Enhanced biohydrogen production from macroalgae by zero-valent iron nanoparticles: Insights into microbial and metabolites distribution

Accepted Manuscript Enhanced biohydrogen production from macroalgae by zero-valent iron nanoparticles: insights into microbial and metabolites distribution Yanan Yin, Jianlong Wang PII: DOI: Reference:

S0960-8524(19)30343-8 https://doi.org/10.1016/j.biortech.2019.02.128 BITE 21156

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

22 February 2019 26 February 2019 28 February 2019

Please cite this article as: Yin, Y., Wang, J., Enhanced biohydrogen production from macroalgae by zero-valent iron nanoparticles: insights into microbial and metabolites distribution, Bioresource Technology (2019), doi: https:// doi.org/10.1016/j.biortech.2019.02.128

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Enhanced biohydrogen production from macroalgae by zero-valent iron

nanoparticles:

insights

into

microbial

and

metabolites

distribution

Yanan Yin1,2, Jianlong Wang1,2,3 * 1 Tsinghua University -- Zhang Jiagang Joint Institute for Hydrogen Energy and Lithium-Ion Battery Technology, INET, Tsinghua University, Beijing 100084, PR China 2 Collaborative Innovation Center for Advanced Nuclear Energy Technology, INET, Tsinghua University, Beijing 100084, P. R. China 3 Beijing Key Laboratory of Radioactive Waste Treatment, INET, Tsinghua University, Beijing 100084, P. R. China

Corresponding author Professor Jianlong Wang Energy Science Building, Tsinghua University, Beijing 100084, PR China E-mail: [email protected]

∗ Corresponding author. Tel.: +86 10 62784843; Fax: +86 10 62771150. E-mail address: [email protected] 1

Abstract: In this work, effect of Fe0 nanoparticles (Fe0 NPs) on macroalgae fermentation was explored. Hydrogen production was significantly enhanced by 6.5 times comparing with control test, achieving 20.25 mL H2/g VSadded with addition of 200 mg/L Fe0 NPs. In-depth analysis of substrate conversion showed that both hydrogen generation and acids accumulation were promoted with Fe0 NPs supplementation. Microbial analysis demonstrated that both hydrogen-producing strains belonging to genus Clostridium and Terrisporobacter sp. favourable for acids formation were enriched with Fe0 NPs supplementation, while species Acinetobacter lwoffii beneficial to organics mineralization was eliminated. Complex substrate compositions resulted in more prevalent cooperative relationships among species in the system. This study suggested that Fe0 NPs plays a crucial role in macroalgae fermentation by affecting the microbial distribution, subsequently influencing the products distribution and energy conversion.

Key words: Biohydrogen, macroalgae, Zero-valent iron nanoparticles, metabolic network, microbial distribution

2

1. Introduction Dark fermentation is considered as a promising method for hydrogen generation because of the mild reaction conditions, easy operation and wide ranges of substrate sources (Wang and Yin, 2017). Among various feedstocks, macroalgae owns great potential for its high carbohydrates composition, high growth rate, easier hydrolysis without lignin, no occupation of farmland and low requirement of cultivation conditions. Besides, its excellent ability of fixing CO2 further promoted algae cultivation as energy crop (Wei et al., 2013; Ren et al., 2019). Furthermore, with the application of algae in adsorbing contaminants in marine environment (Wang and Chen, 2009), macroalgae cultivated in polluted seawater can also be a good source for energy recovery. However, the application of fermentative hydrogen production from macroalgae is restricted by the low hydrogen yield. Besides the explorations of pretreatment methods (Yin and Wang, 2018), enhancing hydrogen yield through process improvement seems more energy saving and environmentally friendly. Iron has been widely reported to be helpful in enhancing the hydrogen production through dark fermentation (Yang and Wang, 2018). Ferrous ion (Fe2+) is the active site of functional proteins for hydrogen formation, like ferredoxins and hydrogenases. Fe2+ is also helpful to motivate the expression of genes responsible for hydrogenase synthesis (Zhao et al., 2017). In addition, recent studies have found that zero-valent iron (Fe0) could bring more benefit. Besides the beneficial Fe2+ obtained by iron corrosion, Fe0 could also help to eliminate the residual oxygen present in the system, and help to maintain a low oxidation–reduction potential (ORP), making it more favorable for hydrogen-producing metabolism (Yang and Wang, 2018). Furthermore, Fe0 has also been reported to be able to catalyse the dissociation of propionate, forming hydrogen and acetate (Zhen et al., 2015). Comparing with Fe0 scraps, Fe0 nanoparticles (Fe0 NPs) can be better dispersed in fermentation broth, and much bigger surface for reactions. Yang and Wang (2018) explored 0-600 mg/L Fe0 NPs on hydrogen generation from grass, obtained highest hydrogen yield of 64.7 mL/g dry grass with 400 mg/L Fe0 NPs addition. Camacho et al. (2018) studied hydrogen production from organic market 3

