Wenlu Song,Rui Huang,Hao Guo,Chunguang Yin,Chuanling Wang,Jun Cheng,*,Weijuan Yang

1 Department of Life Science and Engineering, Jining University, Jining 273155, China

2 The Electrical Engineering College, Guizhou University, Guiyang 550025, China

3 State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China

4 ShanDong Fanpu Analytic Co., Ltd, Jining 272000, China

Keywords:Chlorella pyrenoidosa Steam pretreatment Cell wall disruption Lipids extraction

ABSTRACT Steam pretreatment was employed to disrupt microalgal cells for lipids extraction.Effects of steam pretreatment on microstructure of microalgal cells were investigated through scanning electron microscopy(SEM)and transmission electron microscopy(TEM).Effect of treatment on lipid extraction was also studied.Microalgal cell walls were distorted after steam pretreatment due to the hydrolysis of organic macromolecules contained in cell wall.Maximum curvature was increased from 1.88 × 10-6 m-1 to 1.43 × 10-7 m-1 after treatment with the steam at 130 °C.The fractal dimension of microalgal cells increased from 1.25 to 1.30 after pretreatment for 15 min, and further increased to 1.47 when the pretreatment time was increased to 60 min.Increased steam pretreatment temperature and time enhanced the hydrolysis of organic macromolecules, and finally destroyed microalgal cell walls at pretreatment temperature of 130°C and pretreatment time of 60 min.Lipid extracted from wet microalgal was significantly increased (2.1-fold) after pretreatment.

1.Introduction

The concern regarding alternate sources of fossil fuel is mounting day-by-day due to the global warming and environmental pollution.Known as the third-generation biofuel, microalgae have an efficiency and ability in mitigating carbon dioxide emissions and produce oil with a high productivity which has a lot of potential applications in producing biofuel [1,2].Therefore, Microalgae are excellent candidates for biodiesel production because of their high growth rates, high cellular concentration of lipids, and potential advantages over other sources of biofuel[3,4].However,the industrial performance of biofuel production from microalgae is still challenged with the difficulty in lipids extraction and microalgae cultivation [5,6].One of the prominent challenges is to develop a cost-effective method to extract lipid from microalgae biomass.

To extract lipid using microalgae by traditional technique,microalgae need to be dried.Due to its high water content, drying wet microalgae requires a large amount of energy[7].On the other hand, when extracting lipid from microalgae cells with solvents,the hard and dense cell wall hinders the extraction of the lipid[8].Thus, it is necessary to find an efficient way of disrupting the cell walls of microalgae.At present, the methods of rupturing microalgal cell walls fall into two categories:chemical and physical[9].The chemical method resorts to solvents such as sodium hydroxide, sulfuric acid, nitric acid and hydrogen peroxide.On the other hand, the physical method makes use of microwave,ultrasonic wave, hydrodynamic cavitation, or high-pressure homogenization [10-13].The physical methods can destroy the cell wall without the need for solvents.However, compared with physical methods, chemical methods consume less energy but demand huge amount of chemical reagents.Moreover,the solvents may interact with the lipid,reducing its quality and extraction efficiency.The processes involved in physical methods are simple,and the lipid do not require post-extraction processing after destroying the cell wall.However, the high energy consumption in physical methods needs to be reduced.

Steam pretreatment has been widely used in biomass fermentation [14,15].It breaks down cellulose macromolecule, and it enhances the efficiency of producing hydrogen and methane by biomass fermentation.Steam was employed to pretreatment Nannochloropsis oculate for lipid extraction.A high lipid yield of 29.34%was reported at a temperature of 120 °C, pressure of 103.43 kPa and a time interval of 30 min.Steam pretreatment of Nannochloropsis oculata biomass was shown to increase the lipid yields effectively[8].Lee et al.[16]also achieved high percentage of lipid recovery using steam pretreatment.This indicated the high thermal stress during steam pretreatment ruptured microalgae cell and released the intracellular lipids of microalgae.However, how the steam pretreatment affects the microstructure of microalgae was not investigated.

