Determine the optimal parameters for biogas production from common reed (Phragmites australis)

doi:10.21203/rs.3.rs-3087709/v1

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Determine the optimal parameters for biogas production from common reed (Phragmites australis)

https://doi.org/10.21203/rs.3.rs-3087709/v1

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published 14 Nov, 2023

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A series of batch assays have been conducted to investigate the optimal factors that can be adopted to improve the anaerobic digestion (AD) performance of Phragmites australis (P. australis) and increase biogas production. The assays were carried out using 125 ml microcosm reactors with a working volume of 80 ml and incubated at mesophilic conditions 37 C. The effect of particle size (10, 5, 2, and < 1 mm) and alkaline pre-treatment of P. australis using various concentrations of sodium hydroxide concentration (0.5, 1, 2, and 4%) on biogas production were examined. Furthermore, the best pre-treatment incubation time (12, 24, 48, 72, 96, and 120 hours) and the optimal inoculum to substrate ratio (ISR: 4:1, 2:1, 1:1, 1:2 and 1:4) were also assessed. The results revealed that the highest biogas production from P. australis was achieved at: particle size < 1 mm; 2% NaOH concentration for pre-treatment; 120 hours treatment incubation time; and ISR of 1:2.

Anaerobic digestion

Phragmites australis

Pre-treatment

Inoculum to substrate ratio

Over the past few decades, overconsumption of fossil fuels has led to increased greenhouse gas emissions, aggravating global warming, and transforming it into a paramount environmental concern worldwide. Hence, adopting renewable energy sources, such as solar, wind, and biofuel, has become crucial as an alternative to fossil fuels [1]. Consequently, biogas, one of the biofuel types, has received increasing interest in recent years [2]. For example, in the European Union, biogas production reached the equivalent of 10.9 million tonnes of oil in 2010 [3]. Furthermore, according to the International Energy Agency [4], in 2018, the consumption of 36 million tonnes of fossil fuel was displaced by using biogas. Biogas is a mixture of gases (50–75% methane, 25–50% carbon dioxide, and 0–10% other gases) that is produced through the anaerobic digestion (AD) of organic substrates [5, 6]. Biogas resulting from AD is usually used in producing electricity and heating or as fuel for transport after an upgrade to biomethane. Furthermore, the waste from the digestion of organic matter can be used as valuable fertilizer [7–10].

Energy crops have been widely used in AD plants as feedstocks for biogas production [11, 12]. However, the competition of these crops with food and feed production and on agricultural lands is the crucial obstacle that affected their position as a significant supplier of biomass for AD [13, 14]. This led to the development of the trend towards the use of alternative substrates such as municipal organic waste, industrial food waste, animal manure, agricultural waste, perennial grasses, and wetland plants [15–18].

P. australis is one of the perennial grasses characterised by high productivity ranging from 3 t/ha/y up to 30 t/ha/y[19] and does not compete for arable lands, so it is probably to be one of the promising feedstocks in the field of biogas production [20–23]. P. australis is a tall grass; its length often ranges from 1–3 m and may reach 10 m in the tropics [24]. It is a widespread plant worldwide; the total area covered by P. australis beds worldwide is about 10 million hectares [21, 25]. Wetlands are the main habitats for P. australis, in which the water table is usually slightly under the soil surface to 1 m above the soil surface [26].

In general, there are several key parameters that should be considered to examine the potential for biogas production using P. australis substrates, such as substrate particle size, substrate pre-treatment, pre-treatment incubation time, and inoculum to substrate ratio. As a lignocellulosic biomass, P. australis has a complex molecular structure causing difficulty in their digestion. Therefore, the pre-treatment step is essential to increase the biodegradability of these substrates and enhance biogas production. Physical (mechanical) pre-treatment, such as grinding, assists in reducing particle sizes of the substrate, thus decreasing the degree of crystallinity of the cellulose, and increasing the surface area that can be reached by hydrolysis enzymes [27–29]. The grinding process is considered as the primary step to prepare the lignocellulosic substrates for anaerobic digestion [30].

However, mechanical treatment cannot remove the lignin, which impedes cellulose bioaccessibility [31]. Hence, other methods, such as chemical treatment, can overcome this obstacle. Alkaline treatment has preferred over other chemical treatments for treating lignocellulosic substrate due to its high ability to solubilise lignin [28, 32, 33], fewer inhibitors generation, and lower requirement for equipment such as complicated reactors [33, 34]. In addition, alkaline pre-treatment can be conducted at ambient temperature and pressure [35, 36].

In addition to the substrate particle size and alkaline pre-treatment, the inoculum-to-substrate ratio (ISR) is considered a crucial parameter that improves the performance of AD and enhances biogas production [37, 38]. Providing optimal quantities of microbial aggregates and their nutrient requirements shortens the start-up period for biogas production and reduces the accumulation of inhibitors [39–41]. The appropriate selection of ISR depends on the substrate type and the digestion conditions [42]. However, when the ISR is lower than the optimum ratio, it will lead to an accumulation of VFA and inhibition of the system. In contrast, when the ISR value is higher than the optimum ratio, it will decrease the amount of nutrients required for the microorganisms [43–45].

