Production of biodiesel from CO2 and organic wastes by fermentation and black soldier fly

Journal Pre-proof Production of biodiesel from CO2 and organic wastes by fermentation and black soldier fly

Wancheng Pang, Dejia Hou, Jingwen Ke, Jiangshan Chen, Mark T. Holtzapple, Jeffery K. Tomberlin, Huanchun Chen, Jibin Zhang, Qing Li PII:

S0960-1481(19)31590-3

DOI:

https://doi.org/10.1016/j.renene.2019.10.099

Reference:

RENE 12468

To appear in:

Renewable Energy

Received Date:

04 April 2019

Accepted Date:

18 October 2019

Please cite this article as: Wancheng Pang, Dejia Hou, Jingwen Ke, Jiangshan Chen, Mark T. Holtzapple, Jeffery K. Tomberlin, Huanchun Chen, Jibin Zhang, Qing Li, Production of biodiesel from CO2 and organic wastes by fermentation and black soldier fly, Renewable Energy (2019), https://doi.org/10.1016/j.renene.2019.10.099

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Journal Pre-proof Production of biodiesel from CO2 and organic wastes by fermentation and black soldier fly Wancheng Panga,#, Dejia Houa,#, Jingwen Kea, Jiangshan Chena, Mark T. Holtzappleb, Jeffery K. Tomberlinc, Huanchun Chena, Jibin Zhangd, Qing Li*,a

a State Key Laboratory of Agricultural Microbiology, College of Science, Huazhong Agricultural University, Wuhan 430070, China b Department of Chemical Engineering, Texas A&M University, 77843 College Station, USA c Department of Entomology, Texas A&M University, 77843 College Station, USA d State Key Laboratory of Agricultural Microbiology, National Engineering Research Center of Microbial Pesticides, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

*Corresponding

authors. Tel.: +86 27 87280802; fax: +86 27 87393882

E-mail addresses: [email protected]. #Wancheng

Pang and Dejia Hou contributed equally to this work and should be

considered co-first authors. 1

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Graphical Abstract

Conversion of CO2 into VFAs and then further into lipids through an anthropogenic carbon cycle.

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Journal Pre-proof Abstract: This study aims to explore the feasibility of transforming CO2 into lipids for biodiesel by integrating anaerobic fermentation and black soldier fly larvae (BSFL) bioconversion. The optimal conditions for CO2 and biowaste fermentation into volatile fatty acids (VFAs) were examined under different pH conditions. Moreover, we also explored the effects of different concentrations of VFAs solutions on BSFL lipid accumulation. The results showed that the optimal pH was 7 for the production of VFAs, and the maximum acetate and butyrate concentrations were 10.11 and 3.56 g/L, respectively. Feeding BSFL with this fermentation broth increased their lipid content by 26.30% compared with the control. Moreover, the optimum concentration of VFAs solution was 26 g/L to enhance the lipid content of BSFL. This work provides an innovative route of great potential to reduce the cost and energy consumption to fix CO2 in an anthropogenic carbon cycle: the VFAs from CO2 and organic waste do not need to be extracted from fermentation broth, and can be directly used for lipid accumulation in BSFL for biodiesel. Key Words: CO2; organic wastes; black soldier fly; fatty acid; biodiesel;

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1. Introduction The development of highly industrialized world economy has resulted in an enormous burden on conventional energy resources and the environment.[1] In fact, the worldwide use of non-renewable energy such as oil is expected to continue to grow in the coming decades.[2] Meanwhile, the excessive use of fossil fuels has led to emissions of large amounts of nitrogen oxide, smoke, particle matter, carbon monoxide and carbon dioxide (CO2).[3] Therefore, concerns of climate change and the decline in non-renewable fossil resources have motivated the development of renewable and environment-friendly energy, such as biodiesel, which can be an alternative to petrodiesel owing to its high combustion efficiency, renewability, lower emission of pollutants, and complete compatibility with existing diesel engines.[4] Cost analysis showed that 70–85% of biodiesel cost is from the feedstock, primarily cereal and oil crops.[5] However, increasing food prices have rekindled the debate over the competition for agricultural resources between the energy sector and the food industry. Use of limited food resources for biodiesel is obviously not a feasible option for developing countries such as China, as these resources are better used for human consumption. Therefore, many scientists are seeking new approaches to turn nonedible, low-cost and ecofriendly feedstocks into biodiesel. On the other hand, increasing global population together with enhanced living standards has led to significant increases in the production of organic wastes,[6] which results in environmental pollution and poses great threats to human health.[7] The bioconversion of these wastes to valuable products seems to be an attractive solution to address both the issues of organic waste management and energy scarcity concomitantly. As a natural process, bioconversion of these wastes with insects, such as black soldier fly larvae (BSFL, Hermetia illucens), can be applied to transform 4