The present study was carried out to investigate the effect of steam pretreatment on the cell walls of microalgae using transmission electron microscope (TEM)and scanning electron microscope(SEM).The dynamic microstructures and fractal dimensional changes of wet microalgal cell treated by steam are reported for the first time.The effects of pretreatment temperature and duration of steam pretreatment were comprehensively investigated.

2.Materials and Methods

2.1.Materials

The microalgae (Chlorella pyrenoidosa) used in the experiments were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan.The microalgae were cultivated in Brostol’s medium containing NaNO3(0.25 g·L-1), K2HPO4·3H2O(0.075 g·L-1), MgSO4·7H2O (0.075 g·L-1), CaCl2·2H2O (0.025 g·L-1),KH2PO4(0.175 g·L-1), NaCl (0.025 g·L-1), FeCl3·6H2O (0.005 g·L-1),A5(1ml·L-1),soil extract(40ml·L-1),Feethylenediaminetetraacetic acid (EDTA) (1 ml·L-1), and distilled water (958 ml·L-1).The soil extract was the supernatant of mud(0.5 kg) and deionized water (1 L) after being boiled for 2 h.The A5 solution contained H3BO3(2.86 g·L-1),MnCl2·4H2O(1.81 g·L-1),ZnSO4·4H2O (0.22 g·L-1), CuSO4·5H2O (79 mg·L-1), and (NH4)6Mo7O24·4H2O (39 mg·L-1).Fe-EDTA solution contained Na2EDTA(10 g·L-1), FeCl3·6H2O (0.81 g·L-1), and HCl (0.1 N; 500 ml·L-1).The microalgae were cultivated in a 2 L bioreactor with an air pump for 15 d.Illumination(2500 lux)was supplied at the surface of the bioreactors with 12/12 h of dark/light cycle.After centrifugation,microalgae slurry with biomass density of 100 g·L-1was harvested [17].

2.2.Steam pretreatment of chlorella cells

The algae slurry of biomass density 100 g·L-1(2 ml) was injected into a 10-ml centrifuge tube and the tube with algae slurry was placed in a high-pressure sterilization pot and subjected to set temperatures for various durations.On cooling to room temperature, 3 ml methyl alcohol and 3 ml of chloroform were added to the tube contents, and the tube was shaken for 5 min.Then, the mixture was centrifuged, and the lower chloroform layer was pipetted into a weighing bottle.To dry the chloroform, the weighing bottle was placed in a drying oven at 60°C for 8 h,resulting in the appearance of the Chlorella bio-oil.

Wet microalgae(10 ml)were lyophilized to determine the lipid content of microalgae.The lyophilized microalgal biomass was extracted twice with the Bligh and Dyer method at room temperature to completely extract lipids from the microalgal biomass[18].

Next,the extracted lipids were converted into fatty acid methyl ester (FAME) for gas chromatography (GC) quantification [19].Firstly,5 ml of KOH-methanol solution(1 mol·L-1KOH)was added and shaken for 5 min.Then,10 ml of sulfuric acid-methanol solution(8% sulfuric acid) was added and heated at 75 °C for 1 h.Thirdly,15 ml of biological lipid obtained by extracting and converting hexyl hydride was added, and the solution was centrifuged at 5000 r·min-1for 3 min to delaminate.Biodiesel was obtained by drying the top layer of liquid.Quantitative analysis was carried out on the biodiesel dissolved in hexyl hydride-containing internal standard.

Lipid recovery was evaluated using the equation below:

Where mwetis the mass of FAMEs converted from lipids extracted from wet microalgal biomass;mlyophilizedis the mass of FAMEs converted from lipids extracted from lyophilized micoralgal biomass.

