Citation

Ren Wei-Tong, Lan Tian, He Zi-Lin, Wang Hua-Zhe, Deng Lin, Ye Shan-Shan, Wu Qing-Lian, Guo Wan-Qian. Weak electric field promotes anaerobic granular sludge formation to enhance medium-chain fatty acids synthesis[J]. Chin. Chem. Lett., 2025, 36(8): 110563  

Wei-Tong Ren, Tian Lan, Zi-Lin He, Hua-Zhe Wang, Lin Deng, Shan-Shan Ye, Qing-Lian Wu, Wan-Qian Guo^*     

The overuse of non-renewable fossil energy sources has caused serious environmental problems, and renewable energy sources access is necessary and urgent [1, 2]. One of the most challenging environmental issues is the waste biomass treatment, as it is necessary to meet environmental protection requirements and achieve the carbon emissions reduction at the same time [3, 4]. Medium-chain fatty acids (MCFAs) are renewable biochemicals that can be obtained from biomass by carbon chain elongation (CE) [5, 6]. MCFAs have multiple uses, that can be utilized as biochemical substitutes for traditional fossil fuels, thus contributing to sustainable development [7]. However, the production process of MCFAs is restricted by numerous limitations, such as the inhibition of microorganisms by the biotoxicity of MCFAs as their carbon chains are extended, and the low production efficiency of traditional anaerobic biotechnology [8]. Granularization of sludge microorganisms to form granular sludges is considered a suitable facilitator because granular sludges can help microorganisms to resist the toxicity of MCFAs and can improve the microbial function activity [9]. Nevertheless, how to promote sludge granulation remains a problem. Electric field (EF) has been widely used to promote aerobic granular sludge granulation [10, 11], but whether EF can be used to promote the granulation process of anaerobic granular sludge (AnGS) remains less studied. Our previous study [12] demonstrated that EF can enhance the CE process by up-regulating the abundance of key functional genes, and if EF can promote sludge granulation and the CE process simultaneously, this would likely increase the MCFAs yields to a greater extent. Therefore, in this study, we operated two continuous fed Expanded Granular Sludge Bed (EGSB) bioreactors, one with EF and one without, to demonstrate the promotion of sludge granulation by EF and the enhancement of MCFAs production efficiency by the AnGS.

The continuous fed EGSB bioreactor without EF was named as R-ctrl, and the reactor applied with a low-intensity EF of 0.05 V was named as R-elec, as shown in Fig. 1. The growth medium in the reactor was 9.6 g COD/L lactate as electron donor (ED) and 2.1 g COD/L acetate as electron acceptor (EA), and other substrates per liter were the same as our previous study [12]. The conversion rate of caproate was the ratio of the concentration of caproate in the product to the total substrate concentration (in g COD/L). Scanning electron microscopes (SEM, Hitachi S-4800, Japan) detection was used to examine the morphology of AnGS. Cyclic voltammetry (CV) was used to characterize the redox properties and electron transfer in the AnGS with the electrochemical workstation (VSP-300, Bio-Logic, France). Electron transfer system (ETS) activity was used to assess the activity of sludge microorganisms. The extracellular polymeric substances (EPS) of the AnGS in two reactors were also extracted and quantified. The concentration of MCFAs and lactate were determined by gas chromatograph (GC) and Waters ACQUITY ultra-performance liquid chromatograph, respectively. In addition, metagenomic sequencing was utilized to reveal the mechanisms of caproate production in terms of both microbial community composition and functional genes. The AnGS samples in both reactors were obtained when the stable granular sludges have formed. Detailed information was summarized in Supporting information.

Figure 1

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Fig. 1. Schematic diagram of the reactor in R-ctrl (a) and R-elec (b).

The changes of the carboxylate concentration in two reactors were shown in Fig. 2. In R-ctrl (Fig. 2a), caproate was detected on day 10, after which the concentration began to rise gradually. The AnGS were observed on day 38, subsequently, caproate began to accumulate rapidly, reaching the maximum concentration of 2470.22 mg/L on Day 50. The highest caproate conversion rate was 47% in R-ctrl. In contrast, the rate of stable granular sludge formation was significantly higher in R-elec (Fig. 2b), and was observed on day 21. Before day 21, a large amount of butyrate had been accumulated in R-elec, but the concentration of caproate was relatively low. With the formation of stabilized AnGS, the caproate concentration started to increase rapidly, and the highest concentration was 3357.50 mg/L, which was 36% increase compared to that in R-ctrl, and the caproate conversion rate was upregulated to 63%. The operational efficacy of both reactors demonstrated that the formation of AnGS would substantially increase the yield of MCFAs, which has also been found in other studies [9, 13]. In this study, the formation of AnGS in R-elec was faster, suggesting that the electric field has a facilitating effect on the formation of AnGS. Aerobic granular sludge granulation can be enhanced by applying EF [11]. However, this study seems to demonstrate that EF can also promote anaerobic granulation processes for the first time.

Figure 2

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Fig. 2. Product concentration (mg/L) in R-ctrl (a), with the granular sludges formed on day 38, and in R-elec (b), with the granular sludges formed on Day 21.

In order to further investigate the mechanisms by which EF promote the formation of AnGS, we performed SEM detection on the AnGS formed in two reactors. As shown in Fig. 3, from the 50× and 1000× images, the morphology of the AnGS in two reactors did not differ much, except that there appeared to be more pores in the AnGS in the R-elec. And when the magnification was 5000×, some differences were observed, there seemed to be more microorganisms in the AnGS formed in the R-elec, mostly rod-shaped microorganisms, which might be the most suitable microorganisms for MCFAs production [4, 14]. Whereas in the AnGS in R-ctrl, some rod-shaped microorganisms were also observed, but not as many as in R-elec. SEM detection demonstrated that the EF promoted AnGS formation possibly by enhancing the aggregation of CE microorganisms, which enables particles to form more conveniently. And the tiny pores formed in the AnGS may be more favorable for microorganisms to carry out behaviors such as transmembrane transport of metabolic intermediates [12].