Journal Pre-proof organic wastes into higher value products. Furthermore, BSFL are often used as the feedstock for biodiesel as they do not require the consumption of food resources and only need small occupation of land. Hence, BSFL are considered as an important potential source of next-generation biodiesel. Many studies have explored the applications of various low-cost organic substrates (e.g., animal manure, restaurant waste, and plant debris) as resources to rear BSFL.[8-9] In fact, the properties of the biodiesel derived from the larval fat meet the European biodiesel standard EN 14214.[10] The advantages of BSFL in the treatment of organic waste have aroused the interests in developing BSFL feedstock for biodiesel. But emissions of CO2 are still inevitable in the BSFL biowaste treatment process, which can cause the loss of carbon. To further reduce greenhouse gas emissions and increase larval biomass, anaerobic fermentation technology provides an effective solution to combine CO2 fixation and BSFL waste treatment for biofuel production. To date, many studies have been carried out to improve the production of volatile fatty acids (VFAs) from organic wastes by syngas anaerobic fermentation; but few studies have explored the VFAs production from fermentation broth for subsequent application.[11-13] Besides, as the concentration of VFAs produced by anaerobic fermentation is relatively low and the components are complex, the cost of conventional extraction and separation remains high. To the best of our knowledge, little information is available on the utilization of these VFAs as the organic carbon sources to feed BSFL. Therefore, we attempted to develop a new route for the subsequent processing of VFAs fermentation broth, namely the accumulation of fat from VFAs in BSFL. We used VFAs fermentation broth from CO2 and agricultural wastes to feed BSFL directly without additional treatments, which could further reduce energy consumption and cost. This experiment used pig 5

Journal Pre-proof manure and rice straw as the sources of carbon and nitrogen for bio-fermentation and H2 was used to reduce CO2 under normal temperature and pressure. In summary, on the basis of common anaerobic fermentation, our experiments introduced a carbon source through the fixation of CO2, further increasing the content of VFAs in the product and making it more applicable for industrialization. To improve the feasibility of converting CO2 into biofuel both technically and economically, we intended to develop a bio-engineering system by integrating CO2 fixation and BSFL. Accordingly, the objectives of this work were to: (1) develop a method for CO2 fixation via anaerobic fermentation of agricultural wastes with H2 as the reducing agent into VFAs; (2) bio-convert the VFAs into lipids through BSFL; and (3) extract the grease from dried BSFL for biodiesel, and the BSFL residue may be used as protein for animal feed. This work provides a new perspective to fix CO2 for biofuel by microbe and BSFL using easily available and potentially polluted agricultural organic wastes as the substrate, which seems to be a promising method to help the mitigation of climate changes and conservation of fossil resources.

2. Materials and methods

CO2/H2

Pig manure

Volatile fatty acids

Feeding black Soldier fly

Rice straw Fig.1. Schematic diagram of experimental flow chart

6

Extract lipids for biodiesel

Journal Pre-proof The entire experimental process of this study is shown in Fig. 1. With agricultural wastes as the substrate and H2 as the reducing agent, CO2 was reduced into VFAs, and then the VFAs were converted into lipids by BSFL, which can be used for biodiesel. 2.1. Conversion of CO2 to VFAs 2.1.1. Inoculum and raw materials Sludge derived from Qinghai Lake, China was used directly as the inoculum. The fresh pig manure sample was obtained from the National Pig Breeding Experimental Center at Huazhong Agricultural University (HZAU), Wuhan, China. The rice straw was from the experimental field of HZAU. The main properties are shown in Table 1. Hermetia illucens has been maintained in colony for more than 10 generations at HZAU. Table 1. Basic characteristics of fermentation substrates Pig manure

Rice straw

Total solids (TS, %)

27.85±0.2

89.29±1.77

Volatile solids (VS, %)

15.67±0.3

45.48±1.25

Moisture content (%)