2.3.Analyses and tests

Transmission electron microscope(TEM,Hitachi H-7650,Japan)and scanning electron microscope(SEM,KYKY-EM3200)were used to observe the cell wall of microalgae and its inner structural changes[19].The preparation of microalgae samples for the imaging experiments included 4 steps.First,double fixation.The sample was first fixed with 2.5% glutaraldehyde in phosphate buffer(pH=7.0)for more than 4 h;washed three times in the phosphate buffer;then post-fixed with 1%OsO4in phosphate buffer(pH=7.0)for 1 h and washed three times in the phosphate buffer.Second,dehydration.The sample was first dehydrated by a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for about 15-20 min at each step, and then transferred to absolute acetone for 20 min.Thirdly,infiltration.The sample was placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 h at room temperature,and then transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3 h and to final Spurr resin mixture for overnight.Last, embedding and ultrathin sectioning.The sample was placed in capsules contained embedding medium and heated at 70 °C for about 9 h.The specimen sections were stained by uranyl acetate and alkaline lead citrate for 15 min respectively and observed.Quantitative analysis was performed to detect the components of fatty acid methyl esters of the lipid extracts with a gas chromatograph (GC, Agilent 7890A, US)equipped with HP-INNOWAX column.Helium was used as a carrier gas at a flow rate set at 1.5 ml·min-1.The temperature increase program was:150°C for 1 min,ramped up to 200°C at a ramp rate of 15 °C·min-1, then ramped up to 250 °C at a ramp rate of 2 °C·min-1, and maintained for 5 min at 250 °C.

3.Results and Discussion

3.1.Effect of steam pretreatment temperature on microalgae lipid extraction

As shown in Fig.1,without steam pretreatment,the lipid recovery was only 37.36%, but with steam pretreatment, it increased to 65.75%, 76.31% and 81.55% at 110, 120 and 130 °C, respectively.Thus, the lipid recovery increased significantly with increase in temperature.It can be speculated that steaming pretreatment elevates lipid extraction efficiency by breaking down the cell walls.Due to the interactions, such as hydrogen bond, between lipid molecule and other organic macromolecules, the lipid recovery of 81.55%was achieved even after cell wall disruption[20].The lipid recovery was slightly increased from 76.31% to 81.55% when the pretreatment temperature increased from 120 °C to 130 °C.The increase rate of lipid recovery was decreased.However, the increase of temperature would significantly increase the energy consumption of steam pretreatment, 130 °C was selected as the optimal treatment temperature.

3.2.Influence of steam pretreatment temperature on the structure of microalgae cell walls

Fig.1.Lipid recovery from Chlorella under steam pretreatment at different temperatures.

As shown in Fig.2, the control cells had regular shapes.After steam pretreatment, the microalgae cells were distorted and the area of intracellular shadows was reduced.Moreover, as the processing temperature rose, the cell distortion became more severe,lots of small fragments appeared,and the intracellular shadow area was further reduced.The structure of a single cell was observed at high magnification(Fig.3).As temperature was increased, the cell became distorted,with thinning cell wall and shrinking inner shadow.Dissolution of intracellular substances was observed at 120 °C, and the dissolution was increased at 130 °C.These results suggest that steam pretreatment might decompose macromolecules such as carbohydrates within the cell and decrease the cell inner shadow area.As shown in the Fig.4, the maximum curvature was increased from 1.88 × 10-6m-1to 1.43 × 10-7m-1after treatment with the steam at 130°C.This indicated the rigidity of cell wall was decreased after the steam pretreatment.This can be explained by the following reasons.The cell wall is mainly composed of cellulose, hemicellulose and lignin [21].The cellulose molecules formed microfibril aggregates that are embedded in the soft matrix of hemicellulose and lignin.Steam destroyed the physical structure of densely crosslinked cellulose, hemicellulose and lignin in lignocellulose and exposed cellulose.Steam pretreatment changed the chemical composition of the cell wall,especially cellulose and hemicellulose.The dense physical structure of cellulose after steam pretreatment disappeared indicating that the steam destroys the hydrogen bond network between the cellulose chains, causing the cellulose chains to be arranged in disorder.In addition, steam treatment results in a high degree of degradation of the hemicellulose of the microalgae, releasing acetic acid and formic acid [22].The produced acid and high temperature promoted the hydrolysis of hemicellulose, which broke the cellulose units of cellulose and hemicellulose by β-(1,4) glycosidic bonds.The depolymerization reaction toke place in the amorphous region.The β-aryl ether bond of lining is partially depolymerized by steam,causing partial lignin to also decompose [23,24].Thus, the steam pretreatment may contribute to hydrolysis of cell wall macromolecules such as pectin and cellulose, making the cell wall thinner and distorted.The hydrolysis of these macromolecules increased as temperature rose, with appearance of cracks in the cell walls, and cytoplasmic dissolution at 130 °C.

