Microwave pretreatment of glycogen-containing cyanobacteria Synechococcus elongatus UTEX 2973 enhances enzymatic saccharification and fermentation for bioethanol production

doi:10.21203/rs.3.rs-5154881/v1

The objective of this study was to demonstrate that microwave pretreatment of glycogen-containing Synechococcus elongatus UTEX 2973 could dramatically enhance subsequent enzymatic saccharification and ethanol fermentation. First, the preliminary experiment showed that microwave pretreatment was significantly more effective than either ultrasound pretreatment or lysozyme pretreatment for enzymatic saccharification of intracellular glycogen of S. elongatus UTEX 2973. Next, to investigate the appropriate microwave pretreatment time for enzymatic saccharification of glycogen in S. elongatus UTEX 2973, a cyanobacterial suspension (100 g/L) was pretreated by microwave (200 W) for 0-200 seconds and subjected to the saccharification assay at low biomass loading (10 g/L). The saccharification percentage was only 18% in the case of pretreatment for 0 seconds. On the contrary, the value increased significantly to almost 100% in the case of microwave pretreatment for 100 seconds and more. Finally, to perform saccharification and ethanol fermentation of microwave-pretreated glycogen-containing S. elongatus UTEX 2973, a cyanobacterial suspension (100 g/L) was pretreated by microwave for 0-150 seconds and subjected to enzymatic saccharification, followed by simultaneous saccharification and ethanol fermentation. When the pretreatment time was 150 seconds, the glucose concentration at the end of saccharification was 44 g/L (saccharification percentage of 94%). And the ethanol concentration was 21 g/L during the simultaneous saccharification and fermentation, which is 88% of the theoretical value.

Cyanobacteria

Glycogen

Microwave

Pretreatment

Saccharification

Ethanol fermentation

Glycogen-containing UTEX 2973 was pretreated with microwave.
Microwave pretreatment (200 W, 150 s) dramatically improved enzymatic saccharification.
Saccharification assay results were 18% without pretreatment and 100% with pretreatment.
Pretreated UTEX 2973 (42% glycogen, 100 g/L) was used for ethanol production.
With microwave pretreatment, ethanol concentration was 21 g/L (88% of the theoretical value).

Conversion of fossil resources to biomass resources is important to reduce global warming. Biomass resources that can be used to produce biofuels and biochemicals include first-generation edible biomass such as starch in corn, and second-generation inedible biomass such as cellulose in the cell wall of lignocellulosic biomass. However, first-generation biomass has the disadvantage of competing with food, and second-generation biomass has the disadvantage of having a complex and robust cell wall structure [1]. On the other hand, photoautotrophic microorganisms such as microalgae and cyanobacteria are considered third-generation biomass, which does not compete with food and has a simpler and more fragile cell wall structure [1–3]. Therefore, microalgae and cyanobacteria have received attention as a promising feedstock for the production of biofuels and biochemicals.

Cell wall structural polysaccharides and intracellular storage polysaccharides become target carbohydrates when microalgae and cyanobacteria are used as feedstocks for biofuels and biochemicals. The cell wall of microalgae is mainly composed of cellulose [4], and the cell wall of cyanobacteria is mainly composed of peptidoglycan [5]. Microalgae and cyanobacteria accumulate starch and glycogen, respectively, as intracellular storage polysaccharides under nitrogen limitation [2–7]. The major carbohydrates of cells are intracellular storage polysaccharides (starch or glycogen), not cell wall structural polysaccharides [3]. The microalgae and cyanobacteria containing these intracellular storage polysaccharides are converted to biofuels and biochemicals by saccharification (hydrolysis to fermentable sugars) and fermentation [1–7].

A large number of papers have been reported on the production of bioethanol from microalgae and cyanobacteria by saccharification and fermentation [5–7]. Most of the reports used microalgae as feedstock for bioethanol production (such as Chlorella, Chlamydomonas, and Scenedesmus), and not so many reports used cyanobacteria as feedstock for bioethanol production. However, cyanobacteria (such as Spirulina and Synechococcus) generally grow faster, have simple nutrient requirement and have less complex cell wall compared to microalgae [4, 5], suggesting the promising feedstock for bioethanol production.

The intracellular polysaccharide glycogen has been hydrolyzed to glucose using acids (such as H₂SO₄) [8–16] or enzymes (amylase) [17–20] in bioethanol production from cyanobacteria. However, acid hydrolysis of glycogen often produces glucose-derived by-products (such as hydroxymethylfurfural (HMF) and formic acid) that inhibit the fermentation performance of microorganisms [4–6, 10]. In addition, pH adjustment with alkali after acid hydrolysis produces a high concentration of salt, which also inhibits the fermentation performance of microorganisms [6, 7, 10]. Therefore, enzymatic saccharification and fermentation are considered ideal for bioethanol production from glycogen-containing cyanobacteria [5].

In the previous reports on bioethanol production from glycogen-containing cyanobacteria by enzymatic saccharification and fermentation, several pretreatments were applied to promote enzymatic saccharification. Namely, treatments to lose the cell wall integrity of the cyanobacteria and to increase the accessibility of the amylolytic enzyme to the glycogen. Specifically, freezing and thawing prior to enzymatic saccharification [17, 18], and the addition of lysozyme (an enzyme that cleaves peptidoglycan in bacterial cell walls) before or during saccharification and fermentation [17, 19, 20].

Recently, it was reported that Synechococcus elongatus UTEX 2973 showed the unique faster growth compared to the conventional cyanobacterial strains [21]. Specifically, S. elongatus UTEX 2973 showed two times faster growth rate conditions compared to S. elongatus PCC 7942, the standard strain [21]. S. elongatus UTEX 2973 also showed rather faster growth under the stress conditions of high temperature (42°C) and high light irradiation (500 µmol⸱m^− 2⸱s^− 1) [22], which are advantageous to avoid contamination risk and cooling costs. In addition to the faster growth, S. elongatus UTEX 2973 had a significantly higher glycogen accumulating property even under nitrogen-rich conditions compared to S. elongatus PCC 7942, the standard strain [22, 23]. For the above reasons, S. elongatus PCC 7942 has attracted attention in the field of third-generation biomass refinery. For example, there are some reports that genetically engineered S. elongatus UTEX 2973 has been used as a microbial cell factory to produce sucrose as a fermentable sugar [23–25], bioplastic polyhydroxybutyrate (PHB) [26, 27], isoprene [28, 29], limonene [30, 31], or other useful chemicals [32–35]. However, there are no reports that the glycogen-containing S. elongatus UTEX 2973 has been used as a biomass feedstock for bioethanol production by enzymatic saccharification and microbial fermentation.

