calibrated rotameter. Sterile air was supplied to the bioreactor at a flow rate of 1.8 L/min (1 vvm).

2.5. Kinetics of C. wangnamkhiaoensis α-amylase production in single-stage steady-state continuous cultures

The effects of glucose, soluble starch, and maltose on α-amylase production were investigated in single-stage continuous cultures in an airlift bioreactor at pH 6.0 ± 0.1, 28 ± 2°C and 1 vvm of sterile air.

Single-stage continuous cultures were initiated with a batch culture and allowed to run for 20 h. Afterwards, sterile culture medium containing 20 g/L glucose, maltose, or soluble starch was continuously supplied into the airlift bioreactor at a known flow rate using a peristaltic pump (Masterflex L/S, USA), until the yeast biomass concentration, residual carbohydrate concentration, and α-amylase activity did not vary significantly with respect to time (p > 0.05). This occurred approximately at 3–4 liquid residence times, indicating that a steady state had been reached.

Different culture medium flow rates were assayed, thus assessing different dilution rates (D), which are equal to the specific growth rate of the yeast at steady state. Culture samples collected at steady state were analyzed immediately to quantify biomass and residual carbohydrate concentrations, and α-amylase activity.

2.6. Analytical methods

The yeast biomass concentration was measured gravimetrically as the cell dry weight. Yeast cells from 5 mL of culture were harvested by filtration on GF/A (Whatman) filters, washed three times with deionized water, and oven-dried at 90°C to reach a constant weight. The obtained filtrates were used to quantify the residual carbohydrate concentration and α-amylase activity.

Glucose, maltose, and starch concentrations were measured by enzymatic methods using glucose (GO), maltose, and starch (GO/P) assay kits (Sigma-Aldrich, St. Louis, MI, USA), according to the procedures outlined by the manufacturer.

α-Amylase activity was measured using the starch azure assay (S7629; Sigma-Aldrich, St. Louis, MI, USA), following the methodology described by Chávez-Camarillo et al. [27]. One unit of α-amylase activity (U) was defined as an increase in one unit of absorbance at 595 nm per hour at 50°C.

The α-amylase volumetric activity (P, U/L) was defined as the number of enzyme units per liter. Likewise, the specific activity of α-amylase or enzyme yield (Y_px, U/g biomass) was expressed as the α-amylase volumetric activity per dry weight biomass concentration (X, g/L) of C. wangnamkhiaoensis yeast (P/X). The α-amylase volumetric productivity (Q_p, U/L·h) was estimated as the product of the α-amylase volumetric activity (P) at the steady-state and operating dilution rate (D). Similarly, α-amylase specific productivity (q_p, U/g biomass·h) was computed as the product of the steady-state α-amylase specific activity (Y_P/X) and operating dilution rate (D) [28].

2.7. Statistical analysis

At least three independent continuous culture experiments were performed. GraphPad Prism software version 8.4 was used to perform the statistical analysis of the experimental data by means of one-way analysis of variance (ANOVA) with Tukey’s test and with a 95% confidence level (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results and discussion

The steady-state biomass and residual substrate concentrations in single-stage continuous cultures are shown in Fig 2. The growth and substrate consumption patterns were consistent.

Download:

Fig 2.

Steady-state biomass (A) and substrate (B) concentrations at different dilution rates by C. wangnamkhiaoensis in continuous culture. ●, glucose; ◼, maltose; ◆, starch.

https://doi.org/10.1371/journal.pone.0264734.g002

The highest steady-state biomass concentration (6.87 ± 0.15 g/L) was achieved at a dilution rate of 0.036 h^-1 using maltose as the carbon source in the feeding medium. However, at higher dilution rates (> 0.036 h^-1), a continuously decreasing biomass concentration with an increasing dilution rate was observed (Fig 2A). In contrast, the biomass concentration at the steady-state was essentially constant in the range of dilution rates from 0.043 to 0.13 h^-1 and from 0.051 to 0.13 h^-1, when glucose (~ 5.09 ± 0.24 g/L) and soluble starch (~ 5.95 ± 0.18 g/L) were used as carbon sources, respectively. However, the biomass concentrations decreased at dilution rates higher than 0.13 h^-1.

The steady-state yields of cell mass from carbohydrates (Y_xs) were practically constant in the range of dilution rates that were examined (Fig 3A). When glucose, maltose, and starch were used as carbon sources, the mean cell yield values were 0.535 ± 0.030, 0.456 ± 0.033, and 0.491 ± 0.035 g biomass/g carbohydrate, respectively. A qualitatively similar behavior was reported for Saccharomycopsis fibuligera when this yeast was grown on a potato extract simulating processing blancher water [20,29]. Additionally, it has been reported that the S. fibuligera biomass yield from starch is approximately 0.55 g/g [29], which is slightly higher than that obtained in this study (0.491 ± 0.035 g/g).