waste with 0-2000 mg/L Fe0 NPs addition, enhanced hydrogen yield of 101.7 mL/g VS was attained at 2000 mg/L Fe0 NPs dose. Great variance of optimum Fe0 NPs dose and hydrogen yield indicates the effect of Fe0 NPs on hydrogen production is greatly affected by specific fermentation conditions, like substrate sources, inoculum, and operational conditions. Unlike the widely studied hydrogen-producing feedstocks (sugars, lignocellulosic biomass), macroalgae is rich in triglyceride, glycogen, fucoidan and trehalose, etc. Its decomposition and conversion process during dark fermentation remains unclear, making it hard to enhance the hydrogen yield accurately targeting substrate treatment. Furthermore, macroalgae is characterized to be rich in sulfur (Suutari et al., 2015), which is the well-known inhibitor for fermentative hydrogen production. Thus, explorations of biomass conversion and enhancing the hydrogen yield through process improvement is worth to be explored. This study explored the effect of Fe0 NPs on macroalgae fermentation. Hydrogen generation, products distribution and energy conversion were examined to reveal the influence of Fe0 NPs doses on energy recovery from macroalgal biomass. Changes of microbial distributions and microbial interactions were analyzed to explore the impacting mechanism. Furthermore, Principal Components Analysis was conducted to reveal the correlations among Fe0 NPs doses, hydrogen production, dominant microbes and volatile fatty acids formation.

2. Material and methods 2.1 Inoculum, macroalgae biomass and Fe0 NPs Anaerobic sludge was taken from a primary digester of a sewage treatment plant. Before inoculation, heat treatment (100 °C, 15 min) was applied to eliminate the hydrogen consumers. (Yin and Wang, 2018). Total solids (TS) and volatile solids (VS) of inoculum are 868.8±56.3 mg/L and 342.1±10.2 mg/L, respectively. Macroalgae Saccharina japonica was used as substrate. Macroalgae was pretreated by heat-acid treatment (1% H2SO4, 121°C, 30 min) to enhance the reaction efficiency during 4

fermentation. Characteristics of macroalgae biomass are as follows: TS=94.12±0.16% dry weight (DW), VS=80.28±0.01% DW, Total chemical oxygen demand (TCOD)= 1201.40±5.11 mg/g VS, hydrogen=5.414±0.006% VS, Carbon=38.686±0.274% VS, Nitrogen=2.550±0.057% VS, Oxygen=9.703±0.922% VS and Energy=13.74 kJ/g VS. Fe0 NPs (Shanghai Macklin Biochemical Co., Ltd, China) with diameter of 50 nm, and 99.9% metal basis were used in this study. 2.2 Experimental setup Batch fermentation was processed in 150 mL Erlenmeyer flasks with 100 mL working volume. Pretreated macroalgae mixture and inoculum were filled in each flask, making the reaction mixture with 10 g-VS/L substrate concentration and 20 % inoculation proportion. 0-800 mg/L Fe0 NPs were added to explore the effect of Fe0 NPs on dark fermentation, operations with each Fe0 concentration were conducted in triplicate. Before fermentation, initial pH was adjusted to 7.0±0.2, pure nitrogen was flushed through the flasks for over 3 min. Then, fermentation was conducted at 36°C, 120 r/min. A group without Fe0 addition was set as blank. It was stored at 4°C until the fermentation of experimental groups terminated. 2.3 Analytical methods TS, VS, TCOD and soluble chemical oxygen demand (SCOD) were measured by standard method (APHA, 1995). Element content of macroalgae was determined by an elemental analyzer (Exeter Analytical CE 440, UK). Volatile fatty acids (VFAs), alcohols and monosaccharides were determined by a HPLC. The column was Aminex HPX–87H, mobile phase was 5mM H2SO4 with a flow rate of 0.5 mL/min. Polysacchiarides and protein were measured by phenol–sulfuric acid method and Lowry method. Total soluble ion present in liquid phase was determined by flame atomic absorption spectrometer (Hitachi ZA3000). All the soluble matters were measured before being filtered through 0.45-um filters. Biogas volume and hydrogen content were determined by water displacement method and a gas chromatograph (112A, Shanghai, China). Dynamic characteristics of 5

