1 Introduction

Biomass-based substitute natural gas (BioSNG) can be obtained from biogas upgrading, which is usually produced from traditional anaerobic digestion (AD) by using biodegradable organic wastes such as livestock manure and household waste as the substrate. The technology is mature, but the amount of the organic wastes is limited for large-scale production of BioSNG. On the other hand, the huge amount of lignocellulosic biomass residues from forest and agricultural activities such as logging residues and straw is available, but difficult to be digested into biogas [1]. The lignocellulosic biomass can be easily gasified into syngas or product gas under high temperature, which is composed of H2, CO, CO2, and N2 with a higher heating value from 4 to 12 MJ/m3 depending on the gasification agent of air, oxygen, or steam.

There are two pathways to convert the product gas into methane, high-temperature catalytic synthesis, and low-temperature AD or biomethanation. The catalytic synthesis process requires harsh reaction conditions such as high temperature over 250℃, high pressure of 3–25 bar [2], expensive catalysts, strict gas composition, and especially clean syngas. The catalytic synthesis has been commercially demonstrated to be able to produce BioSNG from the syngas of biomass steam gasification, but would be difficult to treat the product gas from biomass air gasification due to the high nitrogen content [3,4,5,6]. The disadvantages of catalytic synthesis can be avoided by biomethanation which is particularly suitable for the product gas from cheap biomass air gasification. Thus, large-scale BioSNG production from lignocellulosic biomass at a low cost can be expected if the technology of the PGB can be well developed.

Previous studies [4, 7,8,9,10] have shown that syngas biomethanation is a complex biochemical process involving various intermediate species and different pathways. The key factors that can affect the biochemical process are the operation parameters such as the syngas composition, temperature, inoculum, biosludge enrichment, and partial grinding [11].

CO is one of the important substrates to the biomethanation as an energy carrier, and also an inhibitor to methanogenetic bacteria [12]. On one hand, CO can provide electrons [13] for microorganisms in fermentation and supply carbon source for microbial growth[14]. On the other hand, CO has a toxic effect on a considerable number of microorganisms, leading to methanogenesis inhibition. The pathways of CO biomethanation [15] can be summarized in Fig. 1 based on the literature[16, 17]. Sipma et al. [18] found that CO biomethanation at the mesophilic temperature is mainly completed through Reactions 1 and 4, and acetate is the key intermediate product. At the thermophilic temperature, CO biomethanation passes through Reactions 2 and 5, and H2 is the important intermediate product.

Fig. 1
figure 1

CO biomethanation pathways

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Temperature can increase the biomethanation reaction rate and change the dominant intermediate products. Sipma et al. [18] studied the dependency of CO conversion efficiency on temperature and showed that the yield of CH4 increased gradually with the temperature at 40–55℃, and reached the peak at 55℃. Guiot et al. [19] compared the rates of methane production from sludge particles under mesophilic and thermophilic conditions and concluded that high temperature can lead to less systematic stability and low gas–liquid mass transfer.

Many microorganisms that are able to convert product gas into methane exist in nature such as Methanocalculaceae, Methanocorpusculaceae, Methanomicrobiaceae, Methanoregulaceae, and Methanospirillaceae from Methanosarcinales [20], but the conversion efficiency is often poor. One reason is the low activities of the inoculum, which can be improved by enrichment [21]. Bu et al.[15] found that the biomethanation lag phase before methane starts to be produced was shortened after inoculum enrichment treatment. Navarro et al. [10] found that, during the enrichment time, the inhibition of CO on microbial Methanogenis is gradually decreased in response to a gradually increasing CO consumption rate. However, too long time enrichment may give rise to negative effect as shown by [14] that incubation with syngas or CO caused a rapid decrease in the microbial diversity of the anaerobic consortium during enrichment and a stop in biomethanation. The process of enrichment is governed by both kinetic and thermodynamic control [20].

Biosludge partial grinding can help to increase gas–liquid mass transfer, but also destruct the shield of inoculum sludges particle and increase the toxicity of poisonous gas such as H2S and CO at high partial pressure. Navarro et al. [10] have shown that the maximum methanogenic activity was achieved at 0.2 atm of CO with grinding sludge. Fully exposed sludges may lead to a lower rate of product gas biomethanation.

