Maintaining the Long-Term Stability of Anaerobic Digestion of Maize Straw in a Continuous Plug Flow Reactor by Verifying the Key Role of Trace Elements

A mesophilic anaerobic digestion reactor fed with maize straw was investigated in a plug flow reactor for 150 days. The process performance was evaluated through the stable, unstable, and recovery stages. Results obtained in this study showed that the reactor maintained stable within approximately two months, but the gradual depletion of trace elements (Fe, Co and Ni) and significant accumulation of volatile fatty acids (8.07 g/L) occurred in the reactor by the 120th day. Pig manure containing higher trace elements, therefore, was mixed with maize straw at a ratio of 1:4 on a dry matter basis to feed the reactor. As a result of the consequent reactor recovery, volatile fatty acids decreased significantly, and biogas production increased. During the subsequent stable operation stage, a methane yield of 0.21 L/g-VSadded was obtained and methane content stabilized at 54%. Additionally, the uptake of the elements was determined quantitatively, highlighting the necessity of mixing maize straw with pig manure. Therefore, it is imperative to co-digest maize straw with trace element-rich substrates or supply trace elements to ensure the long-term stability of the anaerobic digestion process.


Introduction
Globally environmental pollution, higher energy demands, and abundant production of organic waste have posed challenges to human beings and the environment. Anaerobic digestion, as a mature technology employed for organic waste matter treatment, has gotten significant attention for reducing environmental pollution by producing renewable energy (CH 4 ), diminishing greenhouse gas emissions by replacing part of fossil fuels, and converting organic waste matter including animal manure, food waste, and crop straw into bioenergy [1][2][3][4]. In China, the production of crop straw is estimated at 8.6 × 10 8 tons in 2020 [5]. Among them, maize straw is the major crop residue. Producing biogas by anaerobic digestion is one of the most practical technologies to utilize maize straw, and it provides considerable benefits for resource sustainability [6]. Although anaerobic digestion is a promising technology for the treatment of maize straw, significant challenges are experienced during the mono-digestion of maize straw in commercial biogas plants. Maize straw is mainly composed of cellulose, hemicellulose, and lignin, which are carbohydrate polymers. The complex structure of the carbohydrate polymers results in slow biodegradation and low conversion efficiency in anaerobic digestion. Furthermore, maize straw lacks essential trace elements, which is a hindrance to anaerobically treating maize straw. Studies have pointed out that the anaerobic digestion of maize silage faces reactor instability, low biogas production, and accumulation of volatile fatty acids owing to a lack of essential trace elements [7].
Principally, trace elements are essential for the growth of methanogens and fundamentally determine the conversion efficiency of an anaerobic process [8]. An appropriate amount of trace elements is conducive to the growth of methanogens. Comparing animal manure, sewage sludge, and municipal solid waste, maize straw contains less amount of trace elements [9,10]. For this reason, maize straws are recommended to be co-digested with animal manure. However, whether the anaerobic digestion feeding with maize straw can be continuously operated still lack direct results.
Plug flow reactor is known for lower cost, more stable performances, and tolerating environmental stress [11]. Previous studies have demonstrated that the Chemical Oxygen Demands (COD) removal efficiency of dairy manure in a plug flow reactor was 16% higher than the conventional completely mixing reactor [12]. In the plug flow reactor, functional microbes exist along the materials flowing. Hydrolysis, acidogenesis, acetogenesis, and methanogenesis may thus occur under a more desired environment. Subsequently, biogas production in a plug flow reactor may have advantageous over a traditional reactor [13]. The role of trace elements in a plug flow reactor would be different but still not yet to be known. The begging question is whether the anaerobic digestion of maize straw and long-term operation without co-digestion and additional essential trace elements would be sustainable. Besides, whether the reactor can be restored by adding other raw materials when volatile fatty acids accumulate and the impact of trace elements dosing has not been reported. Most studies focused on the demand for trace elements in food waste [14], manure [15] and sewage sludge [16] in traditional reactors. However, few studies examined the impact of trace elements on a plug flow reactor. The dosage and demand of trace elements also vary among the different feedstock. If the addition of trace elements is mandatory for maize straw in a plug flow reactor was still uncertain.
Therefore, the main objective of this study is to investigate the performances of the anaerobic mono-digestion of maize straw under a mesophilic condition in a continuous plug flow reactor for a lab-scale long-term operation, and study the changes of Fe, Co and Ni during long-term continuous operation. The results are aimed to provide more accurate technical data for guiding industrial applications.