hydrogen production were analyzed by Modified Gompertz model. Energy conversion efficiency (ECE) was analyzed based on the heating value of substrate and all detected products. Heating values of macroalgae, hydrogen, hydrolysis products and metabolites were calculated according to ref. (Nizami et al., 2009; Xia et al., 2015; Yin and Wang, 2018). Principal Components Analysis (PCA) was adopted to explore the relationships among various parameters in dark fermentation, like cumulative hydrogen production (CHP), Fe0 dosage, hydrogen production rate (Rmax), VFAs and dominant microbes. 2.4 Microbial community analysis At the termination of fermentation, sediments of test groups with no Fe0 addition, highest CHP, maximum Fe0 dose as well as blank group were collected for microbial analysis. The DNA extraction and PCR amplification were performed with soil extraction kit and universal primers (V4-V5 region), respectively. Illumina MiSeq PE250 platform was used for PCR based sequencing. Details of microbial analysis conditions can be referred to (Yin and Wang, 2016). Both α-diversity and β-diversity were investigated to describe the microbial diversity abundance and diversity among four samples. Based on the data from all four samples, interactions among microbes were analyzed by microbial network according to ref. (Faust et al., 2015). Microbial species with abundance over 1% and 20% were chosen for the correlation calculation. Network analysis and construction was performed using the Networkx software by Shanghai Majorbio Biopharm Biotechnology.

3. Results and discussion 3.1 Effect of Fe0 on dark fermentation 3.1.1 Hydrogen production Effect of Fe0 on CHP from macroalgae and the corresponding dynamic analysis 6

are demonstrated in Fig. 1 and Table 1. Hydrogen production was significantly enhanced with the Fe0 NPs addition (Fig. 1). Both hydrogen production potential (P), maximum hydrogen production rate (Rmax) and hydrogen yield (HY) were increased, while lag time (λ) was expressively shortened (Table 1). Highest CHP of 26 mL/100 mL and HY of 20.25 mL H2/g VSadded were obtained in Fe0-200 mg/L group, which were 6.5 times of control test without Fe0 NPs addition. The significant stimulation of hydrogen generation from macroalgae by Fe0 NPs addition may due to: (1) the improved environment for dark fermentation. Fe0 is a kind of reductive materials, the added Fe0 NPs can react with the oxidants present in the broth quickly, result in the decline of oxidation-reduction potential (ORP), creating more favorable environment for hydrogen producers (Feng et al., 2014). (2) Nutrients supplementation for hydrogenproducing metabolism. Iron plays an important role in critical gene expression as well as the synthesis of essential enzymes for hydrogen generation. When Fe0 NPs were added, iron corrosion happened, forming ferrous ions (Fe2+) as well as ferric ions (Fe3+). Fe2+ could motivate the functional genes expression, like hydrogenases and dehydrogenase (Zhao et al., 2017). Besides, Fe2+ also serves as critical proteins working in hydrogen-producing metabolism, like ferredoxins and various hydrogenases (Wu et al., 2017; Zhao et al., 2017). Furthermore, Fe0 could also induce the decomposition of propionate, forming acetate and hydrogen (Zhen et al., 2015). (3) The elimination of inhibitors. Since macroalgae is characterized to be rich in sulfur (Suutari et al., 2015), while sulfide is the well-known inhibitor for fermentative hydrogen production. It has been reported that the sulfide inhibition could be entirely eliminated by sufficient iron ions in dark fermentation system (Dhar et al., 2012). Yang and Wang (2018) also obtained prominent enhanced hydrogen generation from grass by Fe0 NPs addition. The decreased ORP, increased dehydrogenase activity as well as the enriched hydrogen producers were observed in Fe0 added groups. Indicating Fe0 NPs supplementation is a good method in enhancing the hydrogen production through dark fermentation. However, both CHP and HY decreased with a further increase of Fe0 dose. CHP of 14 mL/100 mL and HY of 10.90 mL H2/g VSadded were achieved at Fe0-800 mg/L group, indicating excess Fe0 NPs supplementation may cause toxicity to microbes. 7