72.15±0.2

10.71±1.77

2.1.2. Experimental design and operations Pig manure of 200 g and rice straw of 50 g were incubated in 1 L serum bottles containing 600 mL phosphate buffer solution (including minerals and trace elements).[14] The liquid medium was boiled under autoclave for 20 min. After sterilization, the headspace of each bottle was firstly purged with nitrogen gas for 5 min to create an anaerobic environment and then an exogenous gas mixture of CO2/H2 gases (600 mL/600 mL, v/v) was directly injected into the serum bottles. Then, 10 g inoculum was inoculated into the medium, and the fermentation experiments were 7

Journal Pre-proof carried out at 55°C. NaOH (3 mol/L) and H3PO4 (3 mol/L) were used to adjust the pH to 5, 6, 7, 8, 9. The control group was treated in the same way as the fermentation experiments, but injected with N2 instead of CO2/H2 and without pH regulation (initial pH 6.8). The negative biological control group was treated as above fermentation experiments and the pH was adjusted to 7, but with the following settings: 1) inoculum in media without pig manure and rice straw and with H2/CO2 headspace; and 2) inoculum in media without pig manure and rice straw and with N2 headspace. 2.1.3. Inoculum DNA extraction The total DNA from the sludge of sample was extracted using a FastDNA spin kit for soil (Qbiogene, Irvine, CA) according to the instructions of the manufacturer. 0.5 g fresh sludge was used directly for DNA extraction. The extracted DNA was dissolved in 100 μL DES buffer, quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Germany), and stored at −20 °C until use. 2.1.4. High-throughput sequencing and bioinformatics analysis Gel electrophoresis was applied to test the DNA integrity. The V4–V5 regions of bacterial 16S-rRNA genes were amplified using bacterial primers 515F: (5′GTGCCAGCMGCCGCGG-3′) and 907R: (5′- CCGTCAATTCMTTTRAGTTT-3′). PCR amplifications were conducted in a 25-μL reaction system containing 5 μL of 5× PCR buffer, 5 μL of 5× GC buffer, 5 μL of 10 mmol/ L dNTP, 1 μL of 10 μmol /L forward primer, 1 μL of 10 μmol /L reverse primer, 2 μL of DNA template and ddH2O. The PCR program consisted of an initial 2-min denaturation step at 98 °C, 28 cycles of repeated denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, followed by a final extension step of 5 min at 72 °C.

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Journal Pre-proof The composition of bacterial communities was analyzed using Illumina MiSeq sequencing of 16S rRNA at the whole microbial community level. The Miseq sequencing data were analyzed using the QIIME software package (Quantitative Insights Into Microbial Ecology, v1.8.0).[15] Sequences shorter than 150 bp, those containing more than 6 ambiguous bases, a homopolymer longer than 6 bp, or having one or more mismatching bases in the primer or barcodes, or showing a quality score below 20 were eliminated from further analysis. The data were denoised using the Amplicon Noise program.[16] Finally, high quality sequences were clustered into Operational Taxonomic Units (OTUs) based on 97% sequence similarity with UCLUST. 2.2. Conversion of VFAs to fatty acids by BSFL 2.2.1. Experimental design of VFAs accumulation in BSFL To study the effect of VFAs on the growth and conversion efficiency of BSFL, pig manure and rice straw (PMRS), or rice straw and sand (sand is a blank substrate that does not provide nutrients to the larvae but can improve ventilation to keep larvae survival) (RSS) were mixed at a 4:1 ratio (total mass of 300 g) to be used as substrate to feed 3-day-old BSFL. BSFL were inoculated into substrates at a density of 140 larvae per 100 g of waste, for 12 days at room temperature. Subsequently, BSFL were harvested and inactivated at 105°C for 5 min, and then dried at 55°C for 3 days. After grinding with micro-mill, the dried BSFL powder was stored at 4°C until the extraction of grease. 2.2.2. Feeding of BSFL with different concentrations of acid solution To evaluate the effect of different concentrations of VFAs on the grease yield of BSFL, different concentrations of acid solution were sprayed on the substrate, followed by stirring of the system. The design of the experiment is described in Table 9

Journal Pre-proof 2. The maximum concentration of VFAs produced by anaerobic fermentation in section 2.1.2 was set as the reference gradient. Table 2. Different concentrations of VFAs solution for feeding BSFL Pig manure and rice straw