Download:

Fig 3.

Steady-state biomass yield (A) and biomass productivity (B) at different dilution rates by C. wangnamkhiaoensis in continuous culture. ●, glucose; ◼, maltose; ◆, starch.

https://doi.org/10.1371/journal.pone.0264734.g003

The maximum levels of volumetric productivity of biomass were reached at dilution rates of 0.144, 0.133, and 0.158 h^-1, with values of 0.71 ± 0.036, 0.55 ± 0.015, and 0.9 ± 0.016 g biomass/L·h, when glucose, maltose, and starch were used as substrates, respectively (Fig 3B).

The growth and α-amylase production patterns of C. wangnamkhiaoensis (Figs 2A and 4) clearly suggest that α-amylase production and yeast biomass concentration are not directly related. However, the synthesis of the α-amylase is related to the type of carbon source present in the influent culture medium and to the dilution rate.

Download:

Fig 4.

Steady-state α-amylase volumetric (A) and specific (B) activities versus dilution rate for continuous cultures of C. wangnamkhiaoensis. ◼, maltose; ◆, starch.

https://doi.org/10.1371/journal.pone.0264734.g004

C. wangnamkhiaoensis was capable of producing high α-amylase activity when maltose and starch were used as carbon sources (Fig 4). However, no α-amylase activity was detected when glucose was used as the carbon source in the influent medium. These findings indicate that α-amylase synthesis of C. wangnamkhiaoensis is catabolically repressed by glucose and induced by maltose and starch.

The role of glucose in the production of yeast α-amylases is controversial. α-Amylase production is completely repressed by glucose in Pichia burtonii [30], Candida tropicalis CBS 6948 [31], and Lipomyces starkeyi CBS 1809 [32]. It is partially repressed by glucose in Aureobasidium pullulans N13d [33], Schwanniomyces occidentalis CSIR-Y828, S. occidentalis CSIR Y993, Lipomyces kononenkoae IGC 4052 [34], L. starkeyi 1807 [35], and Schwanniomyces castelli ATCC 26077 [36]. In contrast, no glucose repression of α-amylase was evident with Torulopsis ingeniosa Di Mena, L. starkeyi [37], L. kononenkoae 4052 B [34], Dioszegia fristingensis (T9Df1), Dioszegia sp., and Leuconeusospora sp. T11Cd2 [38].

It is well known that cells can adapt their gene expression and consequently their cell metabolism for optimal utilization of the carbon sources available in the environment. In bacteria, filamentous fungi, yeasts, and metazoa, the presence of glucose in the environment prevents the utilization of other available carbon sources by mechanisms known as glucose repression, or more generally, carbon catabolite repression [39]. Carbon catabolite repression is one of the main regulatory phenomena that helps microbial cells use available carbon sources in the most efficient way [40]; it also involves the regulation of a multitude of genes and proteins involved in carbon source utilization and energy generation [41].

In Saccharomyces cerevisiae or baker’s yeast, which has long served as a model for studying glucose repression, when glucose is present in the growth medium, uptake and catabolism of other carbon sources is repressed via three signaling pathways: inhibition of Snf1 protein kinase, activation of cAMP-dependent protein kinase A (PKA), and the regulation of transporter expression and stability at the plasma membrane by the yeast casein kinases Yck1 and Yck2 [39]. Although glucose repression has been extensively investigated, much still remains to be elucidated in order to achieve a full understanding of this regulatory mechanism.

In contrast, compounds composed of α-1,4-D-glucose units linked by α-D-(1–4) linkages such as maltose and starches, are inducers of α-amylase synthesis in several species of yeasts. These include Cryptococcus gilvescens, Mrakia robertii [38], Pichia membranifaciens [4], Aureobasidium pullulans N13d [33], Pichia burtonii [30], Torulopsis ingeniosa Di Mena [37], Lipomyces starkeyi [32,35], Schwanniomyces castelli [36,42], Endomycopsis fibuligera [42], Candida tropicalis CBS 6948 [31], Schwanniomyces alluvius ATCC 26074 [43], Candida silvanorum, Candida tsukubaensis, Filobasidium capsuligenum, Leucosporidium capsuligenum, and Trichosporon pullulans [44].

Our results clearly show that α-amylase synthesis of C. wangnamkhiaoensis is subject to induction by maltose and starch, as well as carbon catabolite repression by glucose. The potential for both induction and carbon catabolite repression in a cell ensures that inducible enzymes are produced in the correct proportions, only in the presence of the substrate and that neither valuable nutrients, energy nor amino acids are wasted in making unnecessary enzymes, but that when needed, these enzymes can be formed quickly [45].

