Energy Recovery from Herbal Wastewater and Monosodium Glutamate Wastewater through Anaerobic Co-Digestion

: Anaerobic continuous stirring tank reactor (CSTR) was employed for biohydrogen production by anaerobic co-digestion from the mixture of herbal wastewater (HW) and monosodium glutamate wastewater (MGW). A series of blend volumetric proportions (MGW/HW), organic loading rates (OLR) were used as control strategy and evaluated for optimal biohydrogen production. The highest biohydrogen production of 7260±7.7 mL L -1 d -1 was attained at blend proportion of 15%, OLR of 41 g COD L -1 d -1 while the highest biohydrogen yield of 508.9±7.2 mL g COD removed-1 was observed at same conditions. This study demonstrated the feasibility and effectiveness of enhanced biohydrogen production by adding MGW to compensate the low nitrogen content of HW.


Introduction
With the rapid development of pharmaceutical enterprises in China, increasing discharge of pharmaceutical wastewater has generated great environmental pollution to the original water resource system of rivers and lakes. The herbal wastewater (HW), mainly containing chrysophanic acid, sugar, lignin, protein, organic acid, anthraquinone and their corresponding hydrolysates, was characterized by highly concentrated organic compounds, suspended substance and chroma [1]. The activated sludge process based on microbial flora was usually applied to the treatment of HW, due to its easily biodegradability. When C/N ratio is too low, most microbes rapidly consume nitrogen for growth. Although this has a positive effect on methane production rate. However, the lack of carbon type cause that decreases in acid forming, nitrogen accumulates in the form of ammonium ions (NH4) that increase the pH which adversely affects biogas production. [2] Anaerobic digestion refers to the process of degrading organic matter into biogas and carbon dioxide by relying on the biochemical action of facultative anaerobes and obligate anaerobes in the absence of oxygen. This process was widely used as a source of renewable energy from the extensive substrates. The four important stages of anaerobic digestion involved hydrolysis, acidogenesis, acetogenesis and methanogenesis [3,4]. Biohydrogen could be produced by hydrolysis and acidogenesis stage.
Monosodium glutamate wastewater (MGW) characterized by highly concentrated organic matter and ammonia nitrogen was very hard to be treated by activated sludge process, because of the toxicity to microorganisms caused by high concentration ammonia nitrogen. As everyone knows, the microorganisms existed in activated sludge would bear NH 3 /NH 4 -N intoxication which resulting in the decreased treating performance once the ammonia nitrogen concentration exceeded a certain limit [5]. Benabdallah EH, et al [6] observed the 50% inhibition of biogas production when ammoniaion reached concentration of 3860 mg L -1 using municipal solid waste as the substrate to anaerobic degradation of the organic part. The strategy of blending MGW into HW for biohydrogen production by anaerobic digestion could be adopted. Chen H, et al [7] conducted systematic treatment of monosodium glutamate (MSG) wastewater using a lab-scale up-flow anaerobic sludge blanket reactor under various organic loading rates (OLRs). The optimal OLR was lower than 8g-COD/L/d with a maximum CH 4 yield of 0.28±0.03 L/g-COD, which was consistent with highest COD removal efficiency up to 97.9%.
The purpose of this study was to appraise the successive biohydrogen production from the combination of HW and MGW in anaerobic co-digestion process. The optimization of the organic loading rate (OLR) and stirring speed were also investigated to improve biohydrogen production.

Inoculums and Substrates
The raw sludge was obtained from the sludge dewatering room located in Hongsha sewage treatment plant (Sanya, China). The sludge was enriched by aerating intermittently for over 20 d to inactivate the hydrogen-consuming microorganisms, especially methanogens [8]. After enough enrichment, the sludge was inoculated into the anaerobic reactor. The total suspended solid (TSS) and volatile suspended solid (VSS) of inoculum was 6.5±0.76 g L -1 and 4.9±0.34 g L -1 , respectively. The HW and MGW were obtained from Hainan Kangnongtang Traditional Chinese Medicine Co. LTD (Haikou, China) and Hainan MSG Factory (Haikou, China), respectively. The compositions of both wastewaters were listed in Table1. As shown in Table1, the HW has a high C/N ratio of 610.0 whereas a low C/N ratio of 8.2 was presented by MGW. The ammonia nitrogen accounted for almost all of nitrogen in wastewaters. The various C/N could be obtained by adjusting the blend volumetric proportion of MGW to HW.

Experimental equipment
Fig 1 shows the anaerobic continuous stirred tank reactor used for biohydrogen production. The reactor is made of plexiglass with an effective working volume of 1.5L. The CSTR reactor maintains the temperature at 35(±1)°C through the outer thermal circulating water bath heating layer. The reactor is equipped with a pH meter, a temperature measuring and controlling instrument, and an oxidation-reduction potential measuring and controlling instrument. An agitator was installed with initial stirring speed of 100 rpm at the top of reactor. The system pH was automatically controlled at about 5.5 with addition of sodium bicarbonate. Before the start-up, the reactor was flushed with nitrogen for 5 min for air replacement to attain the anaerobic environment.