Hence, this study aims to investigate (I) the potential of P. australis substrate for producing biogas through anaerobic digestion; (II) the optimum particle size (10, 5, 2, and < 1 mm) of P. australis substrate that could produce the highest biogas amount during the anaerobic digestion process; (III) the effect of pre-treatment of P. australis on biogas production using various concentrations of sodium hydroxide (0.5, 1, 2, and 4%); (IV) the best incubation time for NaOH treatment (12, 24, 48, 72, 96, and 120 hours); and (V) the optimal inoculum to substrate ratio (ISR = 4:1, 2:1, 1:1, 1:2 and 1:4) that increase biogas production.

The P. australis samples used in the experiments were collected from a small lake at Forrest Hills site, 1 km southeast of Lancaster University, UK. P. australis plants with a length of about 2.5 meters were selected. The aboveground biomass was harvested at ~ 5–10 cm over the soil surface, and leaves and flowers were cut to get the stalks only. The P. australis stalks were packaged in sealed bags and taken to the laboratory and kept in the refrigerator at 4°C until used in the experiments. The methanogenic inoculum was obtained from an on-farm commercial scale, mesophilic, anaerobic digester (Cockerham Green Energy Limited, UK). The inoculum was sieved through a 1 mm sieve to remove coarse materials.

Only the stalks of P. australis were used in all batch assays. In the assay that investigated the optimal particle size of P. australis for biogas production, the harvested P. australis stalks were chopped manually by scissors into four groups of particles size 10, 5, 2, and < 1 mm. While in the rest of the batch assays, the harvested P. australis stalks were ground by a knife mill into particles size < 1mm, following which the P. australis samples were packed in sealed plastic bags and stored in the refrigerator at 4°C until further use. Characterisation of the substrate and inoculum are presented in Table 1.

Table 1

Inoculum, pre-treated *P. australis* and simulated food waste characteristics (mean ± standard deviation).
Batch experiment	Materials	Parameters
Batch experiment	Materials	TS (%)	VS (% dry basis)	VS (% wet basis)	pH
Batch assay to investigate the optimal particles size of pre-treated P. australis (2% NaOH for 3 days) for biogas production.	Inoculum	6.91 ± 0.01	61.36 ± 0.13	4.24 ± 0.02	8.48 ± 0.01
	Pre-treated P. australis < 1mm	98.12 ± 0.02	99.07 ± 0.02	97.20 ± 0.05	7.00 ± 0.00
	Pre-treated P. australis 2mm	98.54 ± 0.10	99.09 ± 0.01	97.65 ± 0.11	7.00 ± 0.00
	Pre-treated P. australis 5mm	98.35 ± 0.00	98.96 ± 0.02	97.33 ± 0.02	7.00 ± 0.00
	Pre-treated P. australis 10mm	98.73 ± 0.26	98.93 ± 0.00	97.67 ± 0.26	7.00 ± 0.00
Batch assay to investigate the optimal particles size of untreated P. australis for biogas production.	Inoculum	6.90 ± 0.00	62.11 ± 0.49	4.29 ± 0.04	8.51 ± 0.00
	Untreated P. australis < 1mm	98.47 ± 0.02	98.10 ± 0.04	96.60 ± 0.06	7.00 ± 0.00
	Untreated P. australis 2mm	98.56 ± 0.03	98.82 ± 0.05	97.40 ± 0.02	7.00 ± 0.00
	Untreated P. australis 5mm	98.57 ± 0.04	99.03 ± 0.02	97.62 ± 0.02	7.00 ± 0.00
	Untreated P. australis 10mm	98.72 ± 0.05	99.06 ± 0.02	97.79 ± 0.03	7.00 ± 0.00
Batch assay to investigate the optimal inoculum to substrate (< 1 mm pre-treated P. australis with 2% NaOH for 3 days) ratio for biogas production.	Inoculum	6.77 ± 0.10	62.20 ± 0.82	4.21 ± 0.01	8.50 ± 0.00
	Pre-treated P. australis	98.74 ± 0.06	99.17 ± 0.05	97.90 ± 0.06	7.00 ± 0.00
Batch assay to investigate the optimal inoculum to substrate (< 1 mm untreated P. australis) ratio for biogas production.	Inoculum	6.47 ± 0.02	59.83 ± 0.10	3.87 ± 0.00	8.56 ± 0.01
	Untreated P. australis	96.30 ± 0.10	98.24 ± 0.06	94.50 ± 0.09	7.00 ± 0.00
Batch assay to investigate the optimal NaOH concentration for pre-treating of < 1mm P. australis.	Inoculum	6.37 ± 0.06	61.57 ± 0.24	3.92 ± 0.02	8.59 ± 0.00
	Pre-treated P. australis (0.5% NaOH)	98.02 ± 0.10	99.08 ± 0.03	97.12 ± 0.13	7.00 ± 0.00
	Pre-treated P. australis (1% NaOH)	98.04 ± 0.08	99.06 ± 0.00	97.12 ± 0.08	7.00 ± 0.00
	Pre-treated P. australis (2% NaOH)	97.94 ± 0.09	99.09 ± 0.03	97.04 ± 0.12	7.00 ± 0.00
	Pre-treated P. australis (4% NaOH)	97.69 ± 0.03	99.12 ± 0.01	96.84 ± 0.04	7.00 ± 0.00
	Untreated P. australis	98.63 ± 0.07	98.17 ± 0.05	95.96 ± 0.02	7.00 ± 0.00
Batch assay to investigate the optimal incubation time for pre-treating of < 1mm P. australis.	Inoculum	6.59 ± 0.00	60.79 ± 0.07	4.01 ± 0.00	8.56 ± 0.00
	Pre-treated P. australis (2% NaOH for 12 hours)	97.96 ± 0.03	98.76 ± 0.00	96.75 ± 0.04	7.00 ± 0.00
	Pre-treated P. australis (2% NaOH for 24 hours)	97.63 ± 0.14	98.78 ± 0.00	96.43 ± 0.14	7.00 ± 0.00
	Pre-treated P. australis (2% NaOH for 48 hours)	97.89 ± 0.24	98.88 ± 0.01	96.79 ± 0.23	7.00 ± 0.00
	Pre-treated P. australis (2% NaOH for 72 hours)	98.03 ± 0.01	98.94 ± 0.01	96.98 ± 0.00	7.00 ± 0.00
	Pre-treated P. australis (2% NaOH for 96 hours)	98.10 ± 0.05	99.01 ± 0.01	97.13 ± 0.06	7.00 ± 0.00
	Pre-treated P. australis (2% NaOH for 120 hours)	97.99 ± 0.08	99.00 ± 0.00	97.01 ± 0.07	7.00 ± 0.00