Feedstock and Inoculum
The maize straw used in this study was collected from Shang Zhuang Experimental Station of China Agricultural University, Beijing, China. After taking back, the maize straw was ground and sieved using 18# mesh sieve (1.0 mm size). The maize straw was mixed with a liquid fraction of centrifuged biogas plant effluent treating cattle manure to ensure that the total solids (TS) of the mixture was 10% and soaked at 37℃ for one day to prepare the substrate. The biogas slurry used for soaking the maize straw was taken from a full-scale mesophilic biogas plant treating cattle manure. The liquid fraction of the slurry was obtained after centrifuging at 4000 rpm for 20 min, the pH was 8.25, total solids (TS) and volatile solids (VS) were 1.1 and 0.1 g/L, respectively, VFAs were 0.13 g/L. The inoculum was obtained from a pilot-scale anaerobic digestion reactor that treats waste-activated sludge at mesophilic temperature. The pig manure added was taken from a pig farm in Beijing, China. The characteristics of feedstock and inoculum are summarized in Table 1.

Calculation of Trace Element Requirements
The quantitative requirements of trace elements were calculated in Eq. (1). (1) where X is the trace elements concentration, in milligrams per liter; S(X) in is the concentration of Fe, Co, and Ni in the influent, respectively, in milligrams per liter; S(X) eff is the concentration of Fe, Co, and Ni in the effluent, respectively, in milligrams per liter; COD in is the concentration of COD in the influent, in kilograms per liter; and COD eff is the concentration of COD in the effluent, in kilograms per liter. Figure 1 shows the plug flow reactor which had a total volume of 30 L and a working volume of 21 L. Intermittent stirring at 60 rpm was realized by a timer, working for five minutes per half an hour. The reactor was operated at a hydraulic retention time of 42 days. Feeding feedstock and withdrawing effluent were operated once per day. The reactor consisted of a water bath interlayer with a thickness of three centimeters to maintain the temperature at 37℃ by circulating hot water through a water pump (Sensen HQB-2200, China). The reactor was equally divided into four chambers. Each chamber had a port for discharging effluent at the bottom and a gas pipe connected to a gas bag above. The reactor started with an organic loading rate (OLR) of 1.4 g-TS/(L day) and increased to 2.4 g-TS/(L day) for the long-term operation.

Analytical Methods
TS, VS, ammonium nitrogen, total chemical oxygen demand (TCOD), soluble chemical oxygen demand (SCOD), and other biochemical parameters were determined according to the American Public Health Association (APHA) methods. pH was measured by using the pH meter (Mettler Toledo, Switzerland), volatile fatty acids (VFAs) and the components of CH 4 and CO 2 in the biogas were tested by a gas chromatography (Shimadzu GC-2010 plus, Japan), and a gas chromatography (Shimadzu GC-8A, Japan), respectively.

Gas bag 1#
Water batch The trace elements were analyzed by the atomic absorption spectrometer (AAS, PerkinElmer 900 T, USA).

Biogas Production Performance
The biogas production at different stages is shown in Fig. 2 and summarized in Table 3. The biogas production increased gradually during the start-up stage and then increased to a maximum of 0.52 L/g-VS. The average biogas and methane production reached 0.39 and 0.23 L/g-VS with a methane content of 60%, which was similar to a previous study treating maize straw in a plug flow reactor at 38℃ [17]. Similar results in other types of reactors using maize straw as raw material are listed in Table 2. In this study, the biogas production decreased to some degree compared with the relatively stable stage, after 99 days of operation, the accumulation of volatile fatty acids and biogas production declined continuously which was observed when the reactor operated till the 120th day. By stopping feeding on the 120th day for  12 days. Then 2 L of pig manure were added to replace the reactor content. After that, feeding of the reactor was done by a mixture of straw and pig manure substrate at a dry matter ratio of 4:1 for 134 ~ 150 days. The biogas production and methane production resumed to 0.32 and 0.21 L/g-VS gradually till 150th days. The observed performance in the last stage was similar to a previous study for mono-digestion of maize straw at 37 ± 1℃ [18].