Therefore, the aim of the present study is to perform bioethanol production from glycogen-containing S. elongatus UTEX 2973 by enzymatic saccharification and microbial fermentation. In particular, the present study investigates the effect of microwave pretreatment on the subsequent saccharification of glycogen-containing S. elongatus UTEX 2973 (Fig. 1).

Cyanobacteria, amylolytic enzymes and yeast

Synechococcus elongatus UTEX 2973 was obtained from the UTEX Culture Collection of Algae (Austin, TX, USA) and used as the biomass source because this cyanobacterial strain has been reported to accumulate glycogen in the cells during proliferation [21]. S. elongatus UTEX 2973 was maintained photoautotrophically in a 200 mL Erlenmeyer flask containing 100 mL BG-11 medium [36] at 42°C with shaking at 120 rpm under continuous illumination by LED lamps at a light intensity of 170 µmol⸱m^− 2⸱s^− 1 with bubbling air containing 3% CO₂ at a flow rate of 1 vvm.

Amylase mixture consisting of α-amylase and glucoamylase (Magnax JW101, powder) was kindly provided by Rakuto Kasei Industrial Co., Ltd. (Otsu, Japan) and was used for the hydrolysis of cyanobacterial glycogen to glucose. The α-amylase and glucoamylase activities of the amylase mixture were determined to be 560 U/g and 370 U/g, respectively, by the α-amylase assay kit and the saccharification capacity assay kit (Kikkoman Biochemifa Company, Tokyo, Japan) using 2-chloro-4-nitrophenyl-6⁵-azide-6⁵-deoxy-β-maltopentaoside and 4-nitrophenyl-β-D-maltoside, respectively, as substrates. Glucoamylase (30 U/mg) was purchased from TOYOBO Co., Ltd. (Osaka, Japan) and was used for glycogen content measurement and enzyme accessibility assay.

The yeast Kluyveromyces marxianus NBRC 1777 was obtained from the Biological Resource Center, NITE (NBRC, Kisarazu, Japan) and used for fermentation of glucose to ethanol. K. marxianus NBRC 1777 was maintained in yeast extract-peptone-dextrose (YPD) media (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) at 30°C.

Preparation of yeast inoculum for fermentation

To prepare the yeast inoculum for fermentation, K. marxianus NBRC 1777 was cultured aerobically at 30°C for 48 h in a Sakaguchi flask containing 50 mL of YPD medium with shaking at 150 rpm. Yeast cells were collected by centrifugation (16,000 ×g for 5 min at 4°C) and washed three times with distilled water. The resulting yeast pellets were used as inoculum for the ethanol fermentation described in the following section 2.5.

Culture of cyanobacterial biomass as feedstock

The experimental culture of S. elongatus UTEX 2973 was performed photoautotrophically for 7 days in a flattened flask containing 500 mL of BG-11 medium at 42°C under continuous illumination by LED lamps at a light intensity of 350 µmol⸱m^− 2⸱s^− 1, with bubbling air containing 3% CO₂ at a flow rate of 1 vvm. The initial optical density at a wavelength of 730 nm (OD₇₃₀) was 0.05. After 7 days of culture, cyanobacterial cells were harvested by centrifugation (16,000 ×g for 5 min at 4°C) to obtain wet pellets in 50 mL centrifuge tubes, each containing 0.5 g dry cell weight equivalent. Harvested cyanobacterial pellets were stored at -25°C until use.

Pretreatment of cyanobacterial biomass

For microwave pretreatment of cyanobacterial biomass, cyanobacterial wet pellet of 0.5 g dry cell weight equivalent was suspended with pure water to prepare 5 mL of 100 g dry cell weight/L suspension. The cyanobacterial suspension was transferred to a 25 mL sealed reaction vessel for microwave oven (P-25, San-ai Kagaku Co. Ltd., Nagoya, Japan) and reacted for 0-200 seconds using a home microwave oven with a fixed input power of 200 W. In the case of saccharification and fermentation for bioethanol production, 5 mL of the pretreated mixture was transferred to a 50 mL centrifuge tube after microwave pretreatment.

For ultrasonic pretreatment of cyanobacterial biomass, 5 mL of cyanobacterial suspension (100 g dry cell weight/L in water) was transferred to a screw-capped glass vial (diameter, 30 mm; volume, 30 ml) and sonicated for 30 minutes using a 38 kHz sonoreactor (QUAVA mini, Kaijo Corporation, Tokyo, Japan) with a fixed input power of 24 W. The glass vial was positioned at a height of 50 mm from the bottom of the sonoreactor.

For lysozyme pretreatment of cyanobacterial biomass, 5 mL of cyanobacterial suspension (100 g dry cell weight/L in water, pH approximately 7) was transferred to 50 mL centrifuge tubes. Lysozyme (chicken egg white, Nacalai Tesque, Inc. Kyoto, Japan) was added at a final concentration of 1 g/L. The tubes were incubated for 24 hours at 37°C in a shaker at 120 rpm.

Saccharification and ethanol fermentation from pretreated cyanobacterial biomass

Saccharification was performed in 50 mL centrifuge tubes containing 5 mL of pretreated cyanobacterial suspension (100 g dry cell weight/L suspension) by adding an amylase mixture at a final concentration of 0.1 g/L (α-amylase and glucoamylase activities were 56 U/L and 37U/L, respectively). The tubes were incubated for 48 hours at 50°C in a shaker at 120 rpm. At 48 hours after the start of saccharification, simultaneous saccharification and fermentation was performed for an additional 24 hours in the same 50 mL centrifuge tube by inoculating yeast to achieve a final OD₆₀₀ of 20 (equivalent to approximately 10 g dry cell weight/L). No other nutrients, such as peptone and yeast extract, were added. Tubes were sealed with a silicone stopper with a gas check valve to release CO₂. Tubes were incubated at 40°C in a shaker at 120 rpm.

Samples were taken from the tube at 0, 12, 24 and 48 hours during the enzymatic reaction for 48 hours and at 54, 60, 66, and 72 hours during the simultaneous saccharification and fermentation for an additional 24 hours. Samples were centrifuged at 21,500 ×g for 1 minute, and the supernatant was frozen until used for glucose and ethanol measurements by high-performance liquid chromatography (HPLC) as described below.

Analysis

Yeast cell concentration

For K. marxianus NBRC 1777, cell concentration was assessed by measuring the OD₆₀₀ value. The dry cell concentration of K. marxianus NBRC 1777 was estimated using a conversion coefficient of 0.50 g-dry cell weight/L per OD₆₀₀, which was determined experimentally from a correlation between the OD₆₀₀ value and the dry cell weight of K. marxianus NBRC 1777.

Cyanobacterial cell concentration and glycogen content

For S. elongatus UTEX 2973, cell concentration was assessed by measuring the OD₇₃₀ value. The dry cell concentration of S. elongatus UTEX 2973 was estimated using a conversion coefficient of 0.35 g-dry cell weight/L per OD₇₃₀, which was determined experimentally from a correlation between the OD₇₃₀ value and the dry cell weight of S. elongatus UTEX 2973.

The glycogen content of S. elongatus UTEX 2973 was determined by the previously described method [22] with some modifications. Specifically, a cyanobacterial wet pellet of 3.5 mg dry cell weight equivalent (OD₇₃₀unit of 10) in a 1.5 mL tube was suspended in 300 µL (30% w/v) KOH aqueous solution, followed by incubation in a heat block at 95°C for 90 min. Ethanol (1.2 mL) was added to the suspension, and the mixture was incubated on ice for 2 h. After centrifugation at 16,000 ×g for 5 min at 4°C, the resulting pellet was solubilized with 250 µL water. Ethanol (1 mL) was then added to the suspension again, and the mixture was incubated on ice for 2 h. After centrifugation at 16,000 ×g for 5 min at 4°C, the resulting pellet was washed three times with 1 mL ethanol, and then vacuum dried at 60°C for 30 min. The dried sample was reconstituted with 270 µL of 400 mM sodium acetate buffer (pH 4.8). Then, 270 µL of the sample was mixed with 30 µL of glucoamylase solution (133 U/mL) in 100 mM sodium acetate buffer (pH 4.8) in a 1.5 mL tube. This resulted in 300 µL of reaction mixture with an enzyme concentration of 13.3 U/mL (enzyme amount of 4 U/assay). The 1.5 mL tube was incubated at 55°C for 30 minutes. The sample was then heated to 90°C for 5 minutes to inactivate the enzyme. The heated samples were centrifuged at 21,500 ×g for 1 minute and the supernatant was used for glucose measurement. Glucose concentrations were measured by the mutarotase GOD method (LabAssay™ Glucose, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The amount of glycogen was calculated from the glucose contents multiplied by the anhydro correction factors of 162/180. The glycogen content was evaluated as the percentage of the amount of glycogen relative to the dry cell weight of the cyanobacteria.

Enzyme accessibility to glycogen in cyanobacteria

A low biomass loading (10 g dry cell weight/L) saccharification assay was performed to evaluate the accessibility of the amylolytic enzyme to the glycogen in pretreated cyanobacterial biomass. Specifically, a sample was taken from the pretreated cyanobacterial suspension (100 g/L solid loading) and diluted nine times with 100 mM sodium acetate buffer (pH 4.8). Then, 270 µL of the diluted sample was mixed with 30 µL of glucoamylase solution (133 U/mL) in 100 mM sodium acetate buffer (pH 4.8) in a 1.5 mL tube. This resulted in 300 µL of reaction mixture with a substrate concentration of 10 g/L with an enzyme concentration of 13.3 U/mL (enzyme amount of 4 U/assay). The 1.5 mL tube was incubated at 55°C for 30 minutes. The sample was then heated to 90°C for 5 minutes to inactivate the enzyme. The heated samples were centrifuged at 21,500 ×g for 1 minute and the supernatant was used for glucose measurement. Glucose concentrations were measured by the mutarotase GOD method (LabAssay™ Glucose). Glycogen saccharification was evaluated as the percentage of glycogen hydrolyzed to glucose relative to glycogen in the initial cyanobacterial biomass.

The morphology of the pretreated cyanobacterial biomass was observed under a microscope (BZ-X700, KEYENCE, Osaka, Japan).

Glucose and ethanol concentrations during saccharification and fermentation

Glucose and ethanol concentrations were determined by HPLC equipped with a refractive index detector (Shimadzu Co., Kyoto, Japan) using a CARBOSep CHO-682 column (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan). The column was run at 85°C with a water mobile phase and a flow rate of 0.4 mL/min.

Glycogen saccharification was evaluated as the percentage of glycogen hydrolyzed to glucose relative to glycogen in the initial cyanobacterial biomass. Ethanol yield was determined as the ratio of the amount of ethanol produced to the amount of initial cyanobacterial biomass.

Preparation of glycogen-containing S. elongatus UTEX 2973 biomass

To prepare the glycogen-containing S. elongatus UTEX 2973 biomass as a feedstock for subsequent enzymatic saccharification and fermentation, photoautotrophic culture of S. elongatus UTEX 2973 was performed for 7 days in a flattened flask containing 500 mL of BG-11 medium at 42°C under continuous illumination at a light intensity of 350 µmol⸱m^− 2⸱s^− 1, with bubbling air containing 3% CO₂ at a flow rate of 1 vvm. Figure 2 shows the time course of dry cell concentration and glycogen content during photoautotrophic culture of S. elongatus UTEX 2973. The cell concentration increased with culture time and reached a saturation level of 2.5 g-dry cell/L after 3–4 days of culture under the culture conditions investigated. Glycogen content also increased gradually from 23% at 2 days to approximately 40% at 5–7 days under the conditions studied. After 7 days of culture, cyanobacterial cells were harvested in 50 mL centrifuge tubes at 0.5 g dry cell weight equivalent each. The harvested cyanobacterial pellet was stored at -25°C until use. These glycogen-containing S. elongatus UTEX 2973 was used as a biomass source for subsequent experiments.

Screening of pretreatment method of glycogen-containing S. elongatus UTEX 2973 for subsequent enzymatic saccharification

To select the appropriate pretreatment method for enzymatic saccharification of glycogen in S. elongatus UTEX 2973, 5 mL of cyanobacterial suspension (glycogen content of 42% and 100 g dry cell weight/L biomass loading) was pretreated by various pretreatment (lysozyme, ultrasonic, and microwave), and then the pretreated cyanobacterial biomass was subjected to the saccharification assay at low biomass loading (10 g/L). Table 1 shows the saccharification percentage of glycogen in S. elongatus UTEX 2973 after various pretreatment. The saccharification percentage was only 18% in the case of no pretreatment. The saccharification percentage was 33% in the case of ultrasonic pretreatment for 30 minutes. The saccharification percentage was 40% in the case of lysozyme pretreatment for 24 hours. On the contrary, the saccharification percentage was almost 100% in the case of microwave pretreatment for 100 seconds. Thus, we selected microwave as candidate method for pretreatment of S. elongatus UTEX 2973 prior to enzymatic saccharification of intracellular glycogen, since microwave pretreatment showed effective pretreatment in a short time.

Microwave pretreatment of glycogen-containing S. elongatus UTEX 2973 for subsequent enzymatic saccharification

To investigate the appropriate microwave pretreatment time for enzymatic saccharification of glycogen in S. elongatus UTEX 2973, 5 mL of cyanobacterial suspension (glycogen content of 42% and 100 g dry cell weight/L biomass loading) was pretreated by microwave at fixed input power (200 W) for 0, 50, 100, 150, or 200 seconds, and then the pretreated cyanobacterial biomass was subjected to the saccharification assay at low biomass loading (10 g/L). Figure 3 shows photographs and microscopic image of S. elongatus UTEX 2973 suspension after microwave pretreatment. The color of the suspension changed from green to brown regardless of the duration of microwave pretreatment examined. The microscopic image showed that the cyanobacterial cells were aggregated and ballooned with the microwave pretreatment duration up to 200 seconds. Figure 4 shows the relationship between the saccharification percentage of glycogen in S. elongatus UTEX 2973 and the pretreatment time. The saccharification percentage was only 18% in the case of pretreatment for 0 seconds. On the contrary, the saccharification percentage increased significantly with the pretreatment time, reaching almost 100% in the case of microwave pretreatment for 100 seconds and more. Thus, it was found that microwave was effective as a pretreatment of S. elongatus UTEX 2973 prior to enzymatic saccharification of intracellular glycogen, and the microwave pretreatment time of 100–200 was sufficient under the condition investigated in this study.

Saccharification and ethanol fermentation from microwave-pretreated glycogen-containing S. elongatus UTEX 2973

To perform saccharification and ethanol fermentation of microwave pretreated glycogen-containing S. elongatus UTEX 2973, 5 mL of cyanobacterial suspension (glycogen content of 42% and 100 g dry cell weight/L biomass loading) was pretreated by microwave for 0, 50, 100 or 150 seconds, and subjected to enzymatic saccharification for 48 hours at 50°C, followed by simultaneous saccharification and fermentation for another 24 hours at 40°C. Figure 5 shows the time course of glucose and ethanol concentration during enzymatic saccharification and yeast fermentation of glycogen-containing S. elongatus UTEX 2973 pretreated for different times of 0, 50, 100 or 150 seconds. The glucose concentration increased with the reaction time up to 48 hours, and glucose was not detected 6 hours after the start of ethanol fermentation by adding yeast cells. The ethanol concentration was almost constant after 6 hours of fermentation. The glucose concentration and ethanol concentration were higher when the pretreatment time was longer from 0 seconds to 100 seconds, and the profiles almost overlapped between the case of 100 seconds and 150 seconds. When the pretreatment time was 150 seconds, the glucose concentration reached 44 g/L at 48 hours (end of saccharification), which corresponds to a saccharification percentage of 94%. And the ethanol concentration reached 21 g/L at 6 hours after the start of simultaneous saccharification and fermentation, which corresponds to 94% of the theoretical value based on the glucose produced and 88% of the theoretical value based on the glycogen in the cyanobacteria used. Based on these results, it was demonstrated that microwave pretreatment of glycogen-containing S. elongatus UTEX 2973 could be a key for subsequent enzymatic saccharification and fermentation.

The present study is the first study to demonstrate that microwave pretreatment of glycogen-containing S. elongatus UTEX 2973 can dramatically enhance subsequent enzymatic saccharification and ethanol fermentation.

As a cyanobacterial feedstock for bioethanol production, glycogen-containing cyanobacteria have been produced by culture under nitrogen-limited conditions. However, the nitrogen-limited condition generally results in slower growth rate and lower cell concentration of cyanobacteria. Therefore, a strain with fast growth rate and high glycogen accumulation even under nitrogen-rich condition is needed to increase the yield of glycogen-containing cyanobacteria as a feedstock for bioethanol production. Therefore, in the present study, S. elongatus UTEX 2973 was selected as a feedstock for bioethanol production by enzymatic saccharification and fermentation because this strain has been reported to have unique faster growth compared to the conventional cyanobacterial strains [21] and to have 40–50% glycogen content even under nitrogen-rich conditions [22, 23].

Regarding pretreatment in bioethanol production by enzymatic saccharification and fermentation from glycogen-containing cyanobacteria, Rempel et al. reported a simple method, freezing and thawing. Specifically, glycogen-containing A. platensis LEB 52 was pretreated by freezing and thawing (-20°C for 24 hours and 4°C for 24 hours) prior to enzymatic saccharification followed by ethanol fermentation [18]. However, in the present study, the saccharification percentage of glycogen-containing S. elongatus UTEX 2973 was only 18% even after pretreatment by freezing and thawing (Table 1 and Fig. 4). Thus, it was found that freezing and thawing could not sufficiently enhance the enzymatic saccharification of glycogen-containing S. elongatus UTEX 2973.

Möllers et al. reported that glycogen-containing Synechococcus sp. PCC 7002 (100 g dry cell weight/L) was pretreated by freezing and thawing followed by reaction with 0.1 g/L lysozyme for 3 hours at 37°C prior to enzymatic saccharification in bioethanol production by enzymatic saccharification and fermentation. The saccharification percentage was 60–80% [17]. While, in the present study, the cyanobacterial suspension of glycogen-containing S. elongatus UTEX 2973 (100 g dry cell weight/L) was pretreated by freezing and thawing, followed by reaction with 1 g/L lysozyme for 24 hours at 37°C. However, the saccharification percentage was only 40% (Table 1). Thus, it was found that freezing and thawing followed by lysozyme reaction could not sufficiently enhance the enzymatic saccharification of glycogen-containing S. elongatus UTEX 2973.

It is unclear why S. elongatus UTEX 2973 used in the present study was more resistant to the reported pretreatment methods such as freeze-thaw and lysozyme, than the typical cyanobacterial strains such as A. platensis LEB 52 and Synechococcus sp. PCC 7002. It has been reported that S. elongatus UTEX 2973 has unique properties of a fast-growing strain with pronounced stress tolerance to high temperature and high light irradiation [21]. This unique property may be related to the higher robustness of S. elongatus UTEX 2973 against freeze-thaw and lysozyme compared to typical cyanobacterial strains.

Some studies reported that microwave could be used for pretreatment prior to enzymatic saccharification and fermentation of starch-containing microalgae [37–40], which have a much more rigid cell wall structure than cyanobacteria. However, there are still no reports that microwave has been used for pretreatment of glycogen-containing cyanobacteria prior to enzymatic saccharification and fermentation in bioethanol production. Therefore, in the present study, microwave was selected as a pretreatment before enzymatic saccharification of glycogen-containing S. elongatus UTEX 2973 (Table 1). As a result of enzymatic saccharification at 100 g/L loading of pretreated S. elongatus UTEX 2973, the glucose concentration in saccharification step of bioethanol production reached 44 g/L which is 94% of the theoretical value (Fig. 5). This value was equal to or higher than those in other cases of bioethanol production by enzymatic saccharification and fermentation from glycogen-containing cyanobacteria [17, 18, 20].

As for yeast fermentation in bioethanol production from glycogen-containing cyanobacteria, in the present study, the ethanol concentration reached 21 g/L at 6 hours after the start of fermentation, which is 94% of the theoretical value based on the glucose produced during enzymatic saccharification (44 g/L). This result indicates that the enzymatic hydrolysate of glycogen-containing S. elongatus UTEX 2973 contained not only glucose as a carbon source but also other nutrient sources that support yeast fermentation, as reported in the previous works [17, 18, 20]. In addition, this result also indicates that the microwave pretreatment did not produce any inhibitory compounds for yeast fermentation from the glycogen-containing S. elongatus UTEX 2973.

Table 2 summarizes the previous studies on bioethanol production by enzymatic saccharification and fermentation of glycogen-containing cyanobacteria. The ethanol yield per cyanobacterial biomass in the present study was 0.21 g-ethanol/g-DCW. This value was lower than those obtained in the previous reports on bioethanol production by enzymatic saccharification and fermentation using glycogen-containing cyanobacteria (0.32 g-ethanol/g-DCW [20] and 0.27 g-ethanol/g-DCW [17]). On the other hand, the percentage of the theoretical value for ethanol concentration was not so different between the previous studies (93% of the theoretical value [20], 82% of the theoretical value [17]) and the present study (88% of the theoretical value). Judging from the above data, the reason why the ethanol yield per cyanobacterial biomass [g-ethanol/g-DCW] was lower in the present study is that the glycogen content [%, g-glycogen/g-DCW] in the present study (42%) was lower than that in the previous study (60% [17, 20]).

Table 1. Saccharification percentages of glycogen in S. elongatus UTEX 2973 with different pretreatments.
Pretreatment method^a)	Pretreatment condition^b)	Pretreatment time	Saccharification percentage ^c)
No	--	--	18 ± 2%
Lysozyme	With addition of 1 g/L egg white lysozyme, 37°C, 120 rpm	24 hours	40 ± 5%
Ultrasound	Ultrasound bath, 38 kHz, 24 W	30 minutes	33 ± 6%
Microwave	Sealed reaction vessel, home microwave oven, 200 W	100 seconds	101 ± 17%
Cyanobacterial samples after freeze-thawing were used for pretreatment experiments. Pretreatment was performed with 5 mL of cyanobacterial suspension (100 g dry cell weight/L). Saccharification percentage was determined by the low biomass loading saccharification assay using the pretreated cyanobacterial suspension with 10-fold dilution (10 g dry cell weight/L) and sufficient concentration of glucoamylase (13.3 U/mL).

Table 2. Comparison of bioethanol production by enzymatic saccharification and fermentation using glycogen-containing cyanobacteria as feedstock.

Cyanobacteria

strain used

Cyanobacteria

culture medium

(NaNO₃ concentraton)

Glycogen

content in

cyanobacterial biomass

[% of DCW]

Cyanobacterial

biomass loading

[g-DCW/L]

(Pre)treatment

to promote

enzymatic saccharification

Glucose

concentration

[g-glucose/L]

(% of theoretical) ^a)

Ethanol

concentration

[g-ethanol/L]

(% of theoretical) ^b)

Ethanol yield

per cyanobacterial biomass

[g-ethanol/g-DCW]

References

A. platensis

NIES 39

SOT medium

(Limited to 3.0 mM)

60%

150 g/L

CaCl₂

Lysozyme

--- g/L

(--%)

48 g/L

(93%)

0.32 g/g-DCW

[20]

Synechococcus sp.

PCC 7002

Medium A

(Limited to 2.8 mM)

60%

108 g/L

Freeze/thaw

Lyzosyme

65 g/L

(90%)

30 g/L

(82%)

0.27 g/g-DCW

[17]

A. platensis

LEB 52

20% diluted

Zarrouk’s medium

(Limited to 5.9 mM)

46%

170 g/L

Freeze/thaw

71g/L

(82%)

25 g/L

(64%)

0.15 g/g-DCW

[18]

S. elongatus

UTEX 2973

BG-11 medium

(17.6 mM)

42%

100 g/L

Freeze/thaw

Microwave

44 g/L

(94%)

21 g/L

(88%)

0.21 g/g-DCW

This study

a) Theoretical value of glucose concentration [g-glucose/L] = Glycogen content [%]/100 × Cyanobacterial biomass loading [g-DCW/L] × (180/162).

b) Theoretical value of ethanol concentration [g-ethanol/L] = Glycogen content [%]/100 × Cyanobacterial biomass loading [g-DCW/L] × (180/162) × 0.51.

DCW: dry cell weight.

The reason why the glycogen content in the previous study is higher than that in the present study is because cyanobacteria was cultured under nitrogen-limited conditions to induce glycogen accumulation [17, 18, 20, 41, 42].. However, in general, the cell concentration obtained under nitrogen limitation is lower, as described above. Thus, even though the glycogen content [%, g-glycogen/g-DCW] may be high under nitrogen limitation, the lower cyanobacterial cell concentration [g-DCW/L] will lead to the lower glycogen amount per cyanobacterial culture volume [g-glycogen/L], ultimately resulting the lower ethanol amount per cyanobacterial culture volume [g-ethanol/L]. On the contrary, S. elongatus UTEX 2973 shows a significantly higher growth rate [g-DCW/L/h] and a glycogen content of 40–50% even under conditions with sufficient nitrogen. Therefore, in the case of S. elongatus UTEX 2973, the ethanol amount per cyanobacterial culture volume [g-ethanol/L] and the ethanol productivity cyanobacterial culture [g-ethanol/L/h] are considered to become higher. (A simple comparison is difficult because the growth media used for culturing the cyanobacteria differed between the previous studies [17, 18, 20] and the present study.)

The present study demonstrated that microwave pretreatment of glycogen-containing S. elongatus UTEX 2973 could dramatically enhance subsequent the enzymatic saccharification and ethanol fermentation. First, it was shown that microwave pretreatment could enhance the enzymatic saccharification of intracellular glycogen. The saccharification percentage was only 18% in the case of pretreatment for 0 seconds. On the contrary, the value increased significantly to almost 100% in the case of microwave pretreatment for 100 seconds and more. Next, saccharification and ethanol fermentation of microwave-pretreated glycogen-containing S. elongatus UTEX 2973 were performed. When the pretreatment time was 150 seconds, the glucose concentration after 48 hours (end of the saccharification phase) was 44 g/L, corresponding to a saccharification percentage of 94%. And the ethanol concentration was 21 g/L during the simultaneous saccharification and fermentation phase, which is 88% of the theoretical value.

Authorship contribution

Kazuaki Ninomiya: Conceptualization, Funding acquisition, Methodology, Supervision, Visualization, Writing – original draft, Writing – review & editing.

Tomoko Hashitani: Formal Analysis, Investigation, Visualization.

Funding

No funding was received.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

R.P. John, G.S. Anisha, K.M. Nampoothiri, A. Pandey, Micro and macroalgal biomass: A renewable source for bioethanol, Bioresour. Technol. 102 (2011) 186–193. https://doi.org/10.1016/j.biortech.2010.06.139.
G. Markou, I. Angelidaki, D. Georgakakis, Microalgal carbohydrates: an overview of the factors influencing carbohydrates production, and of main bioconversion technologies for production of biofuels, Appl. Microbiol. Biotechnol. 96 (2012) 631–645. https://doi.org/10.1007/s00253-012-4398-0.
S. Aikawa, S.H. Ho, A. Nakanishi, J.S. Chang, T. Hasunuma, A. Kondo, Improving polyglucan production in cyanobacteria and microalgae via cultivation design and metabolic engineering, Biotechnol. J. 10 (2015) 886–898. https://doi.org/10.1002/biot.201400344.
Q. Al Abdallah, B.T. Nixon, J.R. Fortwendel, The enzymatic conversion of major algal and cyanobacterial carbohydrates to bioethanol, Front. Energy Res. 4 (2016) 36. https://doi.org/10.3389/fenrg.2016.00036.
D.M. Arias, E. Ortíz-Sánchez, P.U. Okoye, H. Rodríguez-Rangel, A. Balbuena Ortega, A. Longoria, R. Domínguez-Espíndola, P.J. Sebastian, A review on cyanobacteria cultivation for carbohydrate-based biofuels: Cultivation aspects, polysaccharides accumulation strategies, and biofuels production scenarios, Sci. Total Environ. 794 (2021) 148636. https://doi.org/10.1016/j.scitotenv.2021.148636.
G.E. Lakatos, K. Ranglová, J.C. Manoel, T. Grivalský, J. Kopecký, J. Masojídek, Bioethanol production from microalgae polysaccharides, Folia Microbiol. (Praha). 64 (2019) 627–644. https://doi.org/10.1007/s12223-019-00732-0.
C.E. de Farias Silva, A. Bertucco, Bioethanol from microalgae and cyanobacteria: A review and technological outlook, Process Biochem. 51 (2016) 1833–1842. https://doi.org/10.1016/j.procbio.2016.02.016.
D. Deb, N. Mallick, P.B.S. Bhadoria, Analytical studies on carbohydrates of two cyanobacterial species for enhanced bioethanol production along with poly-β-hydroxybutyrate, C-phycocyanin, sodium copper chlorophyllin, and exopolysaccharides as co-products, J. Clean. Prod. 221 (2019) 695–709. https://doi.org/10.1016/j.jclepro.2019.02.254.
D. Deb, N. Mallick, P.B.S. Bhadoria, Engineering culture medium for enhanced carbohydrate accumulation in Anabaena variabilis to stimulate production of bioethanol and other high-value co-products under cyanobacterial refinery approach, Renew. Energy 163 (2021) 1786–1801. https://doi.org/10.1016/j.renene.2020.10.086.
G. Markou, I. Angelidaki, E. Nerantzis, D. Georgakakis, Bioethanol production by carbohydrate-enriched biomass of Arthrospira (Spirulina) platensis, Energies 6 (2013) 3937–3950. https://doi.org/10.3390/en6083937.
S. Maity, N. Mallick, Bioprospecting marine microalgae and cyanobacteria as alternative feedstocks for bioethanol production, Sustain. Chem. Pharm. 29 (2022) 100798. https://doi.org/10.1016/j.scp.2022.100798.
Y. Huang, X. Chen, S. Liu, J. Lu, Y. Shen, L. Li, L. Peng, J. Hong, Q. Zhang, I. Ostrovsky, Converting of nuisance cyanobacterial biomass to feedstock for bioethanol production by regulation of intracellular carbon flow: Killing two birds with one stone, Renew. Sustain. Energy Rev. 149 (2021) 111364. https://doi.org/10.1016/j.rser.2021.111364.
O.N. Tsolcha, V. Patrinou, C.N. Economou, M. Dourou, G. Aggelis, A.G. Tekerlekopoulou, Utilization of biomass derived from cyanobacteria-based agro-industrial wastewater treatment and raisin residue extract for bioethanol production, Water (Switzerland) 13 (2021). https://doi.org/10.3390/w13040486.
S.E. Karatay, Usage of thermophilic cyanobacterial biomass for bioethanol production, Environ. Prog. Sustain. Energy 34 (2015) 903–907. https://doi.org/10.1002/ep.12056.
T.J. Chow, H.Y. Su, T.Y. Tsai, H.H. Chou, T.M. Lee, J.S. Chang, Using recombinant cyanobacterium (Synechococcus elongatus) with increased carbohydrate productivity as feedstock for bioethanol production via separate hydrolysis and fermentation process, Bioresour. Technol. 184 (2015) 33–41. https://doi.org/10.1016/j.biortech.2014.10.065.
N. Chandra, N. Mallick, Co-production of bioethanol and commercially important exopolysaccharides from the marine cyanobacterium Synechococcus elongatus BDU 10144 in a novel low-cost seawater-fertilizer-based medium, Int. J. Energy Res. 46 (2022) 13487–13510. https://doi.org/10.1002/er.8069.
K.B. Möllers, D. Cannella, H. Jørgensen, N. Frigaard, Cyanobacterial biomass as carbohydrate and nutrient feedstock for bioethanol production by yeast fermentation, Biotechnol. Biofuels 7 (2014) 64. https://doi.org/10.1186/1754-6834-7-64.
A. Rempel, F. de Souza Sossella, A.C. Margarites, A.L. Astolfi, R.L.R. Steinmetz, A. Kunz, H. Treichel, L.M. Colla, Bioethanol from Spirulina platensis biomass and the use of residuals to produce biomethane: An energy efficient approach, Bioresour. Technol. 288 (2019). https://doi.org/10.1016/j.biortech.2019.121588.
S. Aikawa, A. Joseph, R. Yamada, Y. Izumi, T. Yamagishi, F. Matsuda, H. Kawai, J.S. Chang, T. Hasunuma, A. Kondo, Direct conversion of Spirulina to ethanol without pretreatment or enzymatic hydrolysis processes, Energy Environ. Sci. 6 (2013) 1844–1849. https://doi.org/10.1039/c3ee40305j.
S. Aikawa, K. Inokuma, S. Wakai, K. Sasaki, C. Ogino, J.S. Chang, T. Hasunuma, A. Kondo, Direct and highly productive conversion of cyanobacteria Arthrospira platensis to ethanol with CaCl2 addition, Biotechnol. Biofuels 11 (2018) 50. https://doi.org/10.1186/s13068-018-1050-y.
J. Yu, M. Liberton, P.F. Cliften, R.D. Head, J.M. Jacobs, R.D. Smith, D.W. Koppenaal, J.J. Brand, H.B. Pakrasi, Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO₂, Sci. Rep. 5 (2015) 5–7. https://doi.org/10.1038/srep08132.
J. Ungerer, P.C. Lin, H.Y. Chen, H.B. Pakrasi, Adjustments to photosystem stoichiometry and electron transfer proteins are key to the remarkably fast growth of the cyanobacterium Synechococcus elongatus UTEX 2973, MBio 9 (2018) e02327-17. https://doi.org/10.1128/mBio.02327-17.
K. Song, X. Tan, Y. Liang, X. Lu, The potential of Synechococcus elongatus UTEX 2973 for sugar feedstock production, Appl. Microbiol. Biotechnol. 100 (2016) 7865–7875. https://doi.org/10.1007/s00253-016-7510-z.
P.C. Lin, F. Zhang, H.B. Pakrasi, Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, Sci. Rep. 10 (2020) 390. https://doi.org/10.1038/s41598-019-57319-5.
L. Zhang, L. Chen, J. Diao, X. Song, M. Shi, W. Zhang, Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO2, Biotechnol. Biofuels 13 (2020) 82. https://doi.org/10.1186/s13068-020-01720-0.
H. Roh, J.S. Lee, H. Il Choi, Y.J. Sung, S.Y. Choi, H.M. Woo, S.J. Sim, Improved CO₂-derived polyhydroxybutyrate (PHB) production by engineering fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 for potential utilization of flue gas, Bioresour. Technol. 327 (2021) 124789. https://doi.org/10.1016/j.biortech.2021.124789.
S.Y. Lee, J.S. Lee, S.J. Sim, Cost-effective production of bioplastic polyhydroxybutyrate via introducing heterogeneous constitutive promoter and elevating acetyl-Coenzyme A pool of rapidly growing cyanobacteria, Bioresour. Technol. 394 (2024) 130297. https://doi.org/10.1016/j.biortech.2023.130297.
I. Yadav, A. Rautela, A. Gangwar, V. Kesari, A.K. Padhi, S. Kumar, Geranyl diphosphate synthase (CrtE) inhibition Using alendronate enhances isoprene production in recombinant Synechococcus elongatus UTEX 2973: A step towards isoprene biorefinery, Fermentation 9 (2023) 217. https://doi.org/10.3390/fermentation9030217.
I. Yadav, A. Rautela, A. Gangwar, L. Wagadre, S. Rawat, S. Kumar, Enhancement of isoprene production in engineered Synechococcus elongatus UTEX 2973 by metabolic pathway inhibition and machine learning-based optimization strategy, Bioresour. Technol. 387 (2023) 129677. https://doi.org/10.1016/j.biortech.2023.129677.
P.C. Lin, F. Zhang, H.B. Pakrasi, Enhanced limonene production in a fast-growing cyanobacterium through combinatorial metabolic engineering, Metab. Eng. Commun. 12 (2021) e00164. https://doi.org/10.1016/j.mec.2021.e00164.
B. Long, B. Fischer, Y. Zeng, Z. Amerigian, Q. Li, H. Bryant, M. Li, S.Y. Dai, J.S. Yuan, Machine learning-informed and synthetic biology-enabled semi-continuous algal cultivation to unleash renewable fuel productivity, Nat. Commun. 13 (2022) 1–11. https://doi.org/10.1038/s41467-021-27665-y.
C.J. Knoot, Y. Khatri, R.M. Hohlman, D.H. Sherman, H.B. Pakrasi, Engineered production of hapalindole alkaloids in the cyanobacterium Synechococcus sp. UTEX 2973, ACS Synth. Biol. 8 (2019) 1941–1951. https://doi.org/10.1021/acssynbio.9b00229.
Z.A. Dookeran, D.R. Nielsen, Systematic engineering of Synechococcus elongatus UTEX 2973 for photosynthetic production of l-lysine, cadaverine, and glutarate, ACS Synth. Biol. 10 (2021) 3561–3575. https://doi.org/10.1021/acssynbio.1c00492.
T. Sun, Z. Li, S. Li, L. Chen, W. Zhang, Exploring and validating key factors limiting cyanobacteria-based CO₂ bioconversion: Case study to maximize myo-inositol biosynthesis, Chem. Eng. J. 452 (2023) 139158. https://doi.org/10.1016/j.cej.2022.139158.
A. Rautela, I. Yadav, A. Gangwar, R. Chatterjee, S. Kumar, Photosynthetic production of α-farnesene by engineered Synechococcus elongatus UTEX 2973 from carbon dioxide, Bioresour. Technol. 396 (2024) 130432. https://doi.org/10.1016/j.biortech.2024.130432.
M.M. Allen, Simple conditions for growth of unicellular blue-green algae on plates, J. Phycol. 4 (1967) 1–4. https://doi.org/10.1111/j.1529-8817.1968.tb04667.x.
R. Sirohi, S.B. Ummalyma, N.A. Sagar, P. Sharma, M.K. Awasthi, P.C. Badgujar, A. Madhavan, R. Rajasekharan, R. Sindhu, S.J. Sim, A. Pandey, Strategies and advances in the pretreatment of microalgal biomass, J. Biotechnol. 341 (2021) 63–75. https://doi.org/10.1016/j.jbiotec.2021.09.010.
A. Agarwalla, J. Komandur, K. Mohanty, Current trends in the pretreatment of microalgal biomass for efficient and enhanced bioenergy production, Bioresour. Technol. 369 (2023) 128330. https://doi.org/10.1016/j.biortech.2022.128330.
G.S. Ha, M.M. El-Dalatony, M.B. Kurade, E.S. Salama, B. Basak, D. Kang, H.S. Roh, H. Lim, B.H. Jeon, Energy-efficient pretreatments for the enhanced conversion of microalgal biomass to biofuels, Bioresour. Technol. 309 (2020) 123333. https://doi.org/10.1016/j.biortech.2020.123333.
G.S. Ha, S. Saha, B. Basak, M.B. Kurade, G.U. Kim, M.K. Ji, Y. Ahn, E.S. Salama, S. Woong Chang, B.H. Jeon, High-throughput integrated pretreatment strategies to convert high-solid loading microalgae into high-concentration biofuels, Bioresour. Technol. 340 (2021) 125651. https://doi.org/10.1016/j.biortech.2021.125651.
S. Aikawa, Y. Izumi, F. Matsuda, T. Hasunuma, J.S. Chang, A. Kondo, Synergistic enhancement of glycogen production in Arthrospira platensis by optimization of light intensity and nitrate supply, Bioresour. Technol. 108 (2012) 211–215. https://doi.org/10.1016/j.biortech.2012.01.004.
F.G. Magro, A.C. Margarites, C.O. Reinehr, G.C. Gonçalves, G. Rodigheri, J.A.V. Costa, L.M. Colla, Spirulina platensis biomass composition is influenced by the light availability and harvest phase in raceway ponds, Environ. Technol. 39 (2018) 1868–1877. https://doi.org/10.1080/09593330.2017.1340352.

GraphicalAbstract.png

Microwave pretreatment of glycogen-containing cyanobacteria Synechococcus elongatus UTEX 2973 enhances enzymatic saccharification and fermentation for bioethanol production

Status:

Version 1

Abstract

Figures

Highlights

Introduction

Materials and Methods

Cyanobacteria, amylolytic enzymes and yeast

Preparation of yeast inoculum for fermentation

Culture of cyanobacterial biomass as feedstock

Pretreatment of cyanobacterial biomass

Saccharification and ethanol fermentation from pretreated cyanobacterial biomass

Analysis

Yeast cell concentration

Cyanobacterial cell concentration and glycogen content

Enzyme accessibility to glycogen in cyanobacteria

Glucose and ethanol concentrations during saccharification and fermentation

Results

Preparation of glycogen-containing S. elongatus UTEX 2973 biomass

Screening of pretreatment method of glycogen-containing S. elongatus UTEX 2973 for subsequent enzymatic saccharification

Microwave pretreatment of glycogen-containing S. elongatus UTEX 2973 for subsequent enzymatic saccharification

Saccharification and ethanol fermentation from microwave-pretreated glycogen-containing S. elongatus UTEX 2973

Discussion

Conclusions

Declarations

References

Supplementary Files

Status:

Version 1