So far, the research works reported in the literature on AD with gas substates have been focusing on the syngas of H2 + CO or simply on CO. No study on the product gas from biomass air gasification with high N2 content has been found. In this experimental work, the product gas from biomass air gasification is used as the AD substrate. The key biomethanation parameters, temperature, inoculum enrichment, and biosludge partial grinding are studied with respect to their influences on the biomethanation process. The biochemical process will be discussed based on relative abundance of microbial colonies, the biocarbon variation in the microorganisms, and Shannon diversity index (SDI).

2 Material and method

2.1 Experimental apparatus and inoculum

The biomethanation reactor used in this study is a 320-mL serum bottle, which is connected to a 100-mL volume airbag by a 6-mm silicone tube as seen in Fig. 2. The volume of the airbag can be changed in the range of 0–100 mL to maintain a constant pressure of 1 atm in the bottle. Two steel pipes are inserted into the bottle for liquid and gas sampling respectively.

Fig. 2
figure 2

The biomethanation batch bioreactor

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Our previous study [22] has shown that the biosludge from juice wastewater treatment plant as the inoculum gave the best syngas biomethanation performance in comparison with other 5 different biosludges. In this work, the juice biosludge was employed as the inoculum, which was provided by Jiangxi Sheerly Environmental Protection Technology CO. Ltd. The biosludge was obtained from an internal circulation reactor at the mesophilic temperature of 30–40 °C. The total solids (TS) of the anaerobic inoculum were 49.5 ± 1.6 g/L, and the volatile solids (VS) 37.4 ± 1.7 g/L.

The inorganic salts, minerals, vitamins, and other inorganic nutrients required for microbial culture growth were provided by a basal anaerobic (BA) medium. The medium components are shown in Table 1.

Table 1 BA medium composition
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The gas substrate used in this study is a mixture of H2 5%, CO 25%, CO2 20%, and N2 50% in volume, which simulates a typical product gas generated from biomass air gasification [12].

The experimental serum bottle was sterilized at a high temperature of 121 °C for 21 min. After cooling, 15 mL sludge, 60 mL BA, and 0.03 g cysteine hydrochloride were added. The interface was sealed with hot melt adhesive to ensure gas tightness. Before the experimental test, the reactor was flushed with 0.1 L/min product gas for 20 min to exhaust the air in the reactor. The BA was diluted with the laboratory-pure water. The BA and the biosludge were storied in sterilized environment. Cysteine hydrochloride was also pure and sterilized. The above experimental materials were added into the bioreactor medium by a disposable injector through the valve.

After flushing, the entire reactor was filled with the gas at 1 atm. Then, a syringe was used to adjust the gasbag volume to 100 mL through the gas sampling valve. Therefore, the volume of the headspace gas of the bottle is 245 mL and the volume of the gas in the gasbag is 100 mL, leading to the total volume of the gas substrate equal to 345 mL. The total volume of the liquid is 75 mL. During the test, 2 mL gas and 4 mL liquid were siphoned out every 2 days to measure the gas composition and the liquid pH in the first 2 weeks, and then gas and liquid chemical components were measured every 3 days. The pH was then adjusted to 6.5–7.5 by 4 M HCl or 4 M NaOH, which is diluted by the laboratory-pure water and stored in sterilized environment if necessary. The biomethanation reactor was cultured in a shaker-bed at 80 rpm and at two different temperatures of 37 °C and 55 °C in the dark.

2.2 Experimental design

The experimental test was designed to evaluate three factors that might change PGB performance: biosludge enrichment, inoculum partial grinding, and reactor temperature, as presented in Table 2. Six test runs were arranged with or without biosludge enrichment, and with or without inoculum partial grinding under mesophilic and thermophilic conditions, respectively.

Table 2 Experimental conditions
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The purpose of enrichment is not only to adapt microorganisms to the product gas environment but also to consume the original digestible organic substance in the sludge. It has been reported that the utilization of syngas by microorganisms during AD and the organic substance AD are different significantly [23]. A comparative study of the gas substrate consumption rate by microbial flora before and after enrichment can provide a reference for whether it is necessary to pretreat sludge in industrial fermentation processes to achieve strain optimization. In Table 2, Tests 1–4 were used to study the enrichment performance under mesophilic and thermophilic conditions respectively. The enrichment experiment begins after the introduction of product gas and ends when the gas components in the headspace of the reactor are stabilized.