2.1 Pre-treatment

In all the batch assays, except that which investigated the optimal NaOH concentration for P. australis treatment, the required amounts of P. australis samples were treated in a 500 ml bottle by adding 100 ml of 2% NaOH solution per 10 g of ground P. australis. Correspondingly, the untreated P. australis samples were prepared by immersing the ground P. australis in deionised water in a 500 ml bottle. 100 ml of deionised water was added for every 10 g of ground P. australis. Concerning the assay that was used to determine the optimal NaOH concentration for P. australis treatment, the required amounts of P. australis were treated in a 500 ml bottle by adding 100 ml of each 0.5%, 1%, 2% and 4% NaOH solution per 10 g of ground P. australis.

After that, the bottles were covered with Parafilm on the top and incubated for 3 days at room temperature (20 ± 2°C), except in the assay that was used to detect the optimal treatment incubation time, the bottles were incubated for 12, 24, 48, 72, 96, 120 hours. After the treatment incubation time was complete, the pre-treated and untreated P. australis substrates were sieved using a sieve (38-micron Mesh) to separate liquid and solid fractions. The solid fraction was washed with deionized water and then drained, washed, and drained until the pH reached 7. Then the treated and untreated P. australis samples were dried in the drying oven at 65°C for 24 hours.

2.2 Experiments setup

A series of six sets of batch experiments were set up using microcosm vessels (125 ml) as reactors with working volumes of 80 ml. In the first set of batch assays, the reactors were filled with inoculum and pre-treated P. australis samples (with 2% NaOH concentration for 3 days). While in the second set of batch assays, the reactors were filled with inoculum and untreated P. australis samples. The particles size of P. australis samples used in both sets of batch assays were 10, 5, 2, and < 1 mm, and the inoculum to substrate ratio (ISR) used was 4:1 based on gram volatile solid content (g VS of inoculum (wet basis)/ g VS of pre-treated and untreated P. australis (dry basis)).

The third and fourth sets of batch assays used reactors filled with pre-treated P. australis (2% NaOH concentration for 3 days) and untreated P. australis with particle sizes of < 1 mm, respectively. The inoculum and P. australis samples were added using various ISR ratios (4:1, 2:1, 1:1, 1:2, and 1:4 based on g VS content) in both sets of batch assays. While in the fifth set of batch assays, the ISR of 1:2 was used. The P. australis samples with particle size < 1mm used in this set of batch assays were pre-treated with various NaOH concentrations (4%, 2%, 1%, 0.5%, and 0.0%) for 3 days. In the sixth set of batch assays, P. australis samples with a particle size of < 1 mm that had been pre-treated with a concentration of 2% NaOH for incubation periods of 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours were added to the reactors. The ISR used in this set of batch assays was 1:2 based on the g VS content of the inoculum and substrate.

Following the addition of the inoculum and substrates to the reactors, the volumes were completed by adding deionised water until reaching a working volume of 80 ml. Afterwards, the reactors were tightly sealed and purged with nitrogen gas for one minute to eliminate any remaining oxygen from the mixture and the space inside the reactors and ensure the desired anaerobic conditions were attained. Then the reactors were incubated in a water bath at mesophilic conditions (37 ± 1 ⁰C). All reactors were manually shaken once a day.