The α-amylase volumetric (P) and specific (Y_px) activities were found to have a sharp peak at a dilution rate of 0.13 h^-1 (Fig 4) when maltose was used as a carbon source, with maximum α-amylase activity values of 43700 ± 1300 U/L and 10200 ± 300 U/g biomass, respectively (Table 1). This behavior has been interpreted to reflect a balance between induction and catabolite repression. At low dilution rates, the degree of enzyme induction by the substrate present in the chemostat limits the amount of the enzyme synthesized. In contrast, at dilution rates above the inflection point in the plots of enzyme specific activity versus dilution rate, the increase in the concentration of the substrate in the chemostat leads to a progressive increase in the catabolite repression of enzyme synthesis [15,46,47]. According to Lemmel et al. [29], catabolite repression of amylase synthesis has been observed in many carbon sources, including glucose, maltose, and starch at high concentrations.

Download:

Table 1. Kinetic parameters of α-amylase production by C. wangnamkhiaoensis in steady-state continuous cultures.

https://doi.org/10.1371/journal.pone.0264734.t001

In contrast, when soluble starch was used as a carbon source, the volumetric and specific activities of α-amylase were constant over the range of dilution rates from 0.05 to 0.11 h^-1. However, at higher dilution rates (> 0.11 h^-1), the activities increased progressively as the dilution rate increased (Fig 4), so that the highest enzyme activities were obtained at the highest dilution rate (0.24 h^-1). The increase in the specific activity of an enzyme with an increase in the dilution rate is thought to be because the substrate or its metabolites present in the culture induce the synthesis of the enzyme and this induction remains partial at submaximal dilution rates [15,46].

The maximum levels of α-amylase volumetric and specific activity were higher in soluble starch (58400 ± 800 U/L and 16900 ± 200 U/g biomass) than in maltose-supplemented medium (43700 ± 1300 U/L and 10200 ± 300 U/g biomass). This indicates that starch is a better inducer than maltose for the synthesis of C. wangnamkhiaoensis α-amylase (Table 1).

The results of α-amylase volumetric and specific productivities of C. wangnamkhiaoensis at the steady-state of single-stage continuous cultures are shown in Fig 5. When maltose was used as a carbon source, a peak of volumetric and specific productivity of α-amylase was obtained at a dilution rate of 0.133 h^-1, with values of 5810 ± 170 U/L·h and 1370 ± 40 U/g biomass·h, respectively. In contrast, the volumetric and specific productivities of α-amylase were maintained practically constant in the range of dilution rates from 0.05 to 0.11 h^-1. From this last dilution rate, both α-amylase productivities gradually increased as the dilution rate increased (Fig 5). Therefore, the maximum α-amylase volumetric and specific productivities were obtained at the highest dilution rate tested (0.24 h^-1), with values of 14000 ± 200 U/L·h and 4050 ± 60 U/g biomass·h, respectively (Fig 5 and Table 1).

Download:

Fig 5.

Steady-state α-amylase volumetric (A) and specific (B) productivities versus dilution rate for C. wangnamkhiaoensis continuous cultures. ◼, maltose; ◆, starch.

https://doi.org/10.1371/journal.pone.0264734.g005

It is evident that the highest α-amylase productivities were obtained with starch as the carbon source, compared to maltose. This is economically advantageous due to the large availability of starch in nature.

The above results clearly demonstrated that the single-stage steady-state continuous culture is a satisfactory mode of operation of an airlift bioreactor, since it allowed high levels of production of yeast α-amylase. C. wangnamkhiaoensis is an attractive microbial source for α -amylase production.

4. Conclusions

This work studied the regulatory mechanisms of α-amylase synthesis and the growth and α-amylase production kinetics of C. wangnamkhiaoensis in single-stage steady-state continuous cultures in a split-cylinder internal-loop airlift bioreactor. Biomass yield was higher on glucose (0.535 ± 0.030 g/g) than on starch (0.491 ± 0.035 g/g) and maltose (0.456 ± 0.033 g/g); however, the maximum volumetric productivity of biomass was higher on starch (0.90 ± 0.016 g biomass/L·h) than on glucose (0.71 ± 0.036 g biomass/L·h) and maltose (0.55 ± 0.015 g biomass/L·h). No α-amylase production was observed when C. wangnamkhiaoensis was grown on glucose, suggesting that glucose catabolically repressed the synthesis of the enzyme. In contrast, maltose and starch induce the synthesis of high levels of α-amylase, with starch being the strongest inducer. The maximum volumetric activity, maximum specific activity, maximum volumetric productivity, and maximum specific productivity of α-amylase achieved with starch were 33.64 ± 3.37%, 65.69 ± 4.11%, 140.96 ± 6.08%, and 195.62 ± 7.43% higher respectively, than that obtained with maltose. These results are economically significant because starch is a cheap and profuse polysaccharide that can be hydrolyzed to simple sugars by C. wangnamkhiaoensis α-amylase and, subsequently, simple sugars can be used in the production of several high added-value compounds of industrial interest through fermentation processes.