Rice straw and sand

300

300

Substrate mass/g Treatment

PMRS0 PMRS1 PMRS2 PMRS3 PMRS4 RSS0 RSS1 RSS2 RSS3 RSS4

Acetic acid g/L

0

10

20

30

40

0

10

20

30

40

Butyric acid g/L

0

5

6

7

8

0

5

6

7

8

Spray volume/mL

20

20

20

20

20

40

40

40

40

40

The weight gained before and after the feeding for a group of BSFL was calculated and replicate was also performed to indicate the weight gain for each batch study in mean ± standard deviation. The value of this weight gain permitted the determination of BSFL growth rate as shown by Eq. (1). Growth rate = (Final larvae dry weight (g) ― Intital larvae dry weight (g))/ Rearing duration (d) 2.2.3. Pilot scale experiment Rice straw and sand (a total mass of 12.5 kg) were mixed at a 4:1 ratio to feed 3day-old BSFL. BSFL were inoculated into substrates at a density of 140 larvae per 100 g of waste, for 12 days at room temperature. Two groups were set up: one group was sprayed with 1 L of tap water per day, and the other group was sprayed with fermentation broth, which was obtained by expanding the fermentation broth under the optimal initial pH in proportion as described in section 2.1.2. After 12 days, BSFL were harvested and inactivated at 105°C for 5 min, and then dried at 55°C for 3 days. After grinding with micro-mill, the dried BSFL powder was stored at 4°C until the extraction of grease. 2.2.4. Extraction of crude grease 10

Journal Pre-proof To extract crude grease from the BSFL, the grinded BSFL biomass was put into a filter bag and soaked in petroleum ether for 48 h at room temperature. After the filter bag was removed, the BSFL crude fat contents were determined by evaporating the petroleum ether using a rotary evaporator (RE52A, Shanghai). 2.3. Analysis The pH of the fermentation system was determined using a le438 pH electrode (Mettler Toledo, USA). CO2 and H2 compositions were determined with a gas chromatograph (Agilent, 7890B) equipped with a thermal conductivity detector and an analytical column of Agilent Hayesep Q. The concentrations of VFAs were determined with a gas chromatograph (Agilent, 7890A) equipped with a flame ionization detector and a DB-wax capillary column (30 m × 530 μm × 1.0 μm). Before measuring the VFAs content, liquid samples were centrifuged at 10,000 rpm for 10 min and filtered through a 0.45 μm mixed cellulose ester membrane. The composition of the fatty acid methyl ester was determined by GC/MS (Thermo Fisher, Trace 1300, USA) equipped with a polyethylene glycol phase capillary column (Agilent, USA). Analysis of variance was conducted by SPASS. The experiments and sample analyses for all important parameters in this study were performed in triplicates.

3. Results and discussion 3.1. Effects of initial pH on production of VFAs pH was shown to be a key factor affecting the production of VFAs. Some studies have investigated the effect of pH, but the results are inconsistent and the optimal pH differs from one study to another,[11,17] possibly due to the differences in substrate, inoculum and operation conditions. Therefore, for economic and environmental concerns, it is necessary to determine the optimal pH for the bioconversion. In 11

Journal Pre-proof addition, the introduced CO2 and H2 would further generate VFAs. Acetate was the most dominant metabolite produced during anaerobic fermentation. As shown in Fig. 2a, the accumulation of acetate at pH 5, 6, 7, 8 and 9 peaked on the second day of fermentation. According to the peak acetate production, the highest yield was 10.11 g/L at initial pH 7, which was about 42.99% greater than that of the control. Moreover, the yield was 9.02 g/L and 8.86 g/L at initial pH 8 and 9, respectively. The yield at initial pH 6 was 7.45 g/L, which was basically the same to that of the control (7.07 g/L). However, initial pH 5 resulted in the lowest concentration of acetate (5.18 g/L), which was 26.73% lower than that of the control. Acetate concentration increased rapidly in the first 2 days of fermentation, and then slowly decreased with the extension of fermentation time until the end. As shown in Fig. 2b, butyrate is also a predominant metabolite. Similar to the variation of acetate concentration, butyrate concentration also increased rapidly in the first two days of fermentation, and then slowly decreased with the extension of fermentation time until the end. Coincidentally, the highest accumulation of butyrate was also observed on the second day and at initial pH 7 (3.56 g/L). At the initial pH 5, the peak concentration of butyrate was only 2.26 g/L, which was lower than that of the control (2.98 g/L). Unlike acetate, butyrate showed smaller differences between the experimental group and control group. The butyrate at initial pH 7 was 19.46% higher than that of the control, and that at initial pH 5 was 24.16% lower than that of the control. The data indicated that shifting pH from 5 to 11 strongly affected the VFAs content. Fermentation at pH 5 might cause an unfavorable environment for bacterial growth, leading to a lower accumulation of VFAs. The VFAs content showed a decrement pattern from the second day to the end of the experiment in all groups 12

Journal Pre-proof regardless of the initial pH, which might be ascribed to the consumption of the produced VFAs by the growth and metabolic activities of bacteria in the mixed cultures. In addition to acetate and butyrate, there were other carbon-containing organic products such as ethanol, propionate and valerate in the fermentation system, but these materials were lower in content and would not be discussed in detail herein. Initial pH 7 could be considered as the optimal pH for VFAs production from anaerobic fermentation because it resulted in significantly higher concentrations of acetate and butyrate.