Quite a few studies have reported the negative effects of superfluous Fe0 on hydrogen generation. Feng et al. (2014) observed significantly increased abundances of hydrogenotrophic methanogens and homoacetogens with surplus Fe0 addition, which resulted in the decrease of hydrogen generation. Similarly, Zhen et al. (2015) also reported the stimulation of excess Fe0 on acetate and methane formation from the hydrogen consumption. Thus, Fe0 NPs dose is a critical factor in enhancing dark fermentative hydrogen production. However, various optimum Fe0 NPs have been obtained in different studies. Yang and Wang (2018) achieved the highest CHP from grass at 400 mg/L Fe0 NPs addition, while Camacho et al. (2018) obtained highest hydrogen generation from market waste residue with 2000 mg/L Fe0 NPs supplementation. Indicating the proper Fe0 NPs dose depends on the substrate source and operational conditions adopted in specific systems. Concentrations of soluble iron ions present in the broth after fermentation were also determined. In the control test, 18.6 mg/L of iron ions was detected even without Fe0 addition, which may come from the hydrolysis of macroalgae during the fermentation. Concentrations of soluble iron ions in 100-600 mg/L Fe0 added groups all ranged from 30.2-40.5 mg/L, which is the consequence of the interaction effect among Fe0 anaerobic corrosion, biomass adsorption and microbial utilization. In Fe0800 mg/L group, significant higher soluble iron ions concentration of 63.0 mg/L was observed, indicating a stronger corrosive effect happened. This may because of the pH decrease induced by the accumulation of acetate with excess Fe0 NPs (Camacho et al., 2018). Figure 1 Table 1 3.1.2 Biomass hydrolysis and substrate utilization Biomass hydrolysis, organics degradation as well as metabolites formation are accompanied with hydrogen accumulation. Both highest VS degradation efficiency of 29.7% and SCOD degradation efficiency of 35.9% were obtained in control test without 8

Fe0 supplementation (Fig.2A). With the addition of Fe0 NPs, degradation efficiency of both VS and SCOD decreased significantly. Which may because of the low ORP in Fe0 NPs added groups restricted the mineralization process. The decreased VS degradation efficiency may also contributed by the enhanced microbial reproduction and inhibited biomass hydrolysis. It has been reported that iron supplementation could promote the Pyruvate ferredoxin oxidoreductase (PFOR) formation, thus facilitating the microbial anabolism (Furdui and Ragsdale, 2000). Besides, cellulose hydrolysis could also be restricted by iron ions through electrons shuttling (Tejirian and Xu, 2010). Furthermore, with the accumulation of hydrogen, high hydrogen partial pressure also performed restriction effect on the biomass hydrolysis process (Feng et al., 2014). Thus, in 100600 mg/L Fe0 NPs supplemented groups, VS degradation efficiency were all around 10%. However, with a further escalation of Fe0 NPs dose, VS degradation efficiency increased to 21.7%. From one side, the excess Fe0 NPs promoted hydrogen consumption, relieved the inhibition effect of high hydrogen pressure (Zhen et al., 2015). On the other side, excess amount of Fe0 could stimulate the protein hydrolysis, resulting in a higher hydrolysis efficiency (Feng et al., 2014). Besides the hydrolysis of macroalgae during the pretreatment process, biomass was further hydrolyzed during dark fermentation. Changes of polysaccharides and protein in liquid phase after fermentation were the consequences of both microbial utilization and biomass hydrolysis (Fig. 2B). The concentrations of polysaccharides in all groups decreased, indicating the degradation effect of polysaccharides was stronger than the hydrolyzing releasement. Differently, concentrations of protein all increased after fermentation, showing a stronger hydrolysis effect than degradation. Possible reason was that polysaccharides were more easily utilized than protein during hydrogen production. Besides, the accumulated protein may also comprised the extracellular proteins, which were formed accompanying the microbial proliferation. The components of hydrolysate after fermentation mainly included xylose, arabinose and glycerol. No glucose or cellose was detected (Fig. 2C). Xylose is mainly obtained from the hydrolysis of hemicellulose and fucoidan, arabinose is the important component of hemicellulose, and glycerol is usually derived from the hydrolysis of 9