References

1. Farooq MA, Ali S, Hassan A, Tahir HM, Mumtaz S, Mumtaz S. Biosynthesis and industrial applications of α-amylase: a review. Arch Microbiol. 2021;203:1281–92. pmid:33481073
- View Article
- PubMed/NCBI
- Google Scholar
2. Visvanathan R, Qader M, Jayathilake C, Jayawardana BC, Liyanage R, Sivakanesan R. Critical review on conventional spectroscopic α-amylase activity detection methods: merits, demerits, and future prospects. J Sci Food Agric. 2020;100:2836–47. pmid:32031680
- View Article
- PubMed/NCBI
- Google Scholar
3. Paul JS, Gupta N, Beliya E, Tiwari S, Jadhav SK. Aspects and recent trends in microbial α-amylase: a review. Appl Biochem Biotechnol. 2021;193:2649–98. pmid:33715051
- View Article
- PubMed/NCBI
- Google Scholar
4. Salah HA, Temerk HA, Salah NA, Alshehri SRZ, Al-Harbi JA, Mawad AMM, et al. Production and optimization of xylanase and α-amylase from non-Saccharomyces yeasts (Pichia membranifaciens). J Pure Appl Microbiol. 2021;15:452–61.
- View Article
- Google Scholar
5. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial α-amylases: a biotechnological perspective. Process Biochem. 2003;38:1599–616.
- View Article
- Google Scholar
6. Djekrif DS, Gillmann L, Bennamoun L, Ait-Kaki A, Labbani K, Nouadri T, et al. Amylolytic yeasts: Producers of α-amylase and pullulanase. Int J Life-Sci Sci Res. 2016;2:339–354.
- View Article
- Google Scholar
7. Hashim SA, Sohail M. Amylases from yeasts: an update. Trends Life Sci. 2014;3:30–7.
- View Article
- Google Scholar
8. Gen Q, Wang Q, Chi Z-M. Direct conversion of cassava starch into single cell oil by co-cultures of the oleaginous yeast Rhodosporidium toruloides and immobilized amylases-producing yeast Saccharomycopsis fibuligera. Renew Energy. 2014;62:522–6.
- View Article
- Google Scholar
9. Carvalho JK, Panatta AAS, Silveira MAD, Tav C, Johann S, Rodrigues MLF, et al. Yeasts isolated from a lotic continental environment in Brazil show potential to produce amylase, cellulase, and protease. Biotechnol Rep. 2021;30:e00630. pmid:34136364
- View Article
- PubMed/NCBI
- Google Scholar
10. Fossi BT, Tavea F, Ndjouenkeu R. Production and partial characterization of a thermostable amylase from ascomycetes yeast strain isolated from starchy soils. Afr J Biotechnol. 2005;4:14–8.
- View Article
- Google Scholar
11. Hernández-Montañez Z, Juárez-Montiel M, Velázquez-Ávila M, Cristiani-Urbina E, Hernández-Rodríguez C, Villa-Tanaca L, et al. Production and characterization of extracellular α-amylase produced by Wickerhamia sp. X-Fep. Appl Biochem Biotechnol. 2012;167:2117–29. pmid:22678824
- View Article
- PubMed/NCBI
- Google Scholar
12. Yalçin HT, Çorbaci C. Isolation and characterization of amylase producing yeasts and improvement of amylase production. Turk J Biochem. 2013;38:101–8.
- View Article
- Google Scholar
13. Limtong S, Kaewwichian R, Jindamorakot S, Yongmanitchai W, Nakase T. Candida wangnamkhiaoensis sp. nov., an anamorphic yeast species in the Hyphopichia clade isolated in Thailand. Antonie Van Leeuwenhoek. 2012;102:23–8. pmid:22331449
- View Article
- PubMed/NCBI
- Google Scholar
14. Hoskisson PA, Hobbs G. Continuous culture–making a comeback? Microbiology. 2005;151:3153–59. pmid:16207900
- View Article
- PubMed/NCBI
- Google Scholar
15. Matin A. Regulation of enzyme synthesis as studied in continuous culture. In: Calcott PH, editor. Continuous cultures of cells. Boca Raton, USA: CRC Press Inc.; 1981. pp. 69–97.
16. Christiansen T, Nielsen J. Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures. J Biotechnol. 2002;97:265–73. pmid:12084482
- View Article
- PubMed/NCBI
- Google Scholar
17. Bayrock DP, Ingledew WM. Ethanol production in multistage continuous, single stage continuous, Lactobacillus-contaminated continuous, and batch fermentations. World J Microbiol Biotechnol. 2005;21:83–8.
- View Article
- Google Scholar
18. Brethauer S, Wyman CE. Review: Continuous hydrolysis and fermentation for cellulosic ethanol production. Bioresour Technol. 2010;101:4862–74. pmid:20006926
- View Article
- PubMed/NCBI
- Google Scholar
