Journal of Applied Phycology

https://doi.org/10.1007/s10811-018-1550-y

Investigation of β-galactosidase production by microalga Tetradesmus

obliquus in determined growth conditions
Jihed Bentahar 1 & Alain Doyen 2 & Lucie Beaulieu 2 & Jean-Sébastien Deschênes 1

Received: 6 March 2018 / Revised and accepted: 13 June 2018

# Springer Nature B.V. 2018

Abstract
β-Galactosidase is a commercially important enzyme widely used in the food industry for the manufacturing of lactose-
hydrolyzed products and synthesis of probiotic food ingredients. It could also be used to reduce the environmental
impacts of the dairy industry relative to lactose disposal. This enzyme has been isolated from different sources, with
varying properties and potential for diverse applications. Several microalgae have been screened for β-galactosidase
activity, among which the chlorophyte Tetradesmus obliquus showed significant levels. The production of enzymes from
microalgae could emerge as a valuable avenue for the utilization of its biomass. In addition, this particular microalga can
grow mixotrophically on lactose as an organic carbon source, offering additional possibilities for the utilization of
lactose. This study investigates the production of β-galactosidase from T. obliquus under different trophic conditions
and known media composition, in order to assess their influence on its productivity and selectivity. Results show that the
photoautotrophic cultures provide a highest selectivity, while mixotrophic conditions provide higher productivities due
to faster growth and higher biomass yields. Further studies on mixotrophic cultures using lactose revealed no significant
differences on β-galactosidase production when varying the concentrations of organic carbon and nitrogen nutrients. The
age of culture has a strong influence on the enzyme production, suggesting a dependence on the growth phase. Maximal
enzyme productivities obtained in mixotrophic conditions on lactose reach about 12.35 U L−1 day−1 after 7 days, which
is a realistic duration for producing the enzyme at larger scales in bioreactors.

Keywords β-Galactosidase . Lactose utilization . Microalgae . Biomass production . Specific enzymatic activity . Volumetric
enzymatic activity

Introduction in cheese whey (Kim and Rajagopal 2000). Its poor digestion
may cause gastrointestinal problems in about 70% of the world
Lactose, the predominant carbohydrate in milk, dairy products, adult population (Khabarova et al. 2011; Rosolen et al. 2015),
and whey from dairy industries, is a disaccharide consisting of and gastrointestinal symptoms may result as follows: diarrhea,
galactose bound to glucose. It makes up about 4.5–5.0% of the bloating, flatulence, and abdominal discomfort (Shaukat et al.
total solids in fluid milk and about 4.8–4.9% of the total solids 2010). Another problem related to lactose is its low solubility,
which often leads to its crystallization. This constitutes another
problem because of the undesirable granular texture, which
can occur in some dairy products, such as condensed milk,
* Jean-Sébastien Deschênes evaporated milk, dry milk, frozen milk, and ice cream, and
[email protected] confectionery products with a high content of milk (Zarate
1
and Lopez-Leiva 1990). Moreover, lactose is largely respon-
Département de mathématiques, d’informatique et de génie, Collectif sible for the high biological oxygen demand (BOD) and
de recherche appliquée aux bioprocédés et à la chimie de
l’environnement (CRABE), Université du Québec à Rimouski, 300, chemical oxygen demand (COD) leading to the environ-
Allée des Ursulines, Rimouski, Québec G5L 3A1, Canada mental pollution of the by-products of dairy industries
2
Département des sciences des aliments, Institut sur la Nutrition et les (Shukla 1975; González Siso 1996). For example, a dairy
Aliments Fonctionnels (INAF), Faculté des sciences de l’agriculture farm processing 100 t of milk per day produces approxi-
et de l’alimentation, Université Laval, 2425, rue de l’Agriculture, mately the same quantity of organic products in its effluent
Québec City, Québec G1V 0A6, Canada
 J Appl Phycol