Fig.2.Cell structure under TEM after steam pretreatment at different temperatures:(a)Untreated cells,(b)Cell structure under TEM after steam pretreatment at 110°C,(c)Cell structure under TEM after steam pretreatment at120°C, (d) Cell structure under TEM after steam pretreatment at 130 °C.

Fig.3.Single cell structure under TEM after steam pretreatment at different temperatures: (a) Untreated Single cell, (b) Single cell structure under TEM after steam pretreatment at 110 °C, (c) Single cell structure under TEM after steam pretreatment at 120 °C, (d) Single cell structure under TEM after steam pretreatment at 130 °C.

Fig.4.The maximum curvature of microalgae cell after steam pretreatment at different temperatures.

After steam pretreatment, the cell wall of microalgae was hydrolyzed, resulting in the destruction of the cell wall and cell membrane structure.The cell matrix was then released from microalgae cell.The released cell matrix significantly facilitated the subsequent lipid extraction.As a result, the lipid extraction was promoted by the steam pretreatment.

3.3.The influence of steaming pretreatment duration on the extraction of chlorella lipid

Fig.5.Lipid recovery from Chlorella cells at different durations of steam treatment.

As shown in Fig.5, the lipid recovery rose as the duration of steaming treatment increased.With 15-min steam treatment,lipid recovery increased from 37.36% to 52.64%.This is mainly because steaming hydrolyzed the macromolecules,thereby breaking the cell structure, which facilitated the solvent extraction of the lipid.When treated for 60 min, the extraction increased to 81.55%.However, further temperature increases aggravated the hydrolysis of the macromolecules and destroyed the cell walls.At 60 min of steaming, the lipid tended to dissolve into the solvent more easily, increasing its extractability.The extraction rate decreased after a certain point of steaming, because with already broken cell walls, excessive steaming doesn’t increase the lipid recovery.

2 ml of algae slurry was heated to 130 °C in a 10 ml chamber under optimal conditions.Due to the low heat capacity of microalgal biomass, the energy absorbed by biomass was ignored.The energy needed to heat 2 ml of water at ambient temperature and pressure to become saturated steam at the temperature of 130 °C with a specific volume of 0.005 m3·kg-1.Thus, the energy consumption for steam pretreatment was calculated using the following formula:

where 2 g is the mass of water,559.19 kJ·kg-1is the enthalpy of the saturated steam at the temperature of 130°C with a specific volume of 0.005 m3·kg-1, 84 kJ·kg-1is the enthalpy of water at ambient temperature and pressure, and 0.5 is the heat transfer efficiency.As 2 ml of wet microalgal biomass contained 0.2 g of dried microalgal biomass,the specific energy requirement of steam pretreatment was 9.5 MJ·kg-1.This value is significantly lower than the specific energy of physical method (33-529 MJ·kg-1) reviewed by Lee et al.[25].