19. Martin GJO, Knepper A, Zhou B, Pamment NB. Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose. J Ind Microbiol Biotechnol. 2006;33:834–44. pmid:16680457
- View Article
- PubMed/NCBI
- Google Scholar
20. Admassu W, Korus RA, Heimsch RC, Lemmel SA, Growth of Saccharomycopsis fibuligera in a continuous-stirred tank fermentor. Biotechnol Bioeng. 1981;23:2361–71.
- View Article
- Google Scholar
21. Admassu W, Korus RA, Heimsch RC. Two-stage continuous fermentation of Saccharomycopsis fibuligera and Candida utilis. Biotechnol Bioeng. 1983;25:2641–51. pmid:18548599
- View Article
- PubMed/NCBI
- Google Scholar
22. Lemmel SA, Heimsch RC, Edwards LL. Optimizing the continuous production of Candida utilis and Saccharomycopsis fibuligera on potato processing wastewater. Appl Environ Microbiol. 1979;37:227–32. pmid:35096
- View Article
- PubMed/NCBI
- Google Scholar
23. Admassu W, Korus RA, Heimsch RC. Continuous fermentation of Saccharomycopsis fibuligera and Candida utilis. Biotechnol Bioeng. 1984;26:1511–13. pmid:18551685
- View Article
- PubMed/NCBI
- Google Scholar
24. Castañeda-Agulló M. Studies on the biosynthesis of extracellular proteases by bacteria. I. Serratia marcescens, synthetic and gelatin media. J Gen Physiol. 1956;89:369–75. pmid:13286455
- View Article
- PubMed/NCBI
- Google Scholar
25. Chisti Y. Pneumatically agitated bioreactors in industrial and environmental processing: Hydrodynamics, hydraulics, and transport phenomena. Appl Mech Rev. 1998;51:33–112.
- View Article
- Google Scholar
26. Klaic R, Kuhn RC, Foletto EL, Prá VD, Jacques RJS, Guedes JVC, et al. An overview regarding bioherbicide and their production methods by fermentation. In: Gupta VK, Mach RL, Sreenivasaprasad S, editors. Fungal biomolecules: sources, applications and recent developments. Chichester, UK: John Wiley & Sons, Ltd.; 1991. pp. 183–199. https://doi.org/10.1002/9781118958308.ch14
27. Chávez-Camarillo GM, Santiago-Flores UM, Mena-Vivanco A, Morales-Barrera L, Cortés-Acosta E, Cristiani-Urbina E. Transient responses of Wickherhamia sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis. Ann Microbiol. 2018;68:625–635.
- View Article
- Google Scholar
28. Blanch HW, Clark DS. Biochemical Engineering. New York, USA: Marcel Dekker, Inc.; 1997. pp. 162–275.
29. Lemmel SA, Heimsch RC, Korus RA. Kinetics of growth and amylase production of Saccharomycopsis fibuligera on potato processing wastewater. Appl Environ Microbiol. 1980;39:387–93. pmid:16345512
- View Article
- PubMed/NCBI
- Google Scholar
30. Moulin G, Galzy P. Etude de l’α-amylase de la paroi de Pichia burtonii Boidin. Zeit Allgem Mikrobiol. 1978;18:269–74.
- View Article
- Google Scholar
31. Azoulay E, Jouanneau F, Bertrand JC, Raphael A, Janssens J, Lebeault JM. Fermentation methods for protein enrichment of cassava and corn with Candida tropicalis. Appl Environ Microbiol. 1980;39:41–7. pmid:16345495
- View Article
- PubMed/NCBI
- Google Scholar
32. Kelly CT, Moriarty ME, Fogarty WM. Thermostable extracellular α-amylase and α-glucosidase of Lipomyces starkeyi. Appl Microbiol Biotechnol. 1985;22:352–58.
- View Article
- Google Scholar
33. Haifeng LI, Zhenming CHI, Xiaohong W, Chunling MA. Amylase production by the marine yeast Aureobasidium pullulans N13d. J Ocean Univ. 2007;6:60–5.
- View Article
- Google Scholar
34. Horn CH, de Kock A, Du Preez JC, Lategan PM. A comparative study of the amylolytic ability of Lipomyces and Schwanniomyces yeast species. Syst Appl Microbiol. 1988;10:106–10.
- View Article
- Google Scholar
35. Moulin G, Galzy P. Study of an amylase and its regulation in Lipomyces starkeyi. Agric Biol Chem. 1979;43:1165–71.
- View Article
- Google Scholar
36. Sills AM, Zygora PSJ, Stewart GG. Characterization of Schwanniomyces castelli mutants with increased productivity of amylases. Appl Microbiol Biotechnol. 1984;20:124–28.
- View Article
- Google Scholar
37. Moulin P, Galzy P. Amylase activity of Torulopsis ingeniosa Di Menna. Folia Microbiol. 1978;23:424–27. pmid:33881
- View Article
- PubMed/NCBI
- Google Scholar
38. Carrasco M, Villareal P, Barahona S, Alcaíno J, Cifuentes V, Baeza M. Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts. BMC Microbiol. 2016;16:21. pmid:26895625