as a town of 55,000 residents (Sienkiewicz and Riedel Materials and methods

1990). This sugar can be hydrolyzed by either acids or
enzymes. However, in dairy products, enzymatic hydroly- Culture and medium
sis is preferred for its specificity, as their contents in pro-
teins and other organic substances should not be impacted Tetradesmus obliquus was obtained from the Canadian
(Kim and Rajagopal 2000). Phycological Culture Center in Waterloo, Canada (CPCC 5).
β-Galactosidase (EC 3.2.1.23) is widely distributed in It was kept in autoclaved (121 °C, 15 min, 1–2 bar) modified
nature and can be found in various microorganisms, Bold’s Basal medium (BBM), with an adjusted pH of 6.8
plants, and animal organs (Nagy et al. 2001; Haider and (Stein 1973). Seed cultures (for all experiments) were kept
Husain 2007). Depending on its origin, its properties (mo- in exponential growth phase in a Multitron II incubator shaker
lecular weight, amino acid chain length, active site posi- (Infors-ht Switzerland) under continuous orbital agitation
tion, pH and thermal optimum and stability), specificity, (120 rpm), constant temperature (21 °C), ambient air, and light
and structure may differ significantly (Mlichova and intensity of 100 μmol photons m−2 s−1 for 15 days prior to
Rosenberg 2006). A main characteristic for the selection inoculation.
of an enzyme in a particular application is its operational The preparation of all samples was done under sterile con-
pH range: acidic pH enzymes derived from fungi are suit- ditions in a laminar flow biological hood and axenic culture
able for the treatment of acid whey and acid whey perme- conditions were confirmed at the beginning and the end of
ates whereas enzymes with neutral pH from yeasts and each culture using a cytometric flow analysis as described in
bacteria are suitable for the treatment of milk and sweet Deschênes et al. (2015) and Tremblay et al. (2009).
whey (Panesar et al. 2010). The main industrial produc- Concentrated solutions of BBM medium and nitrate were
tions are from microbial origin, as they present the highest prepared to constitute the medium for the experiments,
productivity (Rosolen et al. 2015). For their part, allowing the necessary flexibility for varying the growth con-
microalgae as enzyme producers could have several ad- ditions. Concentrated solutions of pure D-(+)-glucose, pure
vantages over other organisms, as their low nutritional D-(+)-galactose, and pure D-lactose monohydrate (all from
requirements (CO2, cheap nitrogen sources, light from ei- Sigma-Aldrich, Canada) were also prepared (in nano-filtered
ther natural or artificial sources, water, and salts) and syn- water) for the heterotrophic and mixotrophic cultures. All so-
thesis of other valuable by-products might improve pro- lutions were autoclaved separately, then diluted to the desired
cess profitability (Brasil et al. 2017). concentration under sterile conditions.
The present work therefore addresses a first practical
study for the quantification and the optimization of the Experimental design
β-galactosidase enzyme production with Tetradesmus
obliquus (formerly known as Scenedesmus obliquus; All experiments were carried out in 250-mL Erlenmeyer
Wynne and Hallan 2015). In our recent studies (Girard et flasks (75 mL operation volume) with an inoculation concen-
al. 2013, 2014), cultures of this microalgae in the presence tration of 1 × 106 cells mL−1 (day 0), using manual cell counts
of lactose improved biomass productivity compared to au- on a hemacytometer. The first experiment was conducted to
totrophic cultures on CO2. During growth a decrease in verify whether the enzyme is produced in different trophic
lactose concentration was observed over time, along with modes of growth (photoautotrophic, heterotrophic, and
a concomitant increase in glucose and galactose concen- mixotrophic) and if so, how it affects the production of the
trations, revealing β-galactosidase activity. However, this enzyme. Thus, these three conditions were compared in trip-
level of activity was not quantified at the time. The objec- licates using normal BBM concentrations (containing about
tives of this work are to investigate and evaluate the β- 180 mg L−1 of nitrate as the sole nitrogen source) and a lactose
galactosidase production by T. obliquus in different tro- concentration of 40 g L −1 for the heterotrophic and
phic modes under determined culture conditions. As this mixotrophic cultures. This concentration was selected from
microalga can grow on lactose, there would be a particular previous results (Girard et al. 2014), suggesting a certain op-
interest for using this low-cost, largely available carbon timum for cell growth.
source for the production of algal biomass in combination The second experiment evaluated the influence of the dis-
with the β-galactosidase enzyme. The paper is structured solved organic carbon (DOC) source on the production of the
as follows: experimental methods including necessary ad- enzyme in mixotrophic conditions. Glucose, galactose, and
aptations to the standard approach for measuring β- lactose, used as DOC sources, were tested at a concentration
galactosidase are first presented, followed by the experi- of 40 g L−1. All conditions were tested in triplicates, with the
mental results. Influences of the trophic mode of culture, autotrophic condition as control.
type of carbon source, nutrient concentrations, and pro- In a third experiment the data were subjected to a 23 facto-
duction kinetics are investigated. rial analysis of variance to evaluate the influence of nitrogen
J Appl Phycol

