Flashsmart

The biomass component of the biofilm was assessed for the relative percentage of the main biomass building elements: C, H, N, S and O. For an effective quantification of the relative percentage of each element in the microorganisms, the pellet obtained after EPS extraction was washed with mili-Q water to remove the salts from the EPS extraction solution. Cells were afterwards freeze dried and, after this heat treatment, the elemental composition of the cells grown in each bioanode was assessed with the elemental analyzer (Flash Smart, Thermo Scientific, The Netherlands).

We followed a modified version of the method by Gao et al. (Gao et al. 2021 (link)) to quantify the free amino acids (FAAs) content. Briefly, 0.4 g of finely chopped and thoroughly mixed samples were homogenized in 8 mL of sulfosalicylic acid (4%, w/v) and centrifuged at 3 °C for 10 min at 12,000 g. The resulting supernatant was filtered through a 0.22 µm membrane filter after undergoing another round of centrifugation with the same condition. Subsequently, an automatic amino acid analyzer (S-433D, Sykam, Eresing, Germany) was employed to analyze the filtered supernatant for FAA determination.
In accordance with the Chinese National Standard GB/T 5009.5-2016, we conducted an assessment of the protein content. Specifically, we employed a Kjeldahl apparatus (Hanon Technologies CO., Ltd. Shandong, China) for protein content determination. Total sugar and reducing sugar contents were assessed using ultraviolet spectroscopy (Shanghai Metash Instruments CO., Ltd. Shanghai, China). Starch content was determined through an iodine colorimetry method. Additionally, we utilized an Elemental Analyzer (Flash Smart, Thermo Fisher, America) to detect the chemical elements, including carbon (C), hydrogen (H), sulfur (S), and nitrogen (N).

The soil EC was determined in a mixture with soil: water ratio of 1:5 (w/v) using EC meter (DDSJ-308A, Leici, China). The soil total carbon (TC) and total nitrogen (TN) concentrations were measured using a CHNS/O elemental analyzer (FlashSmart, Thermo Fisher, Germany).

Grass seed (3.2 g) was planted into pots of 10.5 cm diameter and 12 cm depth with 300 g of topsoil compost from a well-mixed bag, bought from a local garden centre. Each set of three replicate pots (15 total) was placed in individual trays to ensure that any water drainage did not mix with or contaminate neighbouring pots. The grass was cut with hand scissors to 2 cm shoot length for each harvest. Pots were watered with 40 mL of DI H₂O twice a week to account for water uptake and evaporation. The fertilisation and harvest cycle for the grasses is outlined in Table 2. The grass from each harvest was dried at 65 °C for 48 h and weighed. The nitrogen % of harvests 1, 2 and 3 were analysed using a Thermo Fisher Scientific Flash SMART with a stainless-steel column and prepacked quartz reaction tube.

Combustion heat was calculated to represent the energy stored in substances. The combustion heat of glucose and ethanol are − 2804 kJ/mol and − 1366.8 kJ/mol, respectively. The combustion heat of biomass and yeast extract was estimated by Mendeleev’s empirical formula. Their elemental compositions were measured by Elemental Analysis (Flash Smart, ThermoFisher, China). After calculation, the estimated combustion heats of biomass and yeast extract were − 18,654.11 kJ/kg and − 15,473.58 kJ/kg, respectively. The electrical energy released by MFC was calculated by integrating the power density with time.

In this study, 12 indices of soil samples were determined, namely pH, soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), available nitrogen (AN), available phosphorus (AP), nitrate nitrogen (NO₃⁻-N), ammonium nitrogen (NH₄⁺-N), β-1,4-glucosidase (BG), N-acetyl-β -D-glucosaminidase (NAG), acid phosphatase (LCP), and leucine aminopeptidase (LAP). Soil pH was determined in suspension with a water-to-soil ratio of 2.5:1 using a pH meter (PHC-3C, Leici, Shanghai, China). SOC, TN, TP, AN, and AP were determined using the method described by Bao (2000 ). SOC and TN were determined using an automatic elemental analyzer (FlashSmart, Thermo Fisher, USA). TP, AN, and AP were determined on a continuous flow analyzer (Flowsys, Systea, Italy). Nitrogen and ammonium nitrogen in the soil were extracted with potassium chloride solution according to ISO standards and determined with a continuous flow analyzer (Flowsys, Systea, Italy). Soil enzyme activity was analyzed for BG, NAG, LCP, and LAP according to the method of Jiao et al. (2022 (link)) and determined with UV-visible spectrophotometer (Specord 200 Plus, Analytik, Germany), with active units expressed as IUg⁻¹ units.