- View Article
- PubMed/NCBI
- Google Scholar
39. Simpson-Lavy K, Kupiec M. Carbon catabolite repression in yeast is not limited to glucose. Sci Rep. 2019;9:6491. pmid:31019232
- View Article
- PubMed/NCBI
- Google Scholar
40. Görke B, Stülke J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol. 2008;6:613–24. pmid:18628769
- View Article
- PubMed/NCBI
- Google Scholar
41. Kayikci Ö, Nielsen J. Glucose repression in Saccharomyces cerevisiae. FEMS Yeast Res. 2015;15:fov068. pmid:26205245
- View Article
- PubMed/NCBI
- Google Scholar
42. Clementi F, Rossi J, Costamagna L, Rosi J. Production of amylase(s) by Schwanniomyces castelli and Endomycopsis fibuligera. Antonie Van Leeuwenhoek. 1980;46:399–405. pmid:6160813
- View Article
- PubMed/NCBI
- Google Scholar
43. Calleja GB, Yaguchi M, Levy-Rick S, Seguin JRH, Roy C, Lusena CV. Single-cell protein production from potato starch by the yeast Schwanniomyces alluvius. J Ferment Technol. 1986;64:71–5.
- View Article
- Google Scholar
44. De Mot R, van Oudendijck E, Hougaerts S, Verachtert H. Effect of medium composition on amylase production by some starch-degrading yeasts. FEMS Microbiol Lett. 1984;25:169–73.
- View Article
- Google Scholar
45. Wang DIC, Cooney CL, Demain AL, Dunnill P, Humphrey AE, Lilly MD. Fermentation and Enzyme Technology. John Wiley & Sons. 1979.
46. Dean ACR. Influence of environment on the control of enzyme synthesis. J Appl Chem Biotechnol. 1972;22:245–59.
- View Article
- Google Scholar
47. Toda K. Dual control of invertase biosynthesis in chemostat culture. Biotechnol Bioeng. 1976;18:1117–24. pmid:953171
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Farooq MA, Ali S, Hassan A, Tahir HM, Mumtaz S, Mumtaz S. Biosynthesis and industrial applications of α-amylase: a review. Arch Microbiol. 2021;203:1281–92. pmid:33481073
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Visvanathan R, Qader M, Jayathilake C, Jayawardana BC, Liyanage R, Sivakanesan R. Critical review on conventional spectroscopic α-amylase activity detection methods: merits, demerits, and future prospects. J Sci Food Agric. 2020;100:2836–47. pmid:32031680
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Paul JS, Gupta N, Beliya E, Tiwari S, Jadhav SK. Aspects and recent trends in microbial α-amylase: a review. Appl Biochem Biotechnol. 2021;193:2649–98. pmid:33715051
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Salah HA, Temerk HA, Salah NA, Alshehri SRZ, Al-Harbi JA, Mawad AMM, et al. Production and optimization of xylanase and α-amylase from non-Saccharomyces yeasts (Pichia membranifaciens). J Pure Appl Microbiol. 2021;15:452–61.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref5] 5. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial α-amylases: a biotechnological perspective. Process Biochem. 2003;38:1599–616.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref6] 6. Djekrif DS, Gillmann L, Bennamoun L, Ait-Kaki A, Labbani K, Nouadri T, et al. Amylolytic yeasts: Producers of α-amylase and pullulanase. Int J Life-Sci Sci Res. 2016;2:339–354.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref7] 7. Hashim SA, Sohail M. Amylases from yeasts: an update. Trends Life Sci. 2014;3:30–7.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref8] 8. Gen Q, Wang Q, Chi Z-M. Direct conversion of cassava starch into single cell oil by co-cultures of the oleaginous yeast Rhodosporidium toruloides and immobilized amylases-producing yeast Saccharomycopsis fibuligera. Renew Energy. 2014;62:522–6.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref9] 9. Carvalho JK, Panatta AAS, Silveira MAD, Tav C, Johann S, Rodrigues MLF, et al. Yeasts isolated from a lotic continental environment in Brazil show potential to produce amylase, cellulase, and protease. Biotechnol Rep. 2021;30:e00630. pmid:34136364
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref10] 10. Fossi BT, Tavea F, Ndjouenkeu R. Production and partial characterization of a thermostable amylase from ascomycetes yeast strain isolated from starchy soils. Afr J Biotechnol. 2005;4:14–8.
View Article
Google Scholar