triglyceride (Feng et al., 2014). Thus, the significant higher xylose concentration in control test shows a strong microbial hydrolysis of fucoidan, while hydrolysis of hemicellulos was more dominant in 200-800 mg/L Fe0 NPs added groups. Little xylose was detected in Fe0 NPs added groups, indicating xylose utilization was stimulated by Fe0 NPs supplementation. Metabolites formed during the fermentation mainly included C1-C4 VFAs, little ethanol was detected (Fig. 2D). Total VFAs increased with the increased Fe0 NPs doses, proved the promotion effect of Fe0 NPs on the hydrolysis-acidification of macroalgal biomass (Feng et al., 2014). Lowest total VFAs was achieved in control test. Considering the highest VS and SCOD degradation rate (Fig.2A), and the maximum consumption of polysaccharides and protein (Fig.2B) in control test, most of the organics consumed were mineralized. For Fe0 NPs added groups, VFAs was dominated by acetate, indicating the acetate-type fermentation was dominant. Differently, Ren et al. (2019) found that butyrate-type fermentation was the most appropriate fermentation for hydrogen generation from molasses wastewater. The difference in proper fermentation type may due to the differences in substrate composition. No propionate was detected, which was because the low ORP environment in Fe0 NPs added groups was not favorable for propionic-type fermentation (Yang et al., 2018). Besides, the original propionate could also be decomposed with the stimulation of Fe0, forming acetate and hydrogen (Camacho et al., 2018). Since the propionic-type fermentation is not favorable for hydrogen yield, Fe0 NPs supplementation could be a good method in adjusting the metabolism follow the pathways favorable for hydrogen generation. Figure 2 3.2 Products distributions and energy analysis 3.2.1 Summary of products distributions A COD-based products distribution shows the effect of Fe0 NPs on metabolic shifts (Fig. 3). Soluble metabolites, biomass and mineralization were the top three 10

terminals of biomass after the fermentation. Soluble metabolites included the hydrolysate of biomass as well as the intermediate and terminal metabolites. Biomass was comprised of un-hydrolyzed macroalgal biomass and microbes present in the system. The mineralized part supplied energy for microbial metabolism. Big proportion of soluble metabolites and mineralization indicates the microbes present the system were pretty active. While the high percentage of biomass and mineralization indicates lots of substrate was consumed for microbial reproduction and metabolism. Similarly, studies adopted soluble polysaccharides as substrate also obtained high biomass yield, like 10-61% biomass yield from 20 g/L lactose (Palomo-Briones et al., 2017) and 38% biomass yield from soluble condensed molasses (Lay et al., 2010). Besides, substrate hydrolysis could also be further enhanced to improve the energy conversion efficiency. With the addition of Fe0 NPs, The conversion efficiency of hydrogen from substrate was significantly enhanced. However, even with the optimization of Fe0 NPs supplementation, only 1.7% of substrate was directed to the hydrogen generation. Unlike the system with biomass as substrate, significant higher hydrogen conversion efficiency could be obtained from pure sugars. COD-based hydrogen conversion proportion of 11.9-17.8% was obtained from glucose in our previous studies (Yin et al., 2014; Yin and Wang, 2016; Yin and Wang, 2017). Palomo-Briones et al. (2017) also achieved 8% hydrogen yield from lactose. This phenomenon implies that taking hydrogen as sole target product is neither economic nor realistic to recover energy from biomass-based substrate. Since the compositions of biomass are much more complicate than pure sugars, more various metabolites could be obtained during the fermentation, consequently more energy is directed to the intermediate metabolites other than hydrogen. Thus, to promote the application of macroalgal biomass fermentation, energy present in soluble metabolites should also be recovered. Quite a few studies have explored the metabolites recovery. For example, the recovery of valuable chemicals (Wang et al., 2018; Romero Soto et al., 2019), maximum energy recovery through multi-stage procedures (Trchounian et al., 2017), and high value chemicals production through chain-elongation (Yin et al., 2017). Except for the enhancement of hydrogen conversion in Fe0 NPs added groups, proportions of soluble metabolites were also 11