To speed up the reaction rate of the PGB, the gas–liquid mass transfer and the structure of sludge particles are the key issues [11] that were studied in the experiment by means of biosludge partial grinding. The partial grinding treatment was conducted by stirring directly in the anaerobic environment at a rotation speed of 60r/min for 5 min.

Figure 3a shows the microscopic view on the surface of the sludge particles in a 20 µm scale. The microstructure seems like a big circle membrane, which offers the active sites of microbial flora. The unground sludge particles are smooth and intact as seen in Fig. 3b. For the ground sludge particles as seen in Fig. 3c, the originally intact sludge particle surface has been broken, so that the specific surface area of the sludge particles is increased.

Fig. 3
figure 3

a Surface microstructure of 20 µm sludge particles, b unground sludge particles, and c ground sludge particles

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2.3 Analytical methods

TS and VS were determined by heating the samples in an oven at 105 ℃ overnight until the quality remained unchanged, and then by burning the samples in a furnace at 600 ℃ for 2 h. The solubility of H2, CO, CO2, and CH4 was calculated using Lide’s data [24]. The pH value of the solution was measured by the pH meter and the measurement accuracy is 0.01. The gas components of H2, CO, CO2, and CH4 were analyzed by gas chromatography (Shimadzu GC-2014, Kyoto, Japan) equipped with a thermal conductivity detector and a packed column (TDX- 01, 2 m × 3 mm, Stainless Steel). The carrier gas was argon and the flow rate was 30 mL/min. The injector and detector temperatures are 110 °C and 160 °C, respectively. The column temperature was first held at 70 °C for 3 min, then increased to 100 °C at a rate of 3 °C/min, and finally held at 100 °C for 0.5 min. The liquid composition including acetate, propionate, butyrate, and isobutyrate was measured by means of an Agilent 7820A (Wilmington, Delaware, USA) equipped with a flame ionization detector (FID) and DB-FFAP column (30 m □ 0.25 mm □ 0.25 µm). Nitrogen was used as the carrier gas at flow rate of 40 cm/s. The temperature of inlet and detector was set up to be 250 °C and 300 °C, respectively. The GC oven was programmed to begin at 100 °C, held for 5 min, then increased at a rate of 10 °C/min to 250 °C, and finally held at 250 °C for 12 min.

In the PGB process, the internal community structure of microorganisms plays an important role. The relative abundance and diversity of the internal communities of Archaea were measured by 16S rRNA gene analysis as follows. Samples of the inoculum were collected for microbial community analysis. Total genomic DNA was extracted using the DNeasy PowerSoil DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions[25]. 16S rRNA amplification and quantification were conducted using the SmartChip Real-time PCR system (Wafergen Inc. USA). The 16S rRNA gene V4 ~ V5 hypervariable region of bacteria and archaea was amplified using the universal forward 515F (5′-GTGCCAGCMGCCGCGG-3′) and the reverse 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) primers. 7-nucleotide barcodes were permuted for each sample to allow for the identification of individual samples in a mixture within a single pyrosequencing run. The conventional end-point PCR mixture (final volume 25 µL) contained 17.2 µL ddH2O; 2.5 µL 10 ℃ PCR Buffer (Mg2 + Plus); 2.5 mM dNTP mixture; 10 µM of each primer; 1.5 U of Taq DNA Polymerase; and 1 µL of template DNA[25]. The thermal cycling program included an initial denaturation stage at 95 ℃ for 5 min, followed by 24 cycles of denaturation at 95 ℃ for 30 s, annealing at 56 ℃ for 30 s and extension at 72 ℃ for 90 s, and then a final extension stage at 72 ℃ for 8 min[25]. An AxyPrep DNA Gel Extraction Kit (Axygen, Silicon Valley, USA) was used to purify 4the PCR amplification products. All purified amplified DNA samples were stored at − 20 ℃ for downstream analysis. High-throughput sequencing of 16S rRNA was conducted using the Ion GeneStudio S5 (Thermo Fisher Scientific, Waltham, USA).