Fig.2. Concentrations of acetate (a) and butyrate (b) under different initial pH The above data indicate that the accumulation of VFAs at initial pH 7 was favorable for the subsequent experiment in this study. Hence, gas consumption and VFAs accumulation were analyzed for this specific treatment. Table 3 shows that 32.2% of CO2 was consumed, while the H2 content was only reduced by 2.6% on the first day of fermentation in E-1 group (CO2 and H2 headspace without pig manure and rice straw), probably because CO2 was dissolved into the liquid phase and H2 was almost insoluble in liquid phase. Cumulative CO2 and H2 consumption of 61.3% and 72.6% were observed during the entire experiment. Meanwhile, the concentration of acetate gradually increased from 0.75 to 1.28 g/L but that of butyrate remained stable in the 13

Journal Pre-proof medium after CO2 and H2 were introduced into the reactors. However, in the absence of other substrates, VFAs production hardly increased in E-3 group (N2 headspace without pig manure and rice straw). According to gas consumption and VFAs accumulation in E-1 and E-3, acetate was the major metabolic product when CO2 and H2 were the principal energy source. It has been found that some microflora in lake sediments are able to utilize H2 to reduce CO2 to acetate via the Wood-Ljungdahl pathway (acetyl-CoA pathway).[18] In E-2 group (CO2 and H2 headspace with pig manure and rice straw), on the first day, the CO2 and H2 consumption was observed to be 37.2% and 0.8%, followed by an increment to 74.9% and 84.5% respectively on the second day, which corresponds to the rapid increase in VFAs (Fig. 2a, b). After the fourth day, the H2 and CO2 in the gas phase were rarely consumed, and the content became stable, but the total CO2 and H2 utilization rate was higher than that in the medium without pig manure and rice straw. It was worthy to note that the peak acetate production in E-2 (10.11 g/L) was much higher than that in E-1 (1.31 g/L), indicating that bacterial population not only consumed CO2 and H2, but also converted organics from pig manure and rice straw directly to VFAs. This phenomena on is similar to that observed in a previous study dealing with cow manure as a substrate.[12]

Table 3. Cumulative gas consumption and VFAs accumulation over time in the reaction system of initial pH 7 14

Journal Pre-proof a: H2/CO2 headspace without pig manure and rice straw b: H2/CO2 headspace with pig manure and rice straw c: N2 headspace without pig manure and rice straw E-1a Days