According to the previous works,microwave treatment at 80°C for 10 min was shown to achieve 91.67%of lipid recovery[26].The ultrasonic treatment under 500 W for 30 min was shown to achieve 60.43%of lipid recovery[27].The lipid recovery of 81.55%for steam pretreatment was significantly higher than the ultrasonic treatment.Although, the lipid recovery of steam pretreatment was slightly lower than the microwave treatment,the energy consumptions of steam pretreatment was significantly lower than that for microwave treatment.Meanwhile, the steam pretreatment was easier to scale up compared with the microwave treatment.

3.4.Influence of steaming pretreatment time on the microstructure of chlorella cells

Fig.6.SEM image of Chlorella cells at different stages of steaming: (a) Untreated Chlorella cells, (b) SEM image of Chlorella cells after steam treatment for 15 min,(c) SEM image of Chlorella cells after steam treatment for 30 min, (d) SEM image of Chlorella cells after steam treatment for 45 min, (e) SEM image of Chlorella cells after steam treatment for 60 min.

To examine the microstructural changes in Chlorella cells after steam treatment for different durations (15, 30, 45 and 60 min),SEM was used to obtain pictures of the cell structures.In the untreated cells, the cell surfaces were smooth, and the cells showed complete spherical shapes (Fig.6a).Cells steamed for 5 min had wrinkled surfaces, with evidence of cellular distortion(Fig.6b).As treatment time was extended, the wrinkles became aggravated and cracks could be detected in the cell surfaces(Fig.6c, 6d and 6e).Typical single cell structures were observed under TEM and shown in Fig.7.The structural changes were much clearer, with gradual thinning out of the cell wall until it finally became broken.This may be due to gradual hydrolysis of the cell wall macromolecules, leading to distortion and fracture.Furthermore, fractal dimension was adopted to quantitatively analyze the structural changes in the SEM images.

Fractal dimension was employed to quantify the changes of the microalgal cells.SEM images of the cells were initially binarized with MATLAB and then calculated using the following formula:

where d denotes the fractal dimension, ε denotes the length of a grid side, and N denotes the number of grids that cover the fractal[26].The fractal dimension changes of exploded cells at different preheat times are shown in Fig.8.The fractal dimension increased from 1.25 in untreated cell to 1.47 in cells subjected to 60-min steam treatment.This dimension increase indicates that the degree of damage to the microalgal cells increased as the preheat time was prolonged.This can be explained by the following reasons.Steam pretreatment at prolonged time resulted in severe cellulose degradation and structural changes in cellulose during steam explosions,which can greatly reduce the crystallinity index and degree of polymerization of cellulose,resulting in surface morphology of microalgae [28].

Fig.8.Fractal dimension changes in chlorella cells at different durations of steam treatment.

4.Conclusions

Energy-friendly steam pretreatment is a highly efficient and cost-effective process to disrupt microalgal cells for lipid extraction.Steam pretreatment can hydrolyze cell wall macromolecules of Chlorella, thereby disrupting microlagal cells wall and facilitating lipid extraction.Maximum curvature was increased from 1.88 × 10-6m-1to 1.43 × 10-7m-1after treatment with the steam at 130 °C.Fractal dimension of microalgal cells increased from 1.25 to 1.47 when the pretreatment time was increased to 60 min.simultaneously,the lipid recovery also rose 2.1-folds after steam pretreatment, relative to untreated Chlorella.Steam pretreatment is a promising process to produce biodiesel from microalgae on a large scale.

Fig.7.TEM image of Single Chlorella cell at different stages of steaming:(a)Single cell structure under TEM after steam treatment for 15 min,(b)Single cell structure under TEM after steam treatment for 30 min,(c)Single cell structure under TEM after steam pretreatment for 45 min,(d)Single cell structure under TEM after steam pretreatment for 60 min.

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.

Acknowledgements

This study was supported by the National Key Research and Development Program-China (2017YFE0122800), Shandong Provincial Natural Science Foundation (ZR2019MC060), Key Research and Development Program of Jining City(2018ZDGH024),and a Project of Shandong Province Higher Educational Science and Technology Program (J17KA095).