2.4 Calculation of biocarbon variation in the microorganisms

In order to evaluate the variation of the microbial community during the incubation process, the amount of biocarbon contained in the microorganisms needs to be calculated based on carbon balance over the bioreactor system [26]. The carbon balance can be expressed in Eq. 1

$${C}_{\mathrm{gas}}+{C}_{\mathrm{liquid}}+{C}_{\mathrm{microb}}=T$$
(1)

where \({C}_{\mathrm{gas}} ,{C}_{\mathrm{liquid}},\mathrm{ and }{C}_{\mathrm{microb}}\) are the carbon contained in the gas, liquid, and microorganisms. T is constant once the filling of the gas substrate in the reactor gas bag is completed in a batch experimental test.

The carbon balance at the test initial state is

$${C}_{\mathrm{gas\;}0}+{C}_{\mathrm{liquid\;}0}+{C}_{\mathrm{microb\;}0}=T$$
(2)

The carbon balance on i day of the experiment is

$${C}_{\mathrm{gas\;}i}+{C}_{\mathrm{liquid\;}i}+{C}_{\mathrm{microb\;}i}=T$$
(3)

Since a certain amount of gas and liquid are extracted in each sampling for the measurement of the gas and liquid components, the carbon loss from the reactor system is inevitable and the cumulative loss is significant. The carbon loss from the gas and liquid sampling is calculated according to the following formula. The carbon loss from the gas on i day is

$${C}_{\mathrm{gas\;loss\;}i}={V}_{\mathrm{gas}}\frac{\left({P}_{\mathrm{CO}i}+{P}_{{\mathrm{CO}}_{2i}}+{P}_{\mathrm{CH}4i}\right)}{22.4}$$
(4)

where \({P}_{\mathrm{CO}},{P}_{{\mathrm{CO}}_{2}}, {\mathrm{and }P}_{{\mathrm{CH}}_{4}}\) represent the partial pressure of CO, CO2, and CH4 in the reactor headspace, and \({V}_{\mathrm{gas}}\) the volume of the extracted gas. \({P}_{\mathrm{CO}},{P}_{{\mathrm{CO}}_{2}},{\mathrm{and }P}_{{\mathrm{CH}}_{4}}\) are determined by the product of the total pressure in the reactor headspace and each gas concentration.

In the process of PGB, many volatile fatty acids (VFAs) are produced as the intermediate or end products in the liquid phase. The major components are supposed to be acetic, propionic butyric, and isobutyric acids which are taken into account in the carbon balance calculation base on literature [23].

The carbon loss on i day in the liquid phase is

$${C}_{\mathrm{liquid\;loss\;}i}={V}_{\mathrm{liquid}}\left(\frac{{C}_{{\mathrm{BC}}_{2}{\mathrm{H}}_{4}{\mathrm{O}}_{2}i}}{{M}_{{\mathrm{C}}_{2}{\mathrm{H}}_{4}{\mathrm{O}}_{2}}}+\frac{{C}_{{\mathrm{BC}}_{3}{\mathrm{H}}_{6}{\mathrm{O}}_{2}i}}{{M}_{{\mathrm{C}}_{3}{\mathrm{H}}_{6}{\mathrm{O}}_{2}}}+\frac{{C}_{{\mathrm{BC}}_{4}{\mathrm{H}}_{8}{\mathrm{O}}_{2}i}}{{M}_{{\mathrm{C}}_{4}{\mathrm{H}}_{8}{\mathrm{O}}_{2}}}\right)$$
(5)

where \({C}_{{\mathrm{BC}}_{2}{\mathrm{H}}_{4}{\mathrm{O}}_{2}},{C}_{{\mathrm{BC}}_{3}{\mathrm{H}}_{6}{\mathrm{O}}_{2}},\mathrm{ and }{C}_{{\mathrm{BC}}_{4}{\mathrm{H}}_{8}{\mathrm{O}}_{2}}\) are the concentration of acetic acid, propionic acid, and butyric acid (isobutyric acid) in the liquid, and M is the corresponding molar mass fraction