Table 2

Batch experimental setup- Amount of substrate (pre-treated/untreated *P. australis*), inoculum and water added to achieve respective inoculum to substrate ratio (ISR).
Assay	ISR	Conditions	Inoculum (g)	Treated P. australis (g)	Untreated P. australis (g)	Deionised water (g)
Batch assay to investigate the optimal particles size of pre-treated P. australis (2% NaOH for 3 days) for biogas production.	4:1	Inoculum (control)	74.72	ــــــــــــ	ــــــــــــ	5.28
		< 1 mm	74.72	0.84	ــــــــــــ	4.44
		2 mm	74.72	0.84	ــــــــــــ	4.44
		5 mm	74.72	0.84	ــــــــــــ	4.44
		10 mm	74.72	0.84	ــــــــــــ	4.44
Batch assay to investigate the optimal particles size of untreated P. australis for biogas production.	4:1	Inoculum (control)	74.72	ــــــــــــ	ــــــــــــ	5.28
		< 1 mm	74.72	ــــــــــــ	0.84	4.44
		2 mm	74.72	ــــــــــــ	0.84	4.44
		5 mm	74.72	ــــــــــــ	0.84	4.44
		10 mm	74.72	ــــــــــــ	0.84	4.44
Batch assay to investigate the optimal inoculum to substrate (< 1 mm pre-treated P. australis with 2% NaOH for 3 days) ratio for biogas production.	4:1, 2:1, 1:1, 1:2, and 1:4	Inoculum (control)	67.81	ــــــــــــ	ــــــــــــ	12.19
		4:1	67.81	0.76	ــــــــــــ	11.43
		2:1	67.81	1.52	ــــــــــــ	10.67
		1:1	67.81	3.05	ــــــــــــ	9.14
		1:2	67.81	6.09	ــــــــــــ	6.10
		1:4	67.81	12.19	ــــــــــــ	ـــــــــــ
Batch assay to investigate the optimal inoculum to substrate (< 1 mm untreated P. australis) ratio for biogas production.	4:1, 2:1, 1:1, 1:2, and 1:4	Inoculum (control)	67.81	ــــــــــــ	ــــــــــــ	12.19
		4:1	67.81	ــــــــــــ	0.76	11.43
		2:1	67.81	ــــــــــــ	1.52	10.67
		1:1	67.81	ــــــــــــ	3.05	9.14
		1:2	67.81	ــــــــــــ	6.09	6.10
		1:4	67.81	ــــــــــــ	12.19	ـــــــــــ
Batch assay to investigate the optimal NaOH concentration for pre-treating of < 1mm P. australis.	1:2	Inoculum (control)	67.98	ــــــــــــ	ــــــــــــ	12.03
		0.0% NaOH	67.98	ــــــــــــ	6.01	6.01
		0.5% NaOH	67.98	6.01	ــــــــــــ	6.01
		1% NaOH	67.98	6.01	ــــــــــــ	6.01
		2% NaOH	67.98	6.01	ــــــــــــ	6.01
		4% NaOH	67.98	6.01	ــــــــــــ	6.01
Batch assay to investigate the optimal incubation time for pre-treating of < 1mm P. australis.	1:2	Inoculum (control)	67.98	ــــــــــــ	ــــــــــــ	12.03
		12 hours	67.98	6.01	ــــــــــــ	6.01
		24 hours	67.98	6.01	ــــــــــــ	6.01
		48 hours	67.98	6.01	ــــــــــــ	6.01
		72 hours	67.98	6.01	ــــــــــــ	6.01
		96 hours	67.98	6.01	ــــــــــــ	6.01
		120 hours	67.98	6.01	ــــــــــــ	6.01

2.3 Analytical methods

pH values were measured using of pH meter. Total solids (TS) and volatile solids (VS) of inoculum and substrates were determined based on standard methods [46]. Biogas volume was measured using biogas counters from CJC Labs Ltd. The reactors were connected to the gas counters through a plastic tube. These counters contained reversible buckets and were filled with water to a level 10 mm from the lid. The bucket's volume was calibrated to hold 6 ml of the produced biogas. When produced biogas enters the counters, it will displace the water inside the buckets, causing tipping it and releasing the biogas into a small gas bag attached to the counters. Each tipping for these buckets was recorded at a data acquisition system (DAS) from CJC Labs Ltd, which indicates a volume of 6 ml of the produced biogas. Biogas volume was measured at ambient temperature and corrected for standard temperature (273.15 K) and pressure (1 bar). The cumulative biogas production from reactors was expressed as ml/g VS added (VS of inoculum + VS of the substrate).

2.4 Statistical analyses

IBM SPSS Statistics 27 software was utilized for conducting the statistical analyses. An analysis of variance (ANOVA) was employed to identify significant (p < 0.05) effects of particle size of P. australis, ISR, NaOH concentration used for pre-treating P. australis, and the incubation time for pre-treating of P. australis on gas production. a Games-Howell post-hoc test (p < 0.05) was used to group the levels of variables that exhibited significant effects.

3.1 Effect of particles size on biogas production using pre-treated and treated P. australis

The cumulative biogas production from the digestion of pre-treated P. australis was remarkably different among the various particles size examined (Fig. 1a). The cumulative biogas produced from the digestion of pre-treated P. australis with particles size < 1 mm was significantly higher than that produced from the digestion of pre-treated P. australis with particles size 2, 5, and 10 mm. Meanwhile, it was observed that the cumulative biogas produced from treated P. australis with particles size 2 mm and 5mm was significantly higher than that from treated P. australis with particles size 10 mm (Table 2). This can be attributed to the increased surface area and low cellulose crystallinity of the smaller particles size, which led to the greater reach of hydrolysis enzymes to the cellulose, thus increasing the hydrolysis rate of the cellulose and biogas production [27–30]. As well as, the low mixing rate of the reactors may have caused quick sedimentation of the large particles size (2, 5 and 10 mm) compared to the small particles size of the substrate (< 1 mm), which may be led to the irregular distribution of the microbial consortia and increase temperature stratification and pockets of low temperature within the digester, thus reduce the efficiency of the digestion process and biogas production [47–50].

