A series of studies were conducted to explore the effect of different size ranges and concentrations of magnetite nanoparticles on methane production from fresh and degassed anaerobically-digested sludge under different concentrations of acetate, propionate and glucose. Mesophilic digested sludge was used as fresh digested sludge and degassed digested sludge sat for one month at 36 ± 1oC in an incubator. Magnetite was added at three different size ranges of small-sized (50 - 150 nm) purchased from Sigma Aldrich, medium-sized (168 – 490 nm) synthesized via the hydrothermal method, and large-sized (800 nm - 4.5 µm) synthesized via the co-precipitation method at different concentrations of 0, 2, 7, and 20 mM. Initial concentrations of COD:VS ratios of 2:1, 4:1 and 8:1 of acetate, propionate, and glucose; as well further COD:VS ratios of 3:1 and 6:1 of glucose were added. Methane production was monitored periodically over the incubation period and the cumulative maximum methane production was plotted with respect to time. Three replicates were done for each test. Kinetic parameters of lag phase duration and maximum methane production rate were estimated by fitting the modified Gompertz model to experimental methane production curves by nonlinear regression using SPSS software. The statistical analysis was done using a general linear model (GLM) procedure with SAS software.

In the case of the degassed sludge, only the medium magnetite at 2 and 7 mM significantly enhanced the methane production rate by 12 %, as compared to the control (no magnetite). In addition, 2 mM of both small and medium-sized magnetite reduced the lag phase by 17 %, as compared to the control. Conversely, adding magnetite (regardless of the size and the concentration) to fresh sludge significantly increased the methane production rate by 32 % while simultaneously decreasing the lag phase by 15 % - 41 %, as compared to the control. No significant differences in both cumulative methane production and methane yield were observed in the presence of magnetite using either fresh or degassed sludge.

The effect of magnetite is a function of magnetite concentration as well as substrate concentration and type. Increasing acetate and glucose COD:VS ratios from 2:1 to 4:1 in the presence of magnetite significantly increased the maximum methane production rate up to 2 times and 2.5 times respectively; whereas no significant difference was obtained in the control bottles. In contrast, by increasing the glucose COD:VS ratio from 3 COD: 1 VS to 6 COD: 1 VS, the maximum methane production rate in the presence of magnetite significantly decreased up to 1.9 times; whereas it significantly decreased by 7.6 times in the control bottles. Also, increasing the acetate COD:VS ratio from 4:1 to 8:1 in the presence of magnetite significantly decreased the lag phase duration in the range of 7.7 % to 32.1 %; whereas in the control bottles, the lag phase significantly increased by 35 %. Similarly, increasing the propionate COD:VS ratio from 2:1 to 4:1 and then from 4:1 to 8:1 in the presence of magnetite significantly increased in the lag phase duration around 4.4 days and 6.3 days, respectively; however, in the control bottles the lag phase increased around 6 days and 10 days, respectively. In addition, increasing the glucose concentration from 3 COD: 1 VS to 6 COD: 1 VS in the presence of magnetite significantly extended the lag phase in the range of 2 days to 11 days; whereas in the control bottles the lag phase significantly extended 24 days. On the other hand, the ratios of 4 COD: 1 VS and 8 COD: 1 VS were the most suitable ratios for acetate and propionate when magnetite was added since they achieved a higher methane production rate by (59 % and 32 %) respectively. Magnetite was not able to easily trigger methane production when glucose concentration exceeded the ratio of 4 COD: 1VS, as that increased the VFA production and accumulation rates which cause a pH drop.

Using small-sized, medium-sized and large-sized magnetite, the theoretical calculations revealed that electrons transferred via the magnetite-direct interspecies electron transfer (DIET) method were higher than those transferred via interspecies hydrogen transfer (IET) from acetate degradation at rates 0.0498 x 106 and 0.539 x 106 and 35 x 106, as well from propionate degradation at rates 0.006272 x 106 and 0.0727 x 106 and 4.72 x 106 respectively. This strongly suggests that magnetite serves as electron conduits between electron-donating and electron-accepting microorganisms. In addition, the flux increases with the size of the magnetite particles. However, the size ratio between the particles and the bacterial cells might play a role and affect DIET promotion.

These results confirm that adding magnetite to an anaerobic digester significantly enhances the rate of methane production in anaerobic digestion. However, this positive effect depends on the type and the concentration of substrate as well as the size of magnetite particles.