Sequential generation of hydrogen and lipids from starch by combination of dark fermentation and microalgal cultivation

Abstract

Dark fermentative hydrogen production and microalgal lipid production was successfully combined to enhance the energy conversion from starch with simultaneous treatment of volatile fatty acids in the effluent. This study provided a promising and sustainable route for efficient bioenergy production by the process of sequential dark fermentation and microalgal cultivation.

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In the worldwide energy crisis and environmental pollution, renewable energy sources are in high demand to decrease the greenhouse effect and provide alternative options.^1–3 Hydrogen has been recognized as an ideal alternative energy carrier of the future since it has high energy density and doesn't produce pollutants such as CO₂ and CO.⁴ In industry, hydrogen can be produced by a variety of methods, including coal gasification, steam reforming of natural gas and electrolysis of water.⁵ However, these methods still depend on fossil fuels as a source and are not always environment friendly.

Hydrogen production through biological routes is considered more sustainable compared to the traditional methods.⁶ Among various bio-hydrogen production means, dark fermentative hydrogen production has attracted worldwide attention since the approach could allow simultaneous waste reduction and hydrogen production.⁶ To enhance the commercial viability of bio-hydrogen production, the low-cost and easily available raw materials are critical. Starch is widely distributed in the world, and it has been proven to have high potential for hydrogen production.^7–9 However, the main challenges of this technology comes from the accumulation of volatile fatty acids (VFAs), which could induce the cessation of fermentation and result in low substrate conversion efficiency.^10–12 Moreover, the VFAs in effluent might become a potential threat to the aquatic environment and should be treated before discharge.

Combination of dark fermentation and microalgal cultivation may be a promising strategy to solve above problems. Microalgal biomass can be produced by using organic compounds as energy and carbon sources.^13,14 Several studies have reported that simple dissolved organics (e.g., short-chain fatty acids) can be beneficial to the growth and lipid synthesis of microalgae.^15–17 The effluent from dark fermentation containing abundant VFAs and could serve as an alternative feedstock for microalgal cultivation and subsequently lipid accumulation. However, up to now, the information about the combination of dark fermentation and algal culture for hydrogen production and lipid accumulation using starch is quite limited.

In this study, a combined process of dark fermentation and algal cultivation was employed to convert starch into clean bioenergy. Various types of starch were used to produce hydrogen in dark fermentation, effluents of which were further utilized in the algal lipid production process. The growth, substrate utilization and biochemical compositions of microalgae were investigated. Moreover, the energy conversion efficiency of the combined system was evaluated.

Four types of starch, namely, cassava starch (CAS), corn starch (COS), sweet potato starch (SPS) and potato starch (PS), were used to investigate the influence of starch source on hydrogen production by dark fermentation. Fig. 1A exhibited the variation of cumulative hydrogen with time course. Hydrogen production ceased after 48 h of operation for all the tests. The short fermentation time probably attributed to the product-induced inhibition.¹⁸ The highest cumulative hydrogen was obtained from SPS (994.5 mL L⁻¹ culture) and the lowest was from PS (755.5 mL L⁻¹ culture). The hydrogen production from COS (883.3 mL L⁻¹ culture) was close to that of CAS (827.8 mL L⁻¹ culture). It was worth noting that the accumulative hydrogen achieved in the present study was comparable or even higher than those reported in relevant studies.¹⁹

Fig. 1 (A) Cumulative hydrogen production profiles with various types of starch. (B) Effect of starch type on the yield and production rate of hydrogen.

The yield and production rate of hydrogen were also crucial criteria in comparing the hydrogen production potential of various starch sources. Fig. 1B showed the hydrogen yields and production rates in dark fermentation process. SPS led to the highest hydrogen yield of 198.9 mL g⁻¹ starch and hydrogen production rate of 20.7 mL L⁻¹ h⁻¹, followed by COS (176.7 mL g⁻¹ starch and 18.4 mL L⁻¹ h⁻¹) and CAS (165.6 mL g⁻¹ starch and 17.2 mL L⁻¹ h⁻¹). The lowest hydrogen yield of 151.1 mL g⁻¹ starch and hydrogen production rate of 15.7 mL L⁻¹ h⁻¹ were both obtained when PS was used as the substrate. Previous studies showed that the highest hydrogen yield were 92–194 mL g⁻¹ starch by dark fermentation of starch.^20–22 Notably, hydrogen yields of this study can be compared to the yields obtained in the literature.

However, the highest hydrogen yield obtained with SPS was still below the maximum theoretical yield, which might be due to the formation of a mixture of VFAs. Fig. 2 presented the soluble metabolite composition obtained from different types of starch. Butyrate (680.2–792.1 mg L⁻¹) and acetate (591.7–759.6 mg L⁻¹) appeared to be the major soluble metabolites. Besides, a small amount of ethanol (56.4–76.7 mg L⁻¹) and propionate (10.1–20.2 mg L⁻¹) were observed. The high butyrate concentration indicated that the reaction was a butyrate fermentation type which has been shown as accompanying with high hydrogen production from starch.²³

Fig. 2 Soluble metabolites concentration at the end of tests: (A) dark fermentation. (B) Combined system.

Nevertheless, high VFAs concentrations in the effluents, which could contribute to the high COD and give off an unpleasant odor, might become a potential threat to the environment.²⁴ Thus, in this work, the effluents of dark fermentation were further treated by the microalgae to convert the soluble metabolites into bioenergy.