Operational Performance of the Four Chambers
The volumetric biogas production of the four chambers is illustrated in Fig. 3. It was observed that most of the gas production in the reactor was mainly produced in the 3rd and the 4th chambers. The volumetric biogas production increased to the maximum of 1.1 L/(L day) gradually and then decreased, which was determined at an average value of 0.80 L/(L day) during 43 ~ 90 days. Subsequently, volumetric biogas production continued to drop to the lowest value. The trend changed and the volumetric biogas production increased to a maximum of 0.71 L/(L day) after re-feeding. The volumetric methane production corresponded to the volumetric biogas production changing trend. The biogas composition of the four chambers is shown in Fig. 4. The fluctuation magnitude in the 1st chamber was larger than in other chambers. It may be attributed to the closeness of the 1st chamber to the feeding port, resulting in the impact of the feeding on the microorganisms. During the early 99 days of operation, the methane concentration in the four chambers varied within the range of 50% ~ 70%, the CH 4 /CO 2 in the four chambers first rising, then decreasing gradually and stabilized at an average ratio of 1.66 rapidly from day 43 to day 99. After 99 days, the methane concentration decreased rapidly to 34%, the proportion of CO 2 in biogas components gradually increased and the average CH 4 /CO 2 in the four chambers decreased to the minimum of 0.93 on the 120th day. On the contrary, upon the addition of pig manure after re-feeding, the methane concentration began to show an upward trend and resumed to 54% once again with an average value CH 4 /CO 2 ratio of 3.39.
Based on the results above, there were no significant differences in biogas production and methane concentration among the four chambers. It is speculated that the hydrolysis and methanogenesis in the reactor were not deeply separated. The plug flow reactor was designed to separate hydrolysis and methanogenesis. In principle, hydrolysis acidification occurred in the beginning section of the plug flow reactor, and methane production occurred in the rear section, which resulted in basically less gas production in the beginning part. However, it has been reported that hydrolysis seems not the rate-limiting step of the anaerobic digestion of straw, but the methanogenesis stage [23]. Therefore, the hydrolysis and methanogenesis did not take place in one part of the reactor, but in the entire reactor. This may have resulted in low separation efficiency between hydrolysis and methanogenesis.

System Stability With and Without Pig Manure Supplementation
The VFAs concentration is shown in Fig. 5. VFAs maintained at a low level and floated up and down around 0.5 g/L which did not change significantly, it indicated that the After that, the VFAs concentration continued to increase and was about 10 times higher than the stable stage and reached the maximum value of 8.07 g/L with acetic acid dominating at a concentration of 5.95 g/L, while the concentration of propionic acid was 0.63 g/L till 120 days. Generally, in an anaerobic digestion system, acid inhibition will occur when the total VFAs concentration reach 8 g/L or the acetic acid concentration is above 2 g/L [24]. Hence, it was preliminarily considered that VFAs accumulation occurred in the reactor. The absence of sufficient trace elements was the major reason to check if the anaerobic system will poorly perform and the main cause of adverse reactor performance [25]. Previous studies have demonstrated that acidification occurred and the reactor could not operate normally at 2.5 g-VS/(L day) owing to the lack of trace elements during anaerobic mono-digestion of maize straw after 70 days [18]. Similar results were also obtained in the anaerobic system treating wheat silage [26]. Therefore, it is reasonable to conclude that a lack of trace elements occurred in the reactor, resulting in VFAs accumulation. A previous study treating wheat stillage under mesophilic, also reported that propionic acid and acetic acid exceeded 7000 and 2000 mg/L respectively after the collapse of the reactor operation [26]. In this study, after stopping to feed the reactor for 12 days, VFAs concentrations declined to below 0.5 g/L till the end of the experiment. To make a short conclusion here, the anaerobic digestion using mize straw as solo substrate can not keep stability after a long-term operation. The deficiency of trace elemnets would be the reason behind this phenomena.