The cumulative carbon loss on i day is

$${C}_{i}^{\mathrm{^{\prime}}}=\sum \nolimits_{i=1}^{n}{ ({C}_{\mathrm{gas\;loss\;}i}+C}_{\mathrm{liquid\;loss\;}i})$$
(6)

Therefore, actual amount of carbon on i day is

$${C\mathrm{^{\prime}}}_{\mathrm{gas\;}i}+{C\mathrm{^{\prime}}}_{\mathrm{liquid\;}i}+{C}_{\mathrm{microb\;}i}+{C\mathrm{^{\prime}}}_{i}=T$$
(7)

The total biocarbon change, \(\Delta {C}_{\mathrm{microb}}\), in the microorganism can be calculated by Eq. 7 minus Eq. 2:

$$\Delta {C}_{\mathrm{microb}}=-\left(\Delta {C}_{\mathrm{gas}}+\Delta {C}_{\mathrm{liquid}}+{C}_{i}^{\mathrm{^{\prime}}}\right)$$
(8)

\(\Delta {C}_{\mathrm{gas}}+\Delta {C}_{\mathrm{liquid}}+\Delta {C}_{i}^{\mathrm{^{\prime}}}>0\) indicates a loss of carbon in the inoculated microorganism. \(\Delta {C}_{\mathrm{gas}}+\Delta {C}_{\mathrm{liquid}}+\Delta {C}_{i}^{\mathrm{^{\prime}}}=0\) means that the carbon in the microorganism is in a state of self-equilibrium. \(\Delta {C}_{\mathrm{gas}}+\Delta {C}_{\mathrm{liquid}}+\Delta {C}_{i}^{\mathrm{^{\prime}}}<0\) indicates an increase of biocarbon in the microorganism.

3 Results and discussion

3.1 Effect of enrichment on microorganisms

During the PGB process, the organic matter remaining in the original biosludge is converted to H2, CO2, and CH4, but the direct conversion from the organic matter to CO can be ignored. It has been reported that syngas AD by microorganisms does not affect the hydrolytic acidification of the organic matter[23]. Thus, the variation of CO consumption rate during enrichment can be a valuable index to study the effect of enrichment on microorganisms.

The enrichment performance under both the mesophilic and thermophilic conditions is shown in Fig. 4 in comparison to the cases without enrichment. For all the cases with and without enrichment, CO in the bioreactor headspace is consumed fully within a couple of weeks, but the consumption rate varies significantly depending on whether or not the biosludge has been enriched. For the cases with enrichment, CO is consumed much faster than the cases without enrichment, especially in the beginning of the PGB process. The CO conversion rate of the whole process is increased by 25% at 35℃, which is higher than at 55℃, similar to the previous study [22]. Whether it is mesophilic or thermophilic PGB, enrichment can effectively increase the reaction rate and set up the PGB process quickly as soon as the product gas is introduced.

Fig. 4
figure 4

Effect of enrichment on CO conversion under mesophilic or thermophilic conditions

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From the microbial point of view, the enrichment is a process of directional screening, acclimation, and growth. The microbial colony richness in the original biosludge and the biosludge after enrichment under the mesophilic and thermophilic conditions is presented in Fig. 5 by means of 16S rRNA analysis. The abundance and SDI of Archaea can be measured by 16S rRNA gene analysis for each test sample. The relative abundance is thus calculated based on their common sequence of length, which reflect the variation in the microorganism. It can be seen that the microbial colonies in the original biosludge from juice wastewater treatment plant are mainly Methanosaeta, Thermoprotei, Methanobacterium, and Micrarchaeles methane flora and thermophilic methane flora which can convert CO into VFAs and methane. However, the degree of microorganism adaptation to the product gas is fairly low and the CO selectivity is poor. After enrichment, the richness of each colony in the sludge is changed significantly. In particular, the Methanobacterium relative abundance is increased from 14% in the original biosludge to 28–31% after enrichment under the mesophilic condition, and becomes the dominant strain. On the other hand, Thrmautoropics is turned to be the dominant strain after enrichment under the thermophilic condition although the Methanobacterium relative abundance is increased up to 21–22%. These results are in a good response to the enrichment effect indicated in Fig. 4 that the CO consumption rates are increased for both cases of 35℃ and 55℃, but higher at 35℃ than 55℃. The enrichment process can indeed effectively promote the adaptation of the microbiota to the product gas atmosphere.