Similar results were reported by Dubrovskis and Kazulis [51] as they investigated the effect of particle size on biogas production from P. australis samples harvested in the winter and summer seasons. The particle size for P. australis samples harvested in the winter period was 1, 2, 5, 20 mm, while it was 2, 5, 7, 20 mm for P. australis harvested in June. They found that the biogas production from P. australis with particle sizes 1 mm (from winter harvesting) and 2 mm (from summer harvesting) was higher than that produced from P. australis samples with the other particle size. Moreover,Sharma et al. [52] studied the effect of five particle sizes (0·088, 0·40, 1, 6 and 30 mm) on biogas production from seven agricultural and forest residues. They found that the smallest particle size, 0.088 mm, and 0.40 mm presented the greatest quantity of biogas production.

With regards to biogas production from untreated P. australis, it can be seen from Fig. 1(c) that the cumulative biogas produced from untreated P. australis with particles size < 1 mm was significantly higher (p < 0.05) than that produced from untreated P. australis with particles size 2, 5, 10 mm (Table 2), and this was consistent with what was found during the digestion of pre-treated P. australis. However, no significant differences in cumulative biogas production have appeared between the untreated P. australis with particles size 2mm and 5mm and between the untreated P. australis with particles size 5mm and 10 mm (Table 2). Moreover, the digestion of untreated P. australis with particles size 2, 5 and 10 mm showed lower biogas production than inoculum reactors (controls) during the whole digestion period. And that may be due to the low breakdown of the lignin content in untreated P. australis led to limiting the bioavailability of cellulose for microbial [53]. Furthermore, the inadequate mixing rate for the reactors may have resulted in low homogeneity in the distribution of microorganisms, settling of the materials, and created temperature stratification within the reactors, which may be caused some inhibition effects for the digestion process and biogas production [49, 50].

On the other hand, it has appeared from Fig. 1(b and d) that the digestion of pre-treated and untreated P. australis with particle size < 1mm showed the highest biogas production rate along the digestion period in comparison to other particle size. This indicates the faster and higher biodegradability of pre-treated and untreated P. australis at particle size < 1, making it preferable for use to improve and increase biogas production from P. australis. Besides, the high biogas production for all systems with pre-treated P. australis compared to the systems with untreated P. australis made the using of pre-treated P. australis for biogas production more feasible than using of untreated P. australis.

Table 3

Cumulative biogas production (ml/g VS added) and maximum biogas production rate (ml/g VS added/day) from the digestion of pre-treated and untreated *P. australis* at different particles size after 10 days of digestion period (mean ± standard deviation). The letters in parentheses indicate the results of the Games-Howell Pairwise Comparison test. Means that do not share the same letter in each batch assay are significantly different at a 95% confidence level.
Batch assay	Conditions	Cumulative biogas production (ml/g VS added)	Maximum biogas production rate (ml/g VS/day)
Batch assay to investigate the optimal particles size of pre-treated P. australis for biogas production.	Inoculum (control)	14.13 ± 0.05 (D)	10.59 ± 0.00
	< 1 mm	27.97 ± 0.07 (A)	9.79 ± 0.00
	2 mm	21.64 ± 2.38 (B)	6.98 ± 0.01
	5 mm	18.88 ± 0.81 (B)	6.99 ± 0.00
	10mm	16.09 ± 0.81 (C)	6.30 ± 0.00
Batch assay to investigate the optimal particles size of untreated P. australis for biogas production.	Inoculum (control)	13.09 ± 1.01 (B)	10.47 ± 0.00
	< 1 mm	16.67 ± 0.09 (A)	9.72 ± 0.00
	2 mm	11.79 ± 0.81 (BC)	6.24 ± 0.98
	5 mm	10.40 ± 0.80 (CD)	6.24 ± 0.98
	10mm	9.70 ± 0.05 (D)	5.54 ± 0.00

3.2 Effect of ISR on biogas production from pre-treated P. australis (2% NaOH) and untreated P. australis.

After 32 days of digestion period, it is observed that the digestion of pre-treated P. australis at ISR of 1:2 and 1:4 presented the highest cumulative biogas production compared to that from the digestion of pre-treated P. australis at ISR of 1:1, 2:1 and 4:1 (Fig. 2a, Table 3). Moreover, the digestion of pre-treated P. australis at ISR 1:1 showed significantly higher cumulative biogas production than that produced at ISR 2:1, and those two had produced higher cumulative biogas than that produced at ISR 4:1. Similarly, it is appeared from Fig. 2 (c) that the digestion of untreated P. australis showed the highest cumulative biogas production at ISR of 1:4 and 1:2 followed by ISR of 1:1. Whilst the lowest cumulative biogas production was observed at ISR of 2:1 and 4:1 (Table 3).