increased (Fig. 3). Indicating the Fe0 NPs supplementation was conductive to enhance the total energy recovery from macroalgal biomass. Figure 3 3.2.2 Energy conversion of macroalgae Based on the heating value of macroalgae and detected products after fermentation, energy conversion efficiency (ECE) was calculated to illustrate the recoverable energy in this fermentation system (Fig.4). Results showed that recoverable energy significantly increased with the supplementation of Fe0 NPs. Fe0-800 mg/L obtained the highest ECE of 13.9 %, which was 2.0 times of group without Fe0 addition. In the control test, energy in xylose accounted for 80% of all detected energy, indicating irondeficient fermentation could be a feasible method in recovering xylose from macroalgae. With the addition of Fe0 NPs, acetate and butyrate contributed the main energy. When the added Fe0 NPs concentration increased from 100 mg/L to over 200 mg/L, ECE of arabinose increased from 0.1% to around 1%, demonstrating excess Fe0 NPs was preferable for arabinose accumulation. Although the supplementation of Fe0 NPs enhanced total ECE, destinations of over 85% of substrate energy were unclear. As demonstrated in Fig. 3, besides the energy consumed for microbial metabolism and undetermined metabolites, quite amount of energy was stored in biomass, which included both the incompletely hydrolyzed macroalgae and the proliferated microbial biomass. Thus, a more thorough hydrolysis of macroalgae biomass and limited microbial proliferation could be helpful to enhance the total ECE. Measurements have been taken both before and during fermentation process. Like combined pretreatment process employing various advantages of different pretreatment methods (Wang and Yin, 2017; Wang and Yin, 2018; Yin and Wang, 2018), inoculation of functional microbes and addition of hydrolytic enzymes to promote the hydrolysis process during fermentation. As to the limitation of microbial reproduction, microbial immobilization could be a good approach. Furthermore, microbial immobilization is also helpful to maintain an optimal microbial amount for 12

fermentation process, leading to a stable and efficient energy recovery (Wang and Yin, 2017; Wang and Yin, 2018; Yin and Wang, 2018). Besides enhancing the ECE, multi-stage fermentations were also explored to achieve the maximum recovery of gas fuel, like hydrogen-methane fermentation (Liu et al., 2006), and dark-photo hydrogen fermentation (Zhang et al., 2018). To enhance the values of metabolites, recent studies have focused on chain-elongation of fermentation metabolites, forming high value chemicals like caproate, hexanol etc. (Yin et al., 2017; Verbeeck et al., 2018). Thus, lots of efforts are deserved to achieve a complete and high-value conversion of macroalgae biomass. Figure 4 3.3 Microbial analysis Microbial communities of inoculum, and groups with no Fe0 addition, 200 mg/L Fe0 addition and 800 mg/L Fe0 addition after fermentation were analyzed. Table 2 demonstrates the indices of microbial α-diversity, which implies the changes of community diversity and richness with different Fe0 doses after fermentation. The coverage of all samples were over 0.998, showing a high reliability of the results. Inoculum owns the highest Sobs, while Sobs of groups after fermentation significant decreased. Indicating a nature selection happened during the fermentation process, microbes unsuitable to the system were eliminated. For the microbial communities after fermentation, Sobs increased with the increase of Fe0 NPs doses. The community richness represented by Chao and Ace showed the same trend. This may because the lower ORP formed in system with more Fe0 addition was more suitable for the growth of selected microbes, in addition to the formed iron ions, led to both higher Sobs and higher community richness. To compare the microbial communities taking account of both community richness and evenness, microbial diversity was brought forward. Higher microbial diversity was demonstrated by bigger Shannon and smaller Simpson. Similarly, microbial community of inoculum demonstrated the highest microbial diversity. 13

Otherwise, different from the community richness, microbial diversity of Fe0-200 mg/L was higher than Fe0-800 mg/L, indicating microbes were more evenly distributed in group with 200 mg/L Fe0 addition. Table 2 3.3.1 Effect of Fe0 on microbial distribution To take a closer look at the Effect of Fe0 on microbial distribution, β-diversity was analyzed and species-based microbial distributions were demostrated. β-diversity was analyzed by principal co-ordinates analysis (PCoA) to reveal the degree of variances among the four samples (Fig. 5A). Inoculum, group without Fe0 addition and Fe0 added groups were separately located in different areas, showing big differences between each other. As to the Fe0 added groups, no significant difference in microbial communities was formed in Fe0-200 mg/L and Fe0-800 mg/L groups. This phenomenon indicates that both fermentation process and Fe0 addition can cause great changes in microbial distribution, while the dose of Fe0 NPs contributes much less to the change of microbial communities. Species-based microbial distributions of four samples were shown in Fig. 5B. For the inoculum, microbes were relatively diversely distributed. Species Clostridium butyricum, an undefined Bacterium sp. and an undefined Enterobacter sp. showed more occupations of 30.4%, 15.6% and 10.6%, respectively. After fermentation, Clostridium butyricum remained dominant in all groups, while the occupations of undefined Bacterium sp. and Enterobacter sp. decreased to <1%. Indicating these two species were not adaptive to the anaerobic dark fermentation. Besides, species Clostridium sensu stricto became dominant in all groups after fermentation with occupations of 19.8-41.4%. Both Clostridium butyricum and Clostridium sensu stricto are widely reported hydrogen producers (Yin and Wang, 2016; Yin and Wang, 2018). Besides the two species belonging to genus Clostridium, species Exiguobacterium sp. and Acinetobacter lwoffii became dominant with occupations of 19.8% and 19.0% in group without Fe0 addition. With the addition of Fe0 NPs, species Acinetobacter lwoffii was 14