Fig. 5
figure 5

Relative abundance of microbial colonies for the inoculum after enrichment in comparison to the original biosludge

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3.2 Effect of temperature on PGB

Figure 6 shows the gas composition in the bioreactor headspace and the volatile fatty acids (VFAs) in the liquid phase against the time of PGB under (a) the mesophilic and (b) thermophilic conditions, respectively. It can be seen that CO and H2 are rapidly consumed following the methane production as well as the appearance of VFAs intermediate products for both the cases of 35℃ and 55℃. The inoculum used has been enriched so that methane is produced immediately without CO inhibition effect. The biomethanation reaction rates are high under both mesophilic and thermophilic conditions. Comparing the mesophilic condition, however, the methane yield under the thermophilic condition is slightly higher and the biomethanation time is shorter. The CH4 conversion efficiency can be calculated as the actual CH4 yield after biomethanation completion divided by the theoretical CH4 yield calculated with chemical reaction Eqs. 1–5. The methane conversion efficiency is 98.9% and the biomethanation time 8 days under the thermophilic condition, but 75% and 16 days under the mesophilic condition. Thus, it can conclude that the thermophilic condition is superior to the mesophilic condition for PGB, especially when a high-temperature product gas obtained from biomass gasification is considered.

Fig. 6
figure 6

PGB under different experimental conditions of a 35℃ En and b 55℃ En

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It should be noted from Fig. 6 that CO2 content in the headspace increases during the biomethanation process of 6–10 days and then gradually decreases back to the original level after about 20 days. The product gas has a CO-rich gas composition with respect to the methane synthesis from H2 and CO. A certain amount of CO2 should be rejected from the PGB, so that CO2 content should have been increased in the end of the process, which seems contradictory against the result given in Fig. 6.

CO biomethanation at the mesophilic temperature proceeds mainly through the pathway of Reactions 1 and 4 indicated in Fig. 1, and acetate is the key intermediate product. At the thermophilic temperature, CO biomethanation passes through Reactions 2 and 5, and H2 is the key intermediate product. From Reactions 1 and 2, it can be seen that CO2 is produced during the biomethanation process, leading to an increase of CO2 content in the headspace, and more CO2 must be produced at the thermophilic temperature than the mesophilic temperature in agreement with the experimental result in Fig. 6. In order to understand the gradual decreasing of CO2 content to the original level after the methane production is completed, a carbon balance was made over the entire reactor system including the microbial carbon contained in the microorganisms. The biocarbon variation as a function of the reaction time is shown in Fig. 7.

Fig. 7
figure 7

Microbial carbon analysis

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It can be seen from Fig. 7 that the biocarbon content in the microorganisms increases with the biomethanation time, and reaches the platform of the maximum value after 20 days. The biocarbon accumulation in the microorganisms is the reason behind the gradual decreasing of CO2 content in the headspace. Under the thermophilic condition, biocarbon content decreases in the beginning of biomethanation over 6 days. The biosludge used in the experiment was obtained from the mesophilic (30–40℃) bioreactors, which might lose a part of biocarbon due to poor stability of the microorganisms at higher temperature, but then the number of dominant strains gradually increases, so that the biocarbon content begins to rise. As a conclusion, the CO2 reduction or production during biomethanation is also associated to the carbon conversion in the microorganisms.

In the liquid phase, VFAs, as the intermediate product, reach a peak in 4–6 days, in response to the rapid consumption of H2 and CO. Acetate and propionate are the two dominant VFA components produced as the intermediate products during the PGB as seen in Fig. 6. Most of VFAs are converted to methane by the end of biomethanation process. A small part of VFAs remains in the liquid phase due to a minimum concentration limit for the utilization of VFA in solution by Aceticlastic methanogen [27].