This increase in cumulative biogas production at ISR of 1:2 and 1:4 could be attributed to the rise in the substrate proportion, which contributed to providing more carbohydrates (such as cellulose and hemicellulose) that may be degraded by microbial activity into volatile fatty acids (VFAs), which in turn converted by methanogens into biogas. However, the VFAs production from digestion of pre-treated P. australis at ISR of 1:4 may exceed methanogens' consumption capability, which may result in a slight accumulation of VFAs and increased pH in the systems. These conditions may lead to a minor inhibition of microbial consortia and a slowdown in biogas production compared to other ISR, as shown in Fig. 2a [45, 43, 54, 44]. Consequently, the digestion of pre-treated P. australis using ISR of 1:2 is more suitable because it can enhance system stability and avoid the effects of acidification.

On the other hand, the low degradation of untreated P. australis due to the resistance of lignin to hydrolysis enzymes and limiting their accessibility to cellulose and hemicellulose could lead to producing a low amount of VFA, which corresponds to the consumption ability of methanogens bacteria [55, 56]. Therefore, it can be observed from Fig. 2c that the digestion of untreated P. australis at ISR of 1:4 was stable and showed higher biogas production than other ISR.

Liew et al. [57] reported that the solid-state anaerobic digestion of four lignocellulosic substrates (corn stover, wheat straw, yard waste, and leaves) exhibited the highest biogas and methane production at ISR of 1:2 compared to ISR of 1:3, 1:4, and 1:5. Similarly, Xu et al. [58] found the highest production of biogas and methane from the digestion of corn stover was achieved at ISR of 1:2 and 1:4, respectively.

Figure 2b shows that the biogas production rate from the digestion of pre-treated P. australis at ISR 4:1, 2:1, 1:1, and 1:2 was high at the beginning of digestion, but it declined significantly after around 15th days. During this period, the digestion of pre-treated P. australis at ISR 1:2 showed the highest biogas production rate. Besides, the biogas production rate from ISR 1:4 was lower than other ISR at the initial stage of digestion, but it increased to become the highest after 15th days. This may indicate that a slight inhibition occurred at the initial stage of digestion, possibly due to the accumulation of VFAs produced, which may cause an increase in the acidity in the systems. Thus, using an ISR of 1:2 can be beneficial at a large-scale compared ISR of 1:4 because it helps to digest more materials in less time (around 15th days) and thus obtain larger quantities of biogas.

Similarly, the biogas production rate from the digestion of untreated P. australis was higher at ISR 1:2 and 1:4 than that from the other ISR during the digestion period (Fig. 2d). Therefore, the use of ISR 1:2 may consider the best option to enhance biogas production from the digestion of pre-treated P. australis and using ISR 1:2 or 1:4 to promote biogas production from the digestion of untreated P. australis.

Table 4

Cumulative biogas production (ml/g VS added) and maximum biogas production rate (ml/g VS added/day) from the digestion of pre-treated and untreated *P. australis* at different ISR after 32 days of digestion period (mean ± standard deviation). The letters in parentheses indicate the results of the Games-Howell Pairwise Comparison test. Means that do not share the same letter in each batch assay are significantly different at a 95% confidence level.
Batch assay	Conditions	Cumulative biogas production (ml/g VS added)	Maximum biogas production rate (ml/g VS/day)
Batch assay to investigate the optimal ISR to increase biogas production from digestion of pre-treated P. australis (2% NaOH).	Inoculum (control)	14.69 ± 1.13 (E)	12.73 ± 1.39
	ISR = 4:1	28.57 ± 0.85 (D)	10.81 ± 0.02
	ISR = 2:1	38.40 ± 1.49 (C)	11.52 ± 0.00
	ISR = 1:1	54.22 ± 1.10 (B)	8.56 ± 0.00
	ISR = 1:2	78.21 ± 0.36 (A)	6.91 ± 0.00
	ISR = 1:4	73.59 ± 4.97 (A)	5.06 ± 0.27
Batch assay to investigate the optimal ISR to increase biogas production from digestion of untreated P. australis.	Inoculum (control)	12.78 ± 0.01 (D)	12.78 ± 0.00
	ISR = 4:1	14.08 ± 0.97 (C)	9.94 ± 0.01
	ISR = 2:1	14.92 ± 1.57 (C)	8.82 ± 0.96
	ISR = 1:1	21.40 ± 0.57 (B)	6.97 ± 0.00
	ISR = 1:2	25.97 ± 1.51 (A)	4.87 ± 0.46
	ISR = 1:4	28.93 ± 1.55 (A)	3.64 ± 0.27

3.3 Optimal concentration of NaOH for pre-treatment of P. australis to enhance biogas production.

As shown in Fig. 3a, the cumulative biogas production from pre-treated P. australis at various NaOH concentrations (0.5, 1, 2, and 4%) was significantly higher (p < 0.05) than that produced from untreated P. australis (Table 4). This may be due to the low digestibility of untreated P. australis because of the lignin recalcitrance to hydrolysis enzymes, which impeded them from reach to cellulose fibre. This may cause a decrease in the amount of VFAs produced from the degradation of untreated P. australis substrate, thus reducing biogas production. Besides that, it is observed that the digestion of pre-treated P. australis with 1% NaOH concentration showed significantly higher cumulative biogas production than pre-treated P. australis with 0.5% NaOH concentration. As well as the cumulative biogas production from the digestion of pre-treated P. australis with 2% and 4% NaOH concentration was significantly higher (p < 0.05) than that produced from pre-treated P. australis with 0.5% and 1% NaOH concentration. However, no significant difference was observed between the cumulative biogas produced from pre-treated P. australis with 2% and 4% NaOH (Table 4).