Experimental Operating conditions
The experiment was initially controlled at hydraulic retention time (HRT) of 16 h by batch mode for biohydrogen production. The blend proportions of MGW to HW were determined to be five series of 0%, 5%, 10%, 15% and 20%, respectively. The corresponding C/N ratios were 610.0, 73.7, 41.0, 29.2 and 23.2, respectively. The control batch was set for digestion of single MGW. After the optimal blend proportion was determined in terms of maximum biohydrogen production, the different OLRs by decreasing HRTs of 16 h, 12 h, 8 h, and 6 h. All experiments were carried out at the same time in 3 groups of parallel experiments with the same conditions, and the average results were expressed in standard deviation. When the change in biohydrogen production is less than 10% within 5 days, the process is deemed to be in a stable state.

Analytical methods
COD, pH, SS, VSS and alkalinity of inoculum and wastewater were determined in accordance with standard methods [9]. Total nitrogen (TN) content was determined with a TN analyzer (Model NPW-160, HACH, USA), and total phosphorus was measured by a TP meter (Model NPW-160, HACH, USA).
Biogas generated from each reactor was collected using a wet gas meter. Hydrogen content was determined by a gas chromatograph (Shimadzu GC-8A, Japan) equipped with a thermal conductivity detector and a stainless steel column packed with Porapak Q (50-80 meshes). Soluble metabolites (VFAs and ethanol) concentrations were analyzed by another gas chromatograph (Shimadzu GC-14A, Japan) with a flame ionization detector. Fig.2 showed biohydrogen production value at different blending proportion of HW and MGW in a steady state. According to Fig.2, it could be seen that all blends showed a higher final biohydrogen production than that of using HW as substrate alone. The blend has a synergistic effect on enhancement of biohydrogen production if more biohydrogen was produced relative to an estimate the biohydrogen production of single substrate digestion. This obvious synergy could be reflected by the increase in hydrogen production, and the reason may be explained as the addition of MGW supplements the lack of HW nitrogen. An increase in blend proportion of MGW from 5% to 15% during the anaerobic co-digestion led to the increased biohydrogen production. The maximum biohydrogen production of 2468±6.7 mL L -1 d -1 was observed in blend proportion of 15%, which was C/N ratio of 29.2. Compared with the data of 833.3±9.7 mL L -1 d -1 obtained using HW as single substrate, this production gave a 196.2% increase in biohydrogen production. Albeit the highly concentrated organic compounds were considered to be easily degradable, but nitrogen deficiency resulting in imbalance of nutrient ratio restricted the growth and metabolism of microorganisms to some extent [10]. However, a further increase in the blend proportion to 20% with decreased C/N down to 23.2 provoked a drastic reduction biohydrogen production of 1740±7.7 mL L -1 d -1 with ammonia nitrogen concentration of 634 mg L-1. Many researches founded that the free ammonia nitrogen has stronger inhibitory effect than ammonia ion because free ammonia nitrogen was more easily permeable through the cell wall of microorganisms. Nevertheless, it was noteworthy that ammonia nitrogen would be mainly existed in this anaerobic co-digestion in the form of ammonia ion based on ionization balance of NH 3 /NH 4+ at pH 5.5. So, the ammonia nitrogen concentration in influent of reactor was not the limiting parameter for the descendent biohydrogen production. Maybe the blend proportion of 20% was more unfavorable for the microorganism's metabolic activities in this study using HW and MGW as blending substrate, though the corresponding C/N ratio of 23.2 was in the optimal range advised by Parkin GF and Owen WF [11]. The lowest biohydrogen production of 480±4.3 mL L -1 d -1 using MGW as single substrate with ammonia nitrogen concentration of 3300 mg L -1 was obtained indicating the partly inhibitory effect for microorganisms.

Biohydrogen production from various blend proportion
It can be observed from Fig.3 that the biohydrogen yield (based on removed COD) has a similar trend with biohydrogen production at different blend proportions. In the same way, the system possessed the highest biohydrogen yield of 381±3.1 mL g COD removed -1 at 15% blend proportion.The biohydrogen yield obtained could be compared with the value reported in literature. D.Sivaramakrishna, et al [12] used HW and slaughterhouse sludge from anaerobic sequencing batch reactor for biohydrogen production, obtaining highest yield of 387±12 mL g CODremoved-1. Stated thus, the blend proportion of 15% (MGW/HW), in combination with C/N ratio of 29.2 was determined to be the optimal condition from anaerobic co-digestion in terms of highest biohydrogen production and yield in this study.

Biohydrogen production from various OLR
After the determination of optimal blend proportion, the different stirring speed and OLR achieved by decreased HRT were evaluated. The anaerobic co-digestion reactors were operated for ten stages with duration of 68 d. The methane was not detected in the biogas in overall operation, showing the methanogenesis was inhibited effectively. The biohydrogen production and content in biogas were shown in Fig.4. When the OLR was increased from 23.1 g COD L -1 d -1 to 41.0 g COD L -1 d -1 , the incremental biohydrogen production and yield could be observed from 2472±5.2 mL L-1 d-1 to 7260±7.7 mL L -1 d -1 and from 382.0±3.1 mL g COD removed -1 to 508.9±