Microalgae can grow on the effluents of all the starch but their biomass concentration and lipid production exhibited great difference (Fig. 3A and B). SPS effluent was the best substrate with the maximum biomass concentration (1.27 g L⁻¹), total lipid content (39.8%) and lipid productivity (84.2 mg L⁻¹ d⁻¹). COS effluent and CAS effluent were also acceptable feedstock which showed lower biomass concentration (1.13 and 1.09 g L⁻¹), total lipid content (36.4% and 32.3%) and lipid productivity (68.6 and 58.7 mg L⁻¹ d⁻¹), respectively. In addition, the worst growth and lipid accumulation occurred with the PS effluent, and the biomass concentration, total lipid content and lipid productivity were 1.02 g L⁻¹, 30.1% and 51.2 mg L⁻¹ d⁻¹, respectively. These results indicated that the microalgae can not only proliferate in the effluents of starch, but also accumulate high amount of lipids in the microalgal cells.

Fig. 3 (A) Growth profiles of microalgae with different starch effluents. (B) Effect of effluent type on the total lipid content and lipid productivity.

The concentrations of soluble metabolites in final effluents were monitored to investigate the mechanism of substrate utilization. It was found that only a little ethanol (54.3–70.3 mg L⁻¹) can be detected at the end of cultivation (Fig. 2). The residual ethanol in effluent could be further recovered by the pervaporation separation to produce highly concentrated ethanol.^25–27 Almost all the VFAs (acetate, propionate and butyrate) were consumed by the microalgae and the final VFAs concentrations in effluents were below 20 mg L⁻¹. Acetate had low molecular weight, easy degradability and simple structure which enabled its entry into metabolic routes.²⁸ Thus, acetate was a suitable substrate for the growth and lipid synthesis of microalgae. Butyrate and propionate had higher molecular weight and required more steps for conversion to acetyl Co-A.²⁸ Griffiths et al. reported that butyrate could inhibit the growth of Chlorella vulgaris.²⁹ In this study, all the VFAs in effluents were efficiently utilized by the algal cells, which implied that Scenedesmus sp. could be a potential candidate to treat the fatty acid rich wastewater.

Microalgal biomass was mainly composed of lipids, carbohydrates and proteins.³⁰ Algal biochemical composition can be affected by the nutrients constitution and concentration of the culture medium.³¹ The biochemical compositions of microalgae cultured in dark fermentative effluents were investigated and presented in Fig. 4. The protein content ranged from 21.6% to 29.2%, and lipid content ranged from 30.1% to 39.8%. Compared to the change of proteins and lipids, the variation of carbohydrates was slighter (13.7–16.4%). It was interesting to observe that the sum of protein content and lipid content in all tests had no significant difference (57.4–61.4%). This implied that the process of lipid synthesis in algal cells could be related to the metabolism of proteins, and the intracellular proteins might be converted into lipids under nutrients starvation or stress conditions.^32,33

Fig. 4 Biochemical compositions of microalgae with different starch effluents.

The energy conversion efficiency (ECE) and total energy conversion efficiency (TECE) were calculated based on eqn (1) and (2), respectively.

The specific heat values of hydrogen and lipids were 142 and 36.3 kJ g⁻¹, respectively.^34,35 In dark fermentation process, the ECE from CAS, COS, SPS and PS were 13.93%, 15.56%, 16.72% and 13.56%, respectively (Table 1). Energy conversion was markedly improved in the combined system, and the TECE from CAS, COS, SPS and PS reached 30.79%, 36.23%, 41.27% and 29.48%, respectively. The aforementioned results clearly indicated the feasibility and effectiveness of using the proposed combined system of dark fermentation and microalgal cultivation for energy production and fatty acids removal. Compared to dark fermentation alone, the combined system produced 117–147% higher of bioenergy. Most fatty acids in the effluent from dark fermentation could be consumed in the microalgal cultivation process. Thus, this combined system can be deemed as a more efficient and environmentally sustainable process and it provided a dual benefit of simultaneous bioenergy production and waste treatment.

Table 1 Energy conversion efficiencies with various types of starch

Starch type	H2 production			Lipid accumulation			TECE (%)
	H2 (mL L^−1)	HV (kJ)	ECE (%)	Total lipids (g L^−1)	HV (kJ)	ECE (%)
CAS	827.8	10.5	13.93	0.35	12.71	16.86	30.79
COS	883.3	11.2	15.56	0.41	14.88	20.67	36.23
SPS	994.5	12.61	16.72	0.51	18.51	24.55	41.27
PS	755.5	9.58	13.56	0.31	11.25	15.92	29.48

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Conclusions

Bacterial hydrogen production and microalgal cultivation were successfully combined to convert starch into bioenergy. More than 90% of soluble metabolites (mainly acetate and butyrate) produced by hydrogen production process can be consumed in the algal lipid accumulation process, which reduced the potential threat of dark fermentative effluent to the environment. Compared with dark fermentation, the combined system can significantly improve the energy conversion efficiency. This research indicated the viability and potential of utilizing the combined dark fermentation and algal cultivation for efficient energy production at the expense of cheap raw feedstock like starch.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 51478139), the Fundamental Research Founds for Central Universities (No. HIT.BRETIII.201418), the Harbin Innovation Talents Funding of Science and Technology (No. 2014RFQXJ084), the State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2015TS02), and the Shanghai Tongji Gao Tingyao Environmental Science & Technology Development Foundation (STGEF).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra15023j

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