This increase in biogas production as the NaOH concentration used for pre-treatment of P. australis increased (from 0.5 to 4%) can be attributed to the increased lignin removal from P. australis substrate, which enhances the bioaccessibility to cellulose component [10, 59–61], thus increase the biodegradation performance of the substrate and promote biogas production [62].

The study conducted by Zhu et al. [32] showed that the use of 5% NaOH for solid-state pre-treatment of corn stover at ambient temperature (20 ± 0.5°C) for 24 hours presented higher biogas production compared to that produced from the digestion of corn stover that pre-treated with 1%, 2.5%, and 7.5% NaOH concentrations. Xue et al. [63] used NaOH at concentrations of 0%, 1%, 3%, 5%, and 8% in the pre-treatment of Miscanthus reed. They found that 8% NaOH was the best concentration for pre-treatment among the other NaOH concentrations, which increased the biogas production by 56.92%.

As shown in Fig. 3b, the digestion of the pre-treated P. australis with 0.5, 1 and 4% NaOH concentration exhibited the maximum biogas production rate on the first day of digestion (6.16, 6.81, and 7.13 ml/g VS added/day). While the digestion of the pre-treated P. australis with 2% NaOH began to produce 7.13 ml/g VS added/day on first day and rose to reach a maximum rate of 7.78 ml/g VS added/day on day three. However, the biogas production rate from the digestion of the pre-treated P. australis with 2 and 4% NaOH concentration remained for the first nine days in the range of 3.24–7.78 and 3.56–7.13 ml/g VS added/day, respectively. While the range was 2.27–6.16 and 2.59–6.81 ml/g VS added/day from the digestion of the pre-treated P. australis with 2 and 4% NaOH concentration, respectively. This may indicate more hydrolysis performance for P. australis substrate pre-treated with more NaOH concentration owing to enhancing lignin removal. Consequently, the digestion of P. australis pre-treated with 2% and 4% NaOH concentration can provide more biogas than that from P. australis pre-treated with 0.5% or 1% NaOH concentration within the same digestion period. Therefore, using 2% or 4% NaOH concentration is considered more effective for the pre-treatment of P australis substrate. However, since there was no significant difference between cumulative biogas produced from the digestion of pre-treated P. australis with 2% NaOH and 4% NaOH, and to increase the economic feasibly of pre-treatment, a concentration of 2% NaOH will be adopted in our future study for pre-treatment of P. australis biomass.

Table 5

Cumulative biogas production (ml/g VS added) and maximum biogas production rate (ml/g VS added/day) from the digestion of pre-treated *P. australis* with different NaOH concentrations (0.5%, 1%, 2%, and 4%) and untreated *P. australis* after 24 days of digestion period (mean ± standard deviation). The letters in parentheses indicate the results of the Games-Howell Pairwise Comparison test. Means that do not share the same letter in each batch assay are significantly different at a 95% confidence level.
Batch assay	Conditions	Cumulative biogas production (ml/g VS added)	Maximum biogas production rate (ml/g VS/day)
Batch assays to Investigate the optimal NaOH concentration for P. australis pre-treatment.	Inoculum (control)	12.58 ± 0.05 (E)	10.48 ± 0.00
	0.5% NaOH	46.68 ± 1.50 (C)	6.16 ± 0.46
	1% NaOH	58.03 ± 2.62 (B)	6.81 ± 0.46
	2% NaOH	70.01 ± 3.75 (A)	7.78 ± 0.00
	4% NaOH	76.14 ± 2.62 (A)	7.13 ± 0.46
	Untreated P. australis	19.25 ± 0.38 (D)	4.57 ± 0.00

3.4 Optimal incubation time (12, 24, 48, 72, 96, 120 hours) for treatment of P. australis with 2% NaOH concentration.

The cumulative biogas produced from P. australis pre-treated with 2% NaOH at different incubation times (12, 24, 48, 72, 96, and 120 hours) were relatively convergent during most of the digestion period (Fig. 4a). At the end of digestion period, no significant differences were observed in the cumulative biogas produced from the digestion of P. australis pre-treated with 2% NaOH at incubation times of 72, 96 and 120 hours (Table 5). However, the cumulative biogas produced from the digestion of P. australis pre-treated at these three incubation times was significantly higher than that produced from P. australis pre-treated with 2% NaOH at incubation times of 12, 24, and 48 hours (Table 5). This could be attributed to the prolonged incubation time of pre-treatment has provided enough time for more lignin solubilization and removal, thus increasing cellulose availability for hydrolysis enzymes, which leads to increased biogas production [32, 64].

Similar results were found in other studies; for example, Zheng et al. [65] reported that biogas production increased by 72.9% when the corn stover was pre-treated with 2% NaOH for three days at ambient temperature (20°C). Moreover, Chandra et al. [66] found that pre-treatment of wheat straw with 4% NaOH at 37°C for five days achieved an 87.5 % increse in biogas production compared to untreated wheat straw.