[33] View Article

[34] Google Scholar

[ref11] 11. Hernández-Montañez Z, Juárez-Montiel M, Velázquez-Ávila M, Cristiani-Urbina E, Hernández-Rodríguez C, Villa-Tanaca L, et al. Production and characterization of extracellular α-amylase produced by Wickerhamia sp. X-Fep. Appl Biochem Biotechnol. 2012;167:2117–29. pmid:22678824
View Article
PubMed/NCBI
Google Scholar

[36] View Article

[37] PubMed/NCBI

[38] Google Scholar

[ref12] 12. Yalçin HT, Çorbaci C. Isolation and characterization of amylase producing yeasts and improvement of amylase production. Turk J Biochem. 2013;38:101–8.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref13] 13. Limtong S, Kaewwichian R, Jindamorakot S, Yongmanitchai W, Nakase T. Candida wangnamkhiaoensis sp. nov., an anamorphic yeast species in the Hyphopichia clade isolated in Thailand. Antonie Van Leeuwenhoek. 2012;102:23–8. pmid:22331449
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref14] 14. Hoskisson PA, Hobbs G. Continuous culture–making a comeback? Microbiology. 2005;151:3153–59. pmid:16207900
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref15] 15. Matin A. Regulation of enzyme synthesis as studied in continuous culture. In: Calcott PH, editor. Continuous cultures of cells. Boca Raton, USA: CRC Press Inc.; 1981. pp. 69–97.

[ref16] 16. Christiansen T, Nielsen J. Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures. J Biotechnol. 2002;97:265–73. pmid:12084482
View Article
PubMed/NCBI
Google Scholar

[52] View Article

[53] PubMed/NCBI

[54] Google Scholar

[ref17] 17. Bayrock DP, Ingledew WM. Ethanol production in multistage continuous, single stage continuous, Lactobacillus-contaminated continuous, and batch fermentations. World J Microbiol Biotechnol. 2005;21:83–8.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref18] 18. Brethauer S, Wyman CE. Review: Continuous hydrolysis and fermentation for cellulosic ethanol production. Bioresour Technol. 2010;101:4862–74. pmid:20006926
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref19] 19. Martin GJO, Knepper A, Zhou B, Pamment NB. Performance and stability of ethanologenic Escherichia coli strain FBR5 during continuous culture on xylose and glucose. J Ind Microbiol Biotechnol. 2006;33:834–44. pmid:16680457
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref20] 20. Admassu W, Korus RA, Heimsch RC, Lemmel SA, Growth of Saccharomycopsis fibuligera in a continuous-stirred tank fermentor. Biotechnol Bioeng. 1981;23:2361–71.
View Article
Google Scholar

[67] View Article

[68] Google Scholar

[ref21] 21. Admassu W, Korus RA, Heimsch RC. Two-stage continuous fermentation of Saccharomycopsis fibuligera and Candida utilis. Biotechnol Bioeng. 1983;25:2641–51. pmid:18548599
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref22] 22. Lemmel SA, Heimsch RC, Edwards LL. Optimizing the continuous production of Candida utilis and Saccharomycopsis fibuligera on potato processing wastewater. Appl Environ Microbiol. 1979;37:227–32. pmid:35096
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref23] 23. Admassu W, Korus RA, Heimsch RC. Continuous fermentation of Saccharomycopsis fibuligera and Candida utilis. Biotechnol Bioeng. 1984;26:1511–13. pmid:18551685
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref24] 24. Castañeda-Agulló M. Studies on the biosynthesis of extracellular proteases by bacteria. I. Serratia marcescens, synthetic and gelatin media. J Gen Physiol. 1956;89:369–75. pmid:13286455
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref25] 25. Chisti Y. Pneumatically agitated bioreactors in industrial and environmental processing: Hydrodynamics, hydraulics, and transport phenomena. Appl Mech Rev. 1998;51:33–112.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref26] 26. Klaic R, Kuhn RC, Foletto EL, Prá VD, Jacques RJS, Guedes JVC, et al. An overview regarding bioherbicide and their production methods by fermentation. In: Gupta VK, Mach RL, Sreenivasaprasad S, editors. Fungal biomolecules: sources, applications and recent developments. Chichester, UK: John Wiley & Sons, Ltd.; 1991. pp. 183–199. https://doi.org/10.1002/9781118958308.ch14

[ref27] 27. Chávez-Camarillo GM, Santiago-Flores UM, Mena-Vivanco A, Morales-Barrera L, Cortés-Acosta E, Cristiani-Urbina E. Transient responses of Wickherhamia sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis. Ann Microbiol. 2018;68:625–635.
View Article
Google Scholar

[90] View Article

[91] Google Scholar

[ref28] 28. Blanch HW, Clark DS. Biochemical Engineering. New York, USA: Marcel Dekker, Inc.; 1997. pp. 162–275.

[ref29] 29. Lemmel SA, Heimsch RC, Korus RA. Kinetics of growth and amylase production of Saccharomycopsis fibuligera on potato processing wastewater. Appl Environ Microbiol. 1980;39:387–93. pmid:16345512
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref30] 30. Moulin G, Galzy P. Etude de l’α-amylase de la paroi de Pichia burtonii Boidin. Zeit Allgem Mikrobiol. 1978;18:269–74.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref31] 31. Azoulay E, Jouanneau F, Bertrand JC, Raphael A, Janssens J, Lebeault JM. Fermentation methods for protein enrichment of cassava and corn with Candida tropicalis. Appl Environ Microbiol. 1980;39:41–7. pmid:16345495
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref32] 32. Kelly CT, Moriarty ME, Fogarty WM. Thermostable extracellular α-amylase and α-glucosidase of Lipomyces starkeyi. Appl Microbiol Biotechnol. 1985;22:352–58.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref33] 33. Haifeng LI, Zhenming CHI, Xiaohong W, Chunling MA. Amylase production by the marine yeast Aureobasidium pullulans N13d. J Ocean Univ. 2007;6:60–5.
View Article
Google Scholar

[108] View Article

[109] Google Scholar

[ref34] 34. Horn CH, de Kock A, Du Preez JC, Lategan PM. A comparative study of the amylolytic ability of Lipomyces and Schwanniomyces yeast species. Syst Appl Microbiol. 1988;10:106–10.
View Article
Google Scholar

[111] View Article

[112] Google Scholar

[ref35] 35. Moulin G, Galzy P. Study of an amylase and its regulation in Lipomyces starkeyi. Agric Biol Chem. 1979;43:1165–71.
View Article
Google Scholar

[114] View Article

[115] Google Scholar

[ref36] 36. Sills AM, Zygora PSJ, Stewart GG. Characterization of Schwanniomyces castelli mutants with increased productivity of amylases. Appl Microbiol Biotechnol. 1984;20:124–28.
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref37] 37. Moulin P, Galzy P. Amylase activity of Torulopsis ingeniosa Di Menna. Folia Microbiol. 1978;23:424–27. pmid:33881
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref38] 38. Carrasco M, Villareal P, Barahona S, Alcaíno J, Cifuentes V, Baeza M. Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts. BMC Microbiol. 2016;16:21. pmid:26895625
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref39] 39. Simpson-Lavy K, Kupiec M. Carbon catabolite repression in yeast is not limited to glucose. Sci Rep. 2019;9:6491. pmid:31019232
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref40] 40. Görke B, Stülke J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol. 2008;6:613–24. pmid:18628769
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref41] 41. Kayikci Ö, Nielsen J. Glucose repression in Saccharomyces cerevisiae. FEMS Yeast Res. 2015;15:fov068. pmid:26205245
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref42] 42. Clementi F, Rossi J, Costamagna L, Rosi J. Production of amylase(s) by Schwanniomyces castelli and Endomycopsis fibuligera. Antonie Van Leeuwenhoek. 1980;46:399–405. pmid:6160813
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref43] 43. Calleja GB, Yaguchi M, Levy-Rick S, Seguin JRH, Roy C, Lusena CV. Single-cell protein production from potato starch by the yeast Schwanniomyces alluvius. J Ferment Technol. 1986;64:71–5.
View Article
Google Scholar

[144] View Article

[145] Google Scholar

[ref44] 44. De Mot R, van Oudendijck E, Hougaerts S, Verachtert H. Effect of medium composition on amylase production by some starch-degrading yeasts. FEMS Microbiol Lett. 1984;25:169–73.
View Article
Google Scholar

[147] View Article

[148] Google Scholar

[ref45] 45. Wang DIC, Cooney CL, Demain AL, Dunnill P, Humphrey AE, Lilly MD. Fermentation and Enzyme Technology. John Wiley & Sons. 1979.

[ref46] 46. Dean ACR. Influence of environment on the control of enzyme synthesis. J Appl Chem Biotechnol. 1972;22:245–59.
View Article
Google Scholar

[151] View Article

[152] Google Scholar

[ref47] 47. Toda K. Dual control of invertase biosynthesis in chemostat culture. Biotechnol Bioeng. 1976;18:1117–24. pmid:953171
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

Figures

Abstract

1. Introduction

2. Materials and methods

2.1. Microorganism

2.2. Culture media composition

2.3. Inoculum preparation

2.4. Split-cylinder internal-loop airlift bioreactor

2.5. Kinetics of C. wangnamkhiaoensis α-amylase production in single-stage steady-state continuous cultures

2.6. Analytical methods

2.7. Statistical analysis

3. Results and discussion

4. Conclusions

References