E-2b

butyrate g/L 0.22±0.09

CO2 /%

H2/%

1

acetate g/L 0.75±0.19

2

0.82±0.37

3

acetate g/L 2.97±0.18

E-3c CO2 /%

H2/%

32.2±4.6

2.6±0.3

butyrate g/L 1.54±0.16

37.2±2.1

0.8±0.1

acetate butyrate g/L g/L 0.77±0.19 0.19±0.06

0.20±0.11

38.6±6.5

15.0±4.4 10.11±0.89 3.56±0.22

74.9±9.4

84.5±9.1

0.75±0.24 0.22±0.11

0.98±0.22

0.25±0.13

48.3±4.2

37.0±6.6

2.15±0.52

1.47±0.32

77.9±6.5

92.4±9.6

0.82±0.15 0.20±0.06

4

1.16±0.17

0.32±0.10

55.5±7.1

54.5±4.4

2.17±0.64

2.18±0.05

78.1±6.1

94.1±6.7

0.81±0.29 0.22±0.08

5

1.31±0.27

0.29±0.06

60.7±2.3

72.4±5.9

0.94±0.28

0.76±0.16

78.3±7.4

94.9±7.3

0.86±0.17 0.27±0.13

6

1.28±0.30

0.29±0.13

61.3±7.4

72.6±7.1

1.33±0.55

0.75±0.41

78.6±8.2

95.4±6.1

0.86±0.14 0.28±0.11

3.2. Diversity and structure of microbial community High-throughput sequencing of the 16S rRNA genes was conducted to reveal the abundance and diversity of microbial community composition in the inoculums. The obtained sequences were quality-checked and normalized, and then grouped into about 1000 OTUs at 97% similarity. Analysis at the phylum level (Fig. 3a) showed three dominant phyla that can produce VFAs in the inoculums, including Firmicutes (85.1%), Proteobacteria (14.4%) and Bacteroidetes (0.4%),[19] especially Firmicutes, which showed absolute advantages in the sample, and are also the important phylum in anaerobic digestion sludge.[20] To better understand the community structure in these samples, sequences were also classified at the genus level. Bacillus (81.9%), Acetobacter (6.2%), Rheinheimera (4.8%) and Acinetobacter (2.1%) were the dominant bacterial genera in the sample (Fig. 3b). Bacillus, the main genus of Bacillaceae family, is a facultative anaerobe and can produce acetate from glucose and biosolids through anaerobic digestion, and contributes significantly to higher hydrolysis rate and VFAs yield at 55°C.[21] The Acetobacter genus belonging to Acetobacteraceae family is also involved in the production of acetate.[22] The production of butyrate was found to have 15

Journal Pre-proof correlations with Lachnospiraceae[23], which was found in the intestinal tracts with cellulose and hemicellulose degradation enzymes.[24] The genus Parabacteroides is strictly anaerobic bacteria that produce acetic acid as the major product of fermentation of various carbon sources.[25] In addition, some low abundance (<1.0%) acid-producing bacteria were also detected by high-throughput sequencing on the 16S rRNA genes. For example, Blautia

[26]

and Ruminococcus[27], which belong to an

anaerobic microbial group homoacetogen, could produce acetate by utilizing H2 and CO2. Oscillibacter[28] mainly produces butyric acid and acetic acid from carbohydrates, and Roseburia[29] and Butyricimonas[30] are two groups of butyrateproducing bacteria. Among the detected acid-producing bacterial genera, the main metabolism product of most genera is acetic acid, indicating that it is the overwhelming acid in VFAs from anaerobic fermentation.

Fig.3. Microbial community composition at (a) phylum and (b) genus level 3.3. BSFL biomass accumulation 16

Journal Pre-proof BSFL are able to convert organic wastes into insect biomass. Interestingly, the ability of BSFL to reduce levels of fatty acids emitted from swine manure.[31] BSFL are also likely able to convert these small molecular organic acids to biomass. To test this hypothesis, different concentrations of VFAs solutions were used to feed BSFL to investigate their influence on insect biomass accumulation. Fig. 4 shows the fresh mass, dry mass and growth rate of individual BSFL, respectively. Since BSFL showed the same trend of changes in all the three aspects, we only described the case of dry matter mass.

Fig.4. Effects of various concentrations of VFAs on BSFL fresh and dry mass, growth rate in (a) PMRS and (b) RSS There were significant differences in BSFL biomass for the larvae fed with the medium sprayed with VFAs compared to those fed with the control. In PMRS (Fig. 4a), the BSFL biomass increased with increasing concentration of VFAs in the spray solution. BSFL biomass was significantly increased from the PMRS0 group (7.76±0.44 mg) to the PMRS1 group (9.43±0.54 mg) and then to the PMRS2 group (17.79±0.13 mg), while no significant differences were observed among PMRS2, PMRS3 (17.84±0.54 mg) and PMRS4 (20.79±0.23 mg) groups. In addition, the dry

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Journal Pre-proof mass of BSFL also followed this trend, indicating that after the spraying of VFAs solution, BSFL accumulated the VFAs and converted them into lipid. To further highlight the ability of BSFL to use VFAs, we used rice straw and sand as the substrate. As a result, the larvae were able to survive; however, their growth was retarded due to the lack of nutrients. Consequently, BSFL were forced to convert VFAs to accumulate biomass. In RSS (Fig. 4b), larval biomass also increased significantly from RSS0 (1.90±0.05 mg) to RSS1(4.41±0.26 mg) and then to RSS2 (5.69±0.48 mg) groups. Unexpectedly, the dry matter mass of BSFL decreased in the RSS3 (3.03±0.15 mg) and RSS4 (2.97±0.18 mg) groups with higher concentrations of VFAs solution. A pontential explanation might be that the biotoxicity of the solution increased accordingly with increases in VFAs concentration, which inhibits the digestion and growth of BSFL, while due to the relatively abundant nutrients in the PMRS, the BSFL could choose not to convert the VFAs in the solution at a high concentration. Interestingly, the total amount of VFAs added to achieve the maximum biomass of BSFL was the same in both substrates. According to the results of BSFL weight gain and growth rate, it can be concluded that in both substrates, the VFAs concentration of 26 g/L (acetic acid 20 g/L butyric acid 6 g/L) is the optimal concentration for feeding BSFL.