Similarly, it is observed that the biogas production rate from the digestion of P. australis pre-treated at incubation times of 12, 24, 48, 72, 96 and 120 hours was relatively similar in the initial period of digestion (Fig. 4b). However, the digestion of P. australis pre-treated for 72, 96 and 120 hours showed the highest biogas production rate until the end of the digestion period. Hence, it can rely on longer incubation time for pre-treatment of P. australis substrate to promote biogas production.

Table 6

Cumulative biogas production (ml/g VS added) and maximum biogas production rate (ml/g VS added/day) from the digestion of *P. australis* pre-treated with 2% NaOH concentrations at different incubation times (12, 24, 48, 72, 96 and 120 hours) after 24 days of digestion period (mean ± standard deviation). The letters in parentheses indicate the results of the Games-Howell Pairwise Comparison test. Means that do not share the same letter in each batch assay are significantly different at a 95% confidence level.
Batch assay	Conditions	Cumulative biogas production (ml/g VS added)	Maximum biogas production rate (ml/g VS/day)
Batch assays to investigate the optimal incubation time (12,24,48,72,96, and 120 hours) for pre-treating P. australis substrate with 2% NaOH.	Inoculum (blank)	20.53 ± 0.01 (C)	12.32 ± 0.00
	12 hr	64.54 ± 0.01 (B)	7.10 ± 0.00
	24 hr	65.19 ± 1.49 (B)	6.45 ± 0.91
	48 hr	66.76 ± 1.86 (B)	6.45 ± 0.00
	72 hr	71.18 ± 1.79 (A)	6.44 ± 0.01
	96 hr	72.46 ± 1.08 (A)	6.44 ± 0.00
	120 hr	73.78 ± 1.87 (A)	6.44 ± 0.00

The high productivity of P. australis biomass and its ubiquity on non-agricultural land makes it a promising crop for biogas production. However, being a lignocellulosic crop, P. australis is characterised by its recalcitrance to biodegradability due to its high lignin content, which impedes microorganism accessibility to cellulose. Therefore, pre-treatment for P. australis is crucial to improve the AD process and increase biogas production. It was observed that the implementation of mechanical treatment by grinding the reed to a size < 1 mm stimulated biogas production to a higher level than that produced by P. australis with larger particle sizes. This can be attributed to the role of grinding in increasing the surface area that can be reached by hydrolysis enzymes and decreasing cellulose's crystallinity in P. australis.

Furthermore, the use of alkaline chemical treatment to remove the lignin had a significant effect in further increasing cellulose bioaccessibility and improving biogas production from P. australis. It is evidenced in the current study that biogas production from pre-treated P. australis with NaOH was significantly higher than that from untreated P. australis. The 2% NaOH concentration used for P. australis pre-treatment showed the optimum biogas production compared to other NaOH concentrations (0.5%, 1% and 4%). Furthermore, it was found that the longer incubation time of treatment was more appropriate for P. australis pre-treatment at ambient temperature. The long treatment period of 72–120 hr may enhance extra lignin solubilisation and the bioavailability of more cellulose. Finally, the inoculum to substrate ratio, among the other parameters, played an essential role in improving AD performance and enhancing biogas production from P. australis substrate. It is observed that the use of an ISR ratio of 1:2 contributed to an increase in biogas production more than other ISR ratios (4:1, 2:1, 1:1, and 1:4); this may be attributed to the potential of ISR of 1:2 in providing an appropriate balance between the microbial consortia and nutrient amounts needed by these microbes without causing VFA accumulation, pH drop and system failure.

Acknowledgements

The authors thank Mr. Christopher James Parry, the Director of Cockerham Green Energy, in the UK, for his invaluable support and assistance to obtain inoculum/digestates. We extend our thanks to Mr. Tim Gregson and Dr. Clare Benskin, from Lancaster University in the UK for providing technical support.

Funding

This work was sponsored and supported by the Iraqi Ministry of higher education and scientific research and Mosul University, Iraq.

Competing interest

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.

Author Contributions

Ahmed R. Al-Iraqi: Conceptualization, Methodology, Investigation, physio-chemical analysis, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Kirk T. Semple: Conceptualization, Methodology, Investigation, Formal analysis, Validation, Visualization, Resources, Supervision, Writing – review & editing. Andrew M. Folkard: Conceptualization, Methodology, Resources, Visualization, Supervision, Writing – review & editing. Philip A. Barker: Conceptualization, Resources, Visualization, Supervision, Writing – review & editing. Bhushan P. Gandhi: Formal analysis, Writing – review & editing.

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published 14 Nov, 2023

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Editorial decision: Reconsider pending major revisions
07 Sep, 2023
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Reviewers invited by journal
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Determine the optimal parameters for biogas production from common reed (Phragmites australis)

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Pre-treatment

2.2 Experiments setup

2.3 Analytical methods

2.4 Statistical analyses

3 Results and discussion

3.1 Effect of particles size on biogas production using pre-treated and treated P. australis

3.3 Optimal concentration of NaOH for pre-treatment of P. australis to enhance biogas production.

4 Conclusion

Declarations

References

Status:

Journal Publication

Version 1