Deficiency of Trace Elements
Previous studies reported the following trace elements, Fe, Co and Ni being capable of promoting the biosynthesis and methanogenesis of microbial chambers [27,28]. Trace elements are closely related to the activities of microorganisms and are one of the key factors to ensure the stable operation of the reaction [29]. Iron can act as an electron carrier for intracellular redox reactions and promote the increase of methane production [30]. Co is an important metal component of enzyme F420, which can activate the activity of enzyme F420 in the process of anaerobic [31].
Ni is an important component element of the active enzyme of methyl coenzyme reductase (F430 factor) in methanogens chambers [32]. Scherer [33] determined the elemental composition of ten methanogenic species and found that the trace elements content of Fe, Co, and Ni in the chambers of methanogenic were 0.07-0.28%, 10-120 ppm, 65-180 ppm, respectively. It has been reported that Fe, Co, and Ni were the main required trace elements to stimulate the activity of methanogens tested so far in the process of anaerobic digestion [30,34,35]. The lack of Fe, Co, and Ni had remarkable impacts on anaerobic digestion [31]. Figure 6 gives the trace elements' concentration at different stages. In conjunction with the progress of the reaction, Fe, Co, and Ni showed a gradual downward trend and reached the lowest value during 100-120 days. After the operation for 120 days, the concentrations of Fe, Co, and Ni dropped to 0.5, 0.002, and 0.036 mg/L, respectively. VFAs concentration rose to 8.07 g/L, the reactor was acidified. Some studies pointed out that low trace element contents may cause process instabilities, i.e. low biogas production and VFAs accumulation in anaerobic digestion of maize silage as feedstock [7]. Stop feeding the reactor and operation for 12 days and then add pig manure to the feedstock. In Table 1, the contents of Fe, Co, and Ni in maize straw was 407. 0.5 and 3.0 mg/kg-TS, significantly lower than those in pig manure i.e. 4012, 3.0, and 10.0 mg/kg-TS. It was found that these three elements were restored to the initial value of the experiment after re-feeding. However, continuous feeding and discharging, microbial metabolism, and utilization, result in the decrease of trace elements ceaselessly in the reactor. After resuming feeding, the trace elements concentration continued declining, which was in agreement with a previous study pointing that trace elements in the co-digestion of straw and manure keeping a continually decreasing trend for the entire digestion time [36,37].
The decrease of three trace elements when using maize straw as solo substrates is shown in Fig. 7. These three kinds of trace elements gradually decreased from 13.2, 0.032, and 0.284 mg/L to 7.4, 0.01, and 0.063 mg/L on the 99th day, respectively. The reduction of trace elements concentration was accompanied by a high rate of consumption of the essential trace elements, resulting in insufficient trace elements in the reactor and accumulation of VFAs.

Quantitative Estimation of Trace Elements' Requirements
IN this study, the trace elements required to remove per kilogram COD were calculated using Eq. (1). Table 4 lists a comparison of the requirements for Fe, Co, and    Ni between earlier studies and this study. The required concentration of trace elements to remove per kilogram of COD differs significantly, which primarily depends on the substrate and digestion temperature. In this study, the requirements of Fe, Co, and Ni to remove per kilogram COD were 412.0, 0.4 and 2.2 mg under mesophilic. At the same range of temperatures, different substrates such as maize straw (this study), glucose [38] and food waste [39] have different requirements for trace elements. After operation for 120 days, pig manure was mixed as trace element suppliers with maize straw to add to the reactor. Subsequently, biogas production gradually recovered, volatile fatty acids decreased significantly, and the system gradually tended to stabilize.

Conclusion
This study found that the anaerobic digestion process cannot maintain long-term stability when maize straw was fed as the sole substrate. Severe VFAs accumulation induced a decrease in biogas production and process failure. The observed decline of trace elements in the reactor was identified as the main reason for the reduced methanogenic activity. Feeding pig manure increased the amount of trace elements and thus helped to restore the process. For process stability management, the quantitative requirements of trace elements for the anaerobic digestion of maize straw were useful. Overall, when maize straw was used as a substrate in anaerobic treatment, co-digestion with animal manure or direct supplying trace elements are practical options.