not detected, but the occupations of Terrisporobacter sp. significantly increased to 16.7% in 200 mg/L-Fe0 group and 13.2% in 800 mg/L-Fe0 group. Genus Acinetobacter has been widely observed in macroalgae fermentation systems for its high degradability of hemicellulose (Kumar et al., 2014). Wang et al. (2018) has reported an efficient mineralization of organics by genus Acinetobacter, which may be the reason of high mineralization rate observed in Fe0-0 mg/L group. Terrisporobacter sp. is a kind of acetogenic bacterium, which can degrade various carbon sources, like xylose and cellobiose (Deng et al., 2015; Groher and Weuster-Botz, 2016). The increase of Terrisporobacter sp. in Fe0 added groups was corresponded with the high rate of acids formation (Fig. 4). The occupation of Exiguobacterium sp. decreased with Fe0 addition, and disappeared with the excess Fe0 addition. Exiguobacterium sp. has been reported to produce lactate as main liquid metabolite during dark fermentation (Jiang et al., 2013). Lactate-type fermentation has been widely considered as unfavourable metabolic pathways for hydrogen generation (Palomo-Briones et al., 2017). High occupation of Exiguobacterium sp. in Fe0-0 mg/L group may be the reason of its low hydrogen production. The decrease of Exiguobacterium sp. with Fe0 addition indicates Fe0 could promote the hydrogen production by affecting microbial distributions in a system. Figure 5 3.3.2 Microbial network analysis Relationships between different microbial strains are important indicators for the design of synthetic microbial communities (Faust and Raes, 2012). Microbial interactions were analyzed to reveal the relationships among dominant microbes (Fig. 5CD). The color of nodes characterizes the genus of species, and different sizes represent species abundances. Red lines point to cooperative correlation, while green lines represent exclusive correlation between species. In the four samples, 9 species from 2 phyla were selected for their OTU abundances in top 20% of all species. Both positive and negative effects were observed 15

among these species (Fig. 5C). The dominant species Clostridium butyricum in all groups after fermentation showed positive correlation with Clostridium butyricum 5521, Clostridium tertium and Gallicola. All of them were frequently found in hydrogenproducing fermentation systems (Sivagurunathan et al., 2014; Leng et al., 2017). These four strains were negatively correlated with Terrisporobacter sp. and Clostridium malenominatum, both of which were preferable for acids formation and found in Fe0 added groups (Groher and Weuster-Botz, 2016). The positively correlated species Candidatus Caldatribacterium and Proteiniphilum were all found in the inoculum, they are not widely correlated with other species. The correlations among 42 species with OTU abundances in top 1% were displayed in Fig. 5D. Relationships among species belonging to Firmicutes were more abundant than others. Cooperative relationships were more prevalent for species from same phylum, while exclusive correlations were more prevalent for species from different phyla. The cooperative effects among different species mainly comprises complex molecules disintegration, reduction reaction to preserve a low ORP, metabolites consumption to relieve the product-inhibition, etc. (Hung et al., 2011). The exclusive effects mainly includes substrate competition and inhibitive metabolites formation. Unlike the more positive correlations were present in this study, PalomoBriones et al. (2017) found that when lactose was used as sole carbon source for hydrogen production, over 80% of microbial correlations were negative. Indicating more cooperation was needed when complex organics were used as substrate. 3.4 Interactive analysis of principle factors in dark fermentation To explore the correlations among different factors in dark fermentation, Fe0 dosages, kinetic factors of hydrogen production, dominant microbes and VFAs were analyzed by principal components analysis (PCA) (Fig.6). Fe0 is closely located with both hydrogen production and Rmax, indicating Fe0 doses has great effect on hydrogen production from macroalgae. Besides, species Enterococcus sp. and Terrisporobacter sp. are also closely related with Fe0 and hydrogen production. As both two strains were reported to be preferable for acids-forming metabolism, the relationship between this 16