3.3 Effect of inoculum partial grinding on the PGB

The experimental tests described in Fig. 6 were repeated but with the inoculum after partial grinding treatment in order to investigate the effect of inoculum partial grinding on the PGB. The results are shown in Fig. 8 for two cases of (a) at 35℃ and (b) at 55℃. Similar to Fig. 6, CO and H2 in the product gas are rapidly consumed in response to the methane production without an obvious difference in consumption rate. Also, the methane content in the headspace reaches the maximum and the PGB is completed in 8 days under the mesophilic condition and in 16 days under the thermophilic condition, the same to Fig. 6. The same conclusions from Fig. 6 can also be made from Fig. 8 regarding the difference between the mesophilic and thermophilic conditions for the methane conversion efficiency, CO2 variation, etc.

Fig. 8
figure 8

PGB after sludge partial grinding treatment for the two cases of a 35℃ En Gr and b 55℃ En Gr

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Differently from Fig. 6, however, more methane is produced after inoculum partial grinding treatment as shown in Fig. 8. The methane conversion efficiency increases significantly from 75.0% without grinding treatment to 93.4% with grinding treatment under the mesophilic condition, and from 98.9% to over 100% theoretic limit under the thermophilic condition. Clearly, grinding of the wall surface of the biosludge particles can significantly improve the biomethanation conversion efficiency.

In the case of mesophilic condition as shown in Fig. 8a, VFAs content in the liquid phase decrease by 0.11 mmol in response to an increase of methane content by 0.32 mmol over the biomethanation period of time. The biosludge partial grinding treatment is conducive to accelerate the conversion of VFAs to CH4 during the reaction process.

In the case of thermophilic condition as shown in Fig. 8b, the methane yield is high, ends up to 100% and even over 100%, the theoretical limit from day 11 to 25 of the biomethanation process. In the same period of time, \(\Delta {C}_{\mathrm{micro}}\) is lower for the cases with biosludge particle treatment than the cases without the treatment. A part of biocarbon is converted to methane and gives rise to more methane production than the theoretical limit.

The effect of inoculum partial grinding on the PGB can also be analyzed based on the relative abundance of microbial colonies as shown in Fig. 5. Under the mesophilic condition, the relative abundance of Methanosaeta is significantly reduced by the partial grinding treatment, while other bacterial communities are not sensitive to the partial grinding treatment. Methanosaeta is supposed to be more sensitive to CO. Under the thermophilic condition, the relative abundance is slightly reduced except for Thermautotrophics and Thermoprotei. As a conclusion, the inoculum partial grinding treatment can clearly change the microorganism properties for the mesophilic biomethanation, but not for the thermophilic biomethanation.

Figure 9 shows the variation of microbial diversity based on the Shannon index in order to further evaluate the inoculum partial grinding effect. The SDI increases rapidly to a platform as the sample sequence increases. The higher SDI of the sample group means the higher diversity of the microorganisms. For the cases of thermophilic biomethanation, the curves coincide with each other so that a single curve appears for thermophilic biomethanations with and without partial grinding treatment. The diversity analysis suggests that the diversity of microbial colonies is higher at the mesophilic temperature, and the impact of grinding treatment on microbial diversity at the mesophilic temperature is greater than at the thermophilic temperatures. As a conclusion based on the perspective of microorganisms, inoculum partial grinding can effectively reduce the diversity of microorganisms in the mixed bacterium system, thereby improving the PGB efficiency for the mesophilic biomethanation, which is limited for the thermophilic biomethanation.

Fig. 9
figure 9

Microbial diversity (the curves of “55ºC En” and “55ºC En Gr” coincide so that a single curve appears)

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4 Conclusions

In this experimental work, the PGB was studied with respect to the biosludge enrichment and inoculum partial grinding as well as the mesophilic and thermophilic conditions. The major conclusions can be drawn below:

The biosludge enrichment can effectively stop methanogenesis inhibition from the product gas, particularly CO, thus increase the biomethanation reaction rate and shorten the reaction start-up time, which can be attributed to the change of microorganism richness.

The methane conversion efficiency increases significantly from 75.0% without grinding treatment to 93.4% with grinding treatment under the mesophilic condition. The inoculum partial grinding treatment can clearly change the microorganism composition and effectively reduce the diversity of microorganisms in the mixed bacterium system for the mesophilic biomethanation, thereby improving the PGB efficiency, which is limited for the thermophilic biomethanation.