availability (nitrate concentrations of 180 and 360 mg L−1), a sufficient yellow color inactivates the enzyme and stops
carbon availability (lactose concentrations of 20 and the reaction as the pH is increased to 11. The liberated o-
40 g L−1), and age of culture (days 8 and 16) on the production nitrophenol was detected spectrophotometrically at 420 nm
of the β-galactosidase enzyme. All other ion concentrations and was expressed in units (U) of enzyme activity: 1 U is
were as in normal BBM composition. equivalent to 1 nmol o-nitrophenol produced per minute.
A fourth experiment was finally carried out to study the Other adaptations of Miller’s method, initially devel-
kinetics of β-galactosidase production in mixotrophic con- oped for bacteria such as Escherichia coli, include a man-
ditions. Based on the results of the abovementioned exper- datory centrifugation step (3400×g, 6 min) after stopping
iments, a lactose concentration of 20 g L−1 was selected, the enzymatic reaction to remove all cell debris, improving
with other ion concentrations as in standard BBM condi- the absorbance measurement (420 nm in a clear solution)
tions. Due to sample volume restrictions the same condi- for the yellow compound o-nitrophenol. Another measure-
tion was implemented simultaneously in five sets of trip- ment of absorbance at 420 nm was also included for a
licates, in which two measurements (at different days) similar sample without ONPG addition to obtain a correc-
were made for each set. tion for the background color of the sample (mainly from
Measurements were taken at days 8 (growth phase) and 16 alga pigmentation).
(expected stationary phase of culture) in the first three exper- As the ONPG concentration is in excess, the result of
iments to include the stage of culture in the comparisons. In the assay can be assumed to be linearly proportional to the
the last (fourth) experiment, measurements were taken at days amount of enzymes present (Miller 1972). We thus use the
3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 of culture to grasp a more two following quantitative measurements for the enzyme
complete view of the enzyme production throughout the cul- produced: the specific enzymatic activity (SEA) and the
ture stages. (total) volumetric enzymatic activity (VEA). The first
Statistical analyses were systematically conducted on the (Eq. 1) is defined as the amount of enzyme (U) per unit
results using standard analysis of variance (ANOVA) tests for of algal biomass (g cells), which can be used as a measure
comparison of means with the Excel software. Mean values of process selectivity (proportion of enzyme relative to the
and standard deviation of measurements (n = 3) were calculat- total biomass produced, from the same carbon source).
ed for each experiment and statistical significance was deter- The second (Eq. 2) is a measure of process volumetric
mined by the calculation of p values. A p value of less than production (total amount of enzyme produced per unit
0.05 (p < 0.05) was considered to indicate significance. volume of culture).
Results are expressed as means ± SD (the standard deviation).

SEA U ⋅g −1 cells ¼ 1000 ð1Þ
Analytical methods ð OD420 ðwith ONPGÞ−OD420 ðwithout ONPGÞ Þ

t  v  DW  0:0045


Samples were centrifuged (3400×g, 6 min) and then VEA U ⋅L−1 culture ¼ SEA  DW ð2Þ
washed twice with distilled water to remove excess salts,
sugars, or any component that could influence the measure- where OD420 (with ONPG) and OD420 (without ONPG) are
ment of the optical density. Cell enzymatic activity was the absorbance measurements (discussed above), t is the reac-
analyzed based on Miller (1972) with a few adaptations tion time (min), v is the volume of culture used in the assay
for microalgae: a 0.5-mL concentrated cell solution was (mL), DW is the actual dry weight (g L−1) of the sample, factor
mixed with 0.5 mL of Z-buffer (0.43 g of 0.06 M 0.0045 is the absorbance of 1 nmol of o-nitrophenol at 420 nm
Na2HPO 4, 0.28 g of 0.04 M NaH 2PO 4.H2 O, 0.04 g of in a 10-mm light path, and factor 1000 is for conversion of
0.01 M KCl, 0.01 g of 0.001 M MgSO 4 .7H 2 O, and units (mL L−1) after the product of v and DW.
0.135 mL of 0.05 M β-mercaptoethanol in 50 mL distilled Dry weight (DW) measurements were carried out for
water solution with pH adjustment to 7.0). The cell mem- all experiments as follows (Zhu and Lee 1997): a culture
brane was subsequently destroyed over 5 min using 100 μL sample of 10 mL was filtered through a glass-microfiber
of chloroform and 50 μL of 0.1% sodium dodecyl sulfate filter (VWR Grade 691, nominal pore size 1.2 μm) previ-
(SDS) to allow small molecules such as o-nitrophenyl-β- ously dried and weighted. The cells were washed with a
D-galactoside (ONPG) penetrate the cell walls. An enzy- 20-mL solution of 0.5% ammonium formate to ensure the
matic reaction was then started by adding 0.2 mL of ONPG removal of all dissolved compounds that could alter the
(4 mg mL−1) to the mixture incubated at 28 °C. This reac- dry weight measurement. The filtration technique was
tion allows the ONPG molecules (colorless) to be hydro- conducted as described in Strickland and Parsons (1968)
lyzed into galactose (colorless) and o-nitrophenol (yellow and Zhu and Lee (1997). The filters were then dried at
color) in the presence of the β-galactosidase enzyme. The 95 °C until they reach a constant weight, cooled in a
addition of 0.5 mL of 1 M Na2CO3 after the development of desiccator for 5 min, and then weighted.
 J Appl Phycol