3.4. Effect of different concentrations of VFAs solution on BSFL lipid yield 18

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Fig.5. Effects of various concentrations of VFAs on BSFL lipid yield The lipid yields of BSFL fed with different concentrations of VFAs are shown in Fig. 5. In two substrates (PMRS0 and RSS0), the lowest lipid yield was obtained in control treatment (only distilled water was sprayed on the diet) as expected (11.31±1.24% and 6.81±0.28%). In PMRS, the lipid yield of BSFL in the PMRS1– PMRS4 groups was 13.12%, 18.93%, 19.28% and 19.45%, respectively, indicating that the larval lipid yield increased with increasing concentration of VFAs. Thus, there is a positive relationship between VFAs concentration and lipid accumulation. In the RSS, the value rose from 9.09±0.78% to the highest of 14.26±2.19% when the VFAs concentration was increased from 15 g/L to 26 g/L. In RSS3 and RSS4 groups, the lipid yield of BSFL decreased to 7.59% and 7.14%, respectively. This observation is consistent with the results obtained from weight gain and growth rate (Fig. 4). Therefore, it can be speculated that VFAs were introduced into the larvae and converted into fatty acids.

19

Journal Pre-proof Because synthesis of fatty acids is one of the most ubiquitous pathways in organisms, their biosynthesis and regulation have been comprehensively studied in both prokaryotes and eukaryotes.[32] De novo fatty acid synthesis represents a crucial pathway in all living organisms. Although the architecture of the fatty acid system can vary greatly among different organisms, the individual enzymatic steps are essentially the same, and the reactions carried out during the elongation cycle are identical.[33] Therefore, acetate and butyrate may reduce the synthesis of long-chain fatty acids in BSFL. It was reported that [2-14C] acetate added into media as a biosynthetic precursor of fatty acids was successfully incorporated into the internal lipids of B. argentifolii adults,[34] which also indicates that insects are able to convert short-chain organic acids into long-chain fatty acids. 3.5. Fatty acid composition of BSFL BSFL grease is yellow and has a peculiar smell. Nine fatty acids were detected by GC/MS, particularly lauric acid, oleic acid and palmitic acid. The carbon number ranges from 12 to 18 (Table 4). The main saturated fatty acids include lauric acid (12:0), palmitic acid (16:0) and stearic acid (18:0), and the main unsaturated fatty acids are palmitoleic acid (16:1) and oleic acid (18:1). Two fatty acids with odd carbon number were identified from BSFL biodiesel, namely pentadecanoic acid (15:0) and heptadecanoic acid (17:0). Saturated fatty acids were dominant in the fatty acid composition of all BSFL. Saturated fatty acids accounted for 56.54% the total fatty acids in the BSFL resulting from the control. The supplementation of VFAs in the feeding medium increased the saturated fatty acids in the larvae, accounting for 62.65–69.51% (PMRS1–PMRS4) of the total fatty acids, which was higher than that of the control group. From PMRS0 to PMRS1, C14:0 increased from 0.42% to 5.06%, C16:0 from 11.58% to 17.36%, and 20

Journal Pre-proof C16:1 from 2.99% to 7.56%, while C18:0 dropped from 13.04% to 6.70%, and C18:1 from 25.25% to 17.95%. However, fatty acid components were not significantly different among PMRS1 to PMRS4. These results suggest that the converted organic acids in BSFL mainly affect the composition of C16 and C18 but have no obvious relationship with the amount of added VFAs. In previous studies, C16:0, C18:0, elaidic, C18:1, and C18:3 were recognized as the most common fatty acids in biodiesel.[35] BSFL contain high contents of saturated fatty acids such as C16 and C18, indicating their potential to produce high-quality biodiesel. Since saturated fatty acid methyl ester is more oxidatively stable than unsaturated fatty acid methyl ester,[36] the biodiesel produced from the fat of BSFL fed VFAs may have a higher oxidative stability than that produced from the fat of BSFL without the feeding of VFAs. Table 4 Relative contents (% w/w of total fatty acids) of fatty acid composition of BSFL fed on PMRS Fatty acids