two strains and hydrogen production maybe negative. Lag time (λ) is closely located with species and Exiguobacterium sp. and Acinetobacter lwoffii. As Exiguobacterium sp. was reported to follow lactate-type fermentation (Jiang et al., 2013) and Acinetobacter lwoffii contributed to biomass mineralization (Wang et al., 2018), which are all not preferable for efficient hydrogen production. Thus, it can be concluded that λ is positively correlated with these two species. Comparing with the above mentioned four strains, hydrogen producers Clostridium butyricum and Clostridium stricto are farther from Fe0 and hydrogen production. Fig. 5B also shows that Clostridium butyricum was not significantly affected by Fe0 dosages. Formate, acetate and butyrate are all far from each other, indicating the formation of these acids followed different metabolism pathways, and these metabolisms have little effect on each other. Butyrate was more closely correlated with Fe0 and hydrogen production. Although hydrogen can be quickly produced following formate-type and acetate-type fermentation, accumulation of the low-carbon acids will result in a quick decrease of solution pH, causing poisonous effect on microbes. Then, butyrate-type fermentation usually become dominant, eliminating the superfluous H+ and promoting a stable hydrogen production. Thus, acetate is usually positively correlated with hydrogen yield, while butyrate is closely related with cumulative hydrogen production. Figure 6

4. Conclusions This study firstly explored the effect of Fe0 NPs on macroalgae dark fermentation by process analysis. Products distribution and microbial interactions were in-depth analyzed. Results showed that with the supplementation of Fe0 NPs, mineralization effect of organics was impaired with the elimination of species Acinetobacter lwoffii. Both hydrogen generation and acids accumulation were enhanced with the enrichment of Clostridium sp. and Terrisporobacter sp. Positive correlations were more prevalent among species in the system. It can be concluded that Fe0 NPs has significant influence on macroalgae fermentation through changing the microbial distribution and 17

subsequently affecting the metabolic pathways, products distribution and energy conversion.

Acknowledgement The authors would like to gratitude the financial support from the China Postdoctoral Science Foundation (2018M640144) and the National Postdoctoral Program for Innovative Talents.

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Legends Fig. 1 Effect of Fe0 NPs on hydrogen production. Fig. 2 Effect of Fe0 NPs on substrate degradation and metabolites formation. Fig. 3 Effect of Fe0 NPs on products distributions based on COD. Fig. 4 Effect of Fe0 NPs on energy conversion of hydrogen and soluble metabolites Fig. 5 (A) Differences between microbial communities revealed by Principal Coordinates Analysis (PCoA); (B) Comparison of species-based microbial distributions; Microbial interactions among species with OUT abundances in top 20% (C) and top 1% (D). Fig. 6 Principle Components Analysis (PCA) for hydrogen production, Fe0, microbial species and metabolites formation. Table 1 Kinetic analysis for hydrogen production with different Fe0 NPs doses. Table 2 Indices of microbial α-diversity.

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Figure 1

25

Figure 2

26

Figure 3

27

Figure 4

28

Figure 5

29

Figure 6

30

Table 1

Hydrogen yield

Test groups

P

λ

Rmax

R2

Fe0- 0 mg/L

4.00

21.29

1.17

0.9997

3.12

Fe0-100 mg/L

5.00

13.58

1.76

0.9999

3.89

Fe0-200 mg/L

26.05

14.01

5.29

0.9992

20.25

Fe0-400 mg/L

22.02

9.00

2.76

0.9995

17.13

Fe0-600 mg/L

23.03

14.14

4.34

0.9995

17.91

Fe0-800 mg/L

14.01

11.86

1.67

0.9991

10.90

31

(mL/g VSadded)

Table 2

Sample\Estimators

Sobs

Ace

Chao

Shannon

Simpson

Coverage

Inoculum

175

178

178

2.978

0.1242

0.9997

Fe0-0 mg/L

61

86

80

1.765

0.2124

0.9993

Fe0-200 mg/L

77

112

99

2.042

0.1702

0.9989

Fe0-800 mg/L

87

147

116

1.816

0.2438

0.9988

Highlights

Fe0 nanoparticles (Fe0 NPs) could improve H2 production from macroalga. Hydrogen production was enhanced by 6.5 times comparing to control test. Fe0 NPs promoted hydrogen production and acid accumulation. Fe0 NPs enriched Clostridium and Terrisporobacter sp.

Graphical abstract

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