Results Heterotrophic cultures maintain a similar level of productivity

(around 2.9 U L−1 day−1) over either 8 or 16 days.
Influence of the culture mode
Influence of the organic substrate
Experiments demonstrated that β-galactosidase activities were
observed in all trophic modes of growth. A comparison of the Mixotrophic cultures conducted with either pure glucose or
enzymatic activities however revealed significant differences pure galactose as the sole organic carbon source (the individ-
between the cultures. Indeed, the specific production of the ual monosaccharides composing lactose) showed generally
enzyme was always higher in photoautotrophic mode higher enzymatic activities compared to those with lactose
(Fig. 1), whatever the time condition. Mixotrophic cultures (Fig. 2). Furthermore, similarly as in the previous experiment,
then showed higher specific productivities than heterotrophic VEAs were higher in mixotrophic cultures than in the photo-
cultures at day 8 (F = 25.96, p = 0.007), though at day 16, the autotrophic control at day 8 (due to higher biomass concen-
difference was not significant. All cultures showed an increase trations), whereas the SEAs remained higher in photoautotro-
in SEA between days 8 and 16, whether the biomass concen- phic cultures at days 8 and 16 (following a similar profile),
tration increased or not. The VEA, which accounts for the suggesting repeatability. However, at this time, the VEA in the
differences in biomass concentrations, was then higher in photoautotrophic cultures at day 16 was not higher than in the
mixotrophic cultures at day 8, where the difference between mixotrophic cultures at the same time condition. Also, com-
photoautotrophic and heterotrophic cultures was not signifi- paring daily-averaged enzyme productivities, similar results
cant (F = 3.13, p = 0.15) (Fig. 1). After 16 days photoautotro- were observed between photoautotrophic cultures over
phic cultures showed a higher production of the enzyme, 16 days and mixotrophic cultures on lactose over 8 days: the
followed by the mixotrophic and then the heterotrophic latter produced about half the amount of the first in half the
cultures. time. This suggests that long-term cultures are not necessarily
A measure of process productivity (production of enzyme a better option than short-term cultures for producing the
per unit of time) can be obtained by computing the ratio be- enzyme.
tween the VEA and the age of culture. This overall daily- Daily-averaged volumetric enzyme productivities showed
averaged quantity provides additional insights for scale-up per- the highest results with microalgae grown on glucose
spectives and for determining an appropriate operation mode (17.7 U L−1 day−1) and galactose (15.6 U L−1 day−1) over
for an industrial setup. Experiments showed significant differ- 8 days; on lactose, a result of 9.8 U L−1 day−1 was obtained.
ences at this level between trophic modes: photoautotrophic All these (mixotrophic) cultures showed better productivities
cultures show much higher productivities over 16 days over 8 days than over 16 days (9.8 U L−1 day−1 for glucose,
(9.4 U L−1 day−1) than over 8 days (3.7 U L−1 day−1), while 12.7 U L−1 day−1 for galactose, and 8.8 U L−1 day−1 for lac-
mixotrophic cultures show better productivities over 8 days tose). As in the previous experiment (section BInfluence of the
(7.3 U L−1 day−1) than over 16 days (6.6 U L−1 day−1). culture mode^), photoautotrophic (control) cultures showed

Fig. 1 Enzymatic activities and

dry weights of microalga cultures
grown in photoautotrophic,
mixotrophic, and heterotrophic
conditions; results are expressed
as the mean ± SD (n = 3)
J Appl Phycol