PMRS0/%

PMRS1/%

PMRS2/%

PMRS3/%

PMRS4/%

Lauric acid (12:0)

29.44

30.68

33.07

32.41

32.87

Myristic acid (14:0)

0.42

5.06

6.93

8.54

8.26

Pentadecanoic acid (15:0)

0.94

2.34

1.81

1.73

1.98

Palmitic acid (16:0)

11.58

17.36

18.72

19.80

19.17

Palmitoleic acid (16:1)

2.99

7.56

7.82

8.10

8.46

Heptadecanoic acid (17:0)

1.12

0.51

0.73

0.45

0.63

Stearic acid (18:0)

13.04

6.70

6.07

6.44

6.60

Oleic acid (18:1)

25.25

17.95

16.49

17.58

17.97

Linoleic acid (18:2)

0.36

0.23

0.37

0.37

0.49

saturated fatty acid

56.54

62.65

67.33

69.37

69.51

unsaturated fatty acid

28.60

25.74

24.68

26.05

26.92

21

Journal Pre-proof 3.6. Pilot plant test Pilot plant test was carried out to confirm whether this method to convert CO2 and organic wastes into fatty acids is promising for industrial application. Anaerobic fermentation was performed with pig manure and rice straw as the substrate. The fermentation broth was obtained by expanding the fermentation broth under the optimal initial pH in proportion as described in section 2.1.2, and the fermentation was stopped on the second day. At this time, the fermentation broth contained about 10 g/L acetate and 5 g/L butyrate, and the yield of other products was very low and was thus ignored. Table 5 shows the biomass of larvae after 12 days of feeding. Table 5 Biomass of BSFL in pilot experiment Control

Fermentation broth

Fresh mass/mg

6.65±0.22

8.24±0.87

Dry mass/mg

1.85±0.31

2.55±0.19

Growth rate/mg/d

0.07±0.03

0.13±0.09

Lipid yield/%

6.54±0.79

8.26±1.01

The biomass of BSFL was slightly increased compared with the control. To be specific, the dry mass and lipid yield of the experimental group were 23.91% and 26.30% higher than those of the control, respectively. However, the biomass of BSFL was lower than that of lab-scale experiments. Even so, our results still prove the possibility and prospect of pilot-scale production of lipids from CO2 and organic wastes through the bioconversion by microbes and BSFL. Compared to chemical methods with high technical requirements (such as high temperature and pressure, precious metal catalysis),[37-39] this method of converting CO2 into fuel has the advantages of easy operation and mild conditions. In future research, some issues should be further addressed. For instance, CO2 derived from the separation and purification of industrial waste gas[40] and H2 obtained from wastewater[41] or rice 22

Journal Pre-proof straw[42] could be used the exogenous gas, and the equipment and operation should be further optimized to improve VFAs yield and larval biomass. Overall, the method in this study is promising for industrial application.

4. Conclusions This study demonstrates that conversion of CO2 into VFAs and then further into lipids for the production of biodiesel is a promising technology by utilizing low-cost biowaste as the substrate and microbe and BSFL as the biological system. In addition, larvae fed with VFAs showed higher levels of saturated fatty acids, indicating their potential in producing high-quality biodiesel. This integrated process can not only recycle CO2 and organic waste into clean energy, but also bypass the processes of extracting VFAs from fermentation broth, contributing to high practical application potential of bio-energy industry. However, a high biodiesel yield may not be expected since the lipid contents of the harvested biomass were not high enough. Therefore, future research should be focused on improving the content of VFAs or combining various organic wastes to increase the yield of BSFL lipids.

Conflicts of interest All the authors do not have any possible conficts of interest

Acknowledgments This study was financially supported by the National Key Technology Research and Development Program of China (Project No.2018YFD0500203). Fundamental Research Funds for the Central Universities (Project No.2662017JC045 and Project No.2662017JC026), the open funds of the State Key Laboratory of Agricultural Microbiology (Project No. AMLKF201610 and Project No. AMLKF201903).

23

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Journal Pre-proof Highlights • Black soldier fly (BSF) was fed by volatile fatty acids (VFAs) to obtain lipids. • CO2 was fixed for biofuel by integrated anaerobic fermentation and BSF conversion. • Low content VFAs do not require extraction and can be converted by BSF directly. • Lipids from BSF fed with VFAs is ideal feedstock of biodiesel.