Figure 3

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Fig. 3. SEM detection of the AnGS in R-ctrl (a) and in R-elec (b). Three magnifications were selected, 50×, 1000× and 5000×.

CV was used to qualitatively compare the redox capacity in sludge. As shown in Figs. 4a and b, the increased intensity of the current in R-elec indicated that the AnGS in R-elec could improve the extracellular electron transfer capacity [15]. Figs. 4c and d revealed that the pseudocapacitance in R-elec was higher than that in R-ctrl, which indicated that AnGS in R-elec had higher charge−discharge capacity [16]. Besides, ETS was used to evaluate the electron transfer activity. Two time points were selected, on day 20, the sludges exist mainly in suspended form in two reactors, and on day 50, there were stable AnGS in both two reactors. As illustrated in Fig. 4e, the ETS activity of R-elec was higher (131.48% of R-ctrl) than that of R-ctrl when granular sludges were not formed, which could be attributed to the promotion effect performed by the EF [17]. And when AnGS formed, the ETS activity of R-elec was further increased (193.76% of R-ctrl), which demonstrated that the EF enhanced the electron transfer capacity of the AnGS, which is one of the reasons for the easier formation of AnGS and the better CE efficiency in the presence of the EF.

Figure 4

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Fig. 4. CV of the AnGS in R-ctrl (a) and in R-elec (b). The pseudocapacitance in R-ctrl (c) and in R-elec (d). (e) Relative ETS activity was calculated based on R-ctrl on day 20 and day 50, respectively.

EPS has significant effects on microbial aggregation and is important for the structural stability of the granular sludges [18]. Consequently, polysaccharides (PS) and proteins (PN) of LB-EPS and TB-EPS in the AnGS in two reactors were detected, and the results were shown in Fig. 5. In both the LB-EPS and TB-EPS, the concentration of PN and PS were upregulated. PS and PN are considered to be beneficial for improving the structural stability of AnGS [19, 20]. Besides, EPS promotes microbial aggregation through ionic bridging and hydrophobicity, and plays a positive role in the formation of AnGS. The increased EPS concentration in R-elec suggested that EF enabled the formation of a more structurally stable granular sludge.

Figure 5

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Fig. 5. EPS concentration of AnGS in R-ctrl and R-elec.

The above discussion demonstrated that the EF facilitated the formation of AnGS by promoting microbial aggregation and stabilization of the granular structure, which improved the electron transfer capacity to produce more MCFAs. However, the in-depth biological effect mechanisms of the AnGS with EF still need to be further elucidated. Therefore, the different microbial community functions were analyzed and illustrated in Fig. 6. Quorum sensing (QS) is an interbacterial communication mechanism that coordinates population behavior through specific signal molecules, which might in turn regulates the formation of granular sludges and the optimization of structural functions [21, 22]. Although the key gene LuxS encoding for Autoinducer 2 (AI-2) synthesis was detected to be upregulated in R-elec, its transporter-associated key genes such as LsrABCD were not detected to be upregulated. However, the abundance of rbsABCD, the key genes controlling AI-2 transporter, were significantly upregulated in the ABC transporters. It's worth noting that wspA encoding for EPS production [23] was found to be upregulated in R-elec, which was consistent with the results in Fig. 5. This suggested that the microorganisms in R-elec could promote the granulation process by generating more EPS. Bacterial chemotaxis (BC) and flagellar assembly (FA) can help microorganisms respond to the environment by enabling bacteria to aggregate more efficiently in localized areas [24, 25], which in turn form the granular sludges. The decreased BC and FA activity in R-elec suggested that the microorganisms have found more suitable locations for survival and functioning, which also indicated that the AnGS in R-elec was more stable compared to that in R-ctrl.

Figure 6

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Fig. 6. Changes in microbial community functions in R-ctrl and R-elec. TCS: two-component system, QS: quorum sensing, BC: bacterial chemotaxis.

In summary, EF was demonstrated to facilitate the formation of AnGS, which significantly enhanced the MCFAs yield. The AnGS formed in R-elec with EF have higher extracellular electron transfer capacity compared to that in R-ctrl. EF promoted microbial aggregation and the synthesis of EPS, which led to the accelerated formation of granular sludges. By elucidating the mechanisms of the biological effects of AnGS in R-elec, key functional genes of QS and EPS production were upregulated to regulate the microbial community functions. Besides, the key genes of BC and FA were downregulated, which improved particle stability. In conclusion, we developed a method for promoting anaerobic granulation with EF, which might improve the efficiency and stability of the CE process to produce more MCFAs, in turn replace more non-renewable energy sources.

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.

Wei-Tong Ren: Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Tian Lan: Software. Zi-Lin He: Software. Hua-Zhe Wang: Project administration. Lin Deng: Software. Shan-Shan Ye: Software. Qing-Lian Wu: Writing – review & editing, Resources, Project administration, Funding acquisition. Wan-Qian Guo: Resources.

This work was supported by the Natural Science Foundation of Heilongjiang Province (No. LH2023E051), Open Project of State Key Laboratory of Urban Water Resource and Environment (No. HC202241), and Young Scientist Studio of Harbin Institute of Technology.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110563.