Fig. 2 Enzymatic activities and

dry weights of microalga cultures
grown in mixotrophic and
photoautotrophic conditions;
results are expressed as the mean
± SD (n = 3)

higher productivities over 16 days (9.7 U L−1 day−1) than over concentration, and lactose concentration) showed that only
8 days (4.9 U L−1 day−1), where its highest level is similar to the age of culture has a consistent influence on the SEA (F =
that of cultures on lactose. 48.02, p < 0.01) and the VEA (F = 8.90, p < 0.01). Biomass
production is not being affected by increased nitrate concen-
trations at either day 8 or 16. However, increased lactose con-
Influence of carbon and nitrogen concentrations centrations do positively influence the biomass concentration
over the first 8 days of production, whether the nitrate con-
Under the conditions tested (lactose concentrations of 20 and centration is 180 mg L−1 (F = 18.43, p = 0.013) or 360 mg L−1
40 g L−1 and nitrate concentrations of 180 and 360 mg L−1), (F = 31.56, p = 0.005).
results showed similar SEA values for most cultures at either
time condition (Fig. 3). The VEA appears to rise slightly as the
nutrient concentrations are increased (Fig. 3), thought the only Kinetics of enzyme production
case of statistical significance is for a higher lactose concen-
tration at a constant nitrate concentration of 360 mg L−1 at day As the age of culture has a significant influence on most re-
8 (F = 192.6, p < 0.01). Overall results of the statistical analy- sults, characterizing the enzyme production through time was
ses about the influence of factors (age of culture, nitrate deemed of importance. Mixotrophic cultures on lactose

Fig. 3 Enzymatic activities and

dry weights of microalga cultures
grown in mixotrophic conditions
(initial nutrient concentrations
indicated); results are expressed
as the mean ± SD (n = 3)
 J Appl Phycol

Fig. 4 Evolution of cell dry

weights and enzymatic activities
through time in mixotrophic
conditions (initial nutrient
concentrations indicated); results
are expressed as the mean ± SD
(n = 3)

(20 g L−1) were used for this study based on previous volu- Discussion
metric productivities over a rather short period of time (higher
than in the autotrophic case) and low cost of this organic This study investigated β-galactosidase production by
carbon source. Tetradesmus obliquus across a range of DOC and nitrogen
The SEA and VEA evolution through time showed a some- conditions. Results showed that the trophic mode has a signif-
what similar pattern (steady increase followed by a stationary icant influence on both the production of enzyme and bio-
phase), as does the biomass concentration (Fig. 4). At the end mass. The SEA was always higher in photoautotrophic mode,
of the experiment (days 19 and 21), enzymatic activities while the use of a DOC source reduced the specific enzyme
tended to decrease (late stationary phase). To grasp a better production (both in heterotrophic and in mixotrophic modes).
view of the actual enzyme productivity, a daily-averaged vol- This observation contrasts with the case of bacteria E. coli,
umetric enzyme productivity (VEA divided by culture time) where lactose induces a higher specific enzyme production
was computed (Fig. 5): results show an optimum at around (Jacob and Monod 1961; Law et al. 2002). The use of a
day 7 (12.35 U L−1 day−1), confirming the previous apprehen- DOC source however significantly stimulated the growth of
sions in BIntroduction,^ BMaterials and methods,^ and T. obliquus (as observed in Girard et al. 2014): in mixotrophic
BResults.^ cultures, this resulted in higher volumetric enzyme produc-
tions after 8 days and overall higher enzyme productivities
(U L−1 day−1). In heterotrophic cultures, however, the lowest
enzyme productions were observed.
The highest enzyme productivities were observed in
mixotrophic cultures over 8 days and in autotrophic cul-
tures over 16 days. Since higher variability was observed
in autotrophic cultures, mixotrophic conditions appear as a
more viable alternative for producing the enzyme.
Heterotrophic cultures presented the lowest enzyme pro-
duction levels and therefore do not appear suitable for
producing the enzyme.
The supplementation levels of carbon and nitrogen nu-
trients in β-galactosidase productions have not been previ-
ously studied to a great extent (Kazemi et al. 2016). The
present study however showed no significant influence on
the SEA in the range of tested concentrations (20 to
40 g L −1 lactose and 180 to 360 mg L −1 nitrate) in
Fig. 5 Daily-averaged volumetric enzyme productivity in mixotrophic
conditions (initial nutrient concentrations indicated); results are mixotrophic conditions. Most results on the VEA also sug-
expressed as the mean ± SD (n = 3) gested a similar pattern, though there some occurrences of
J Appl Phycol

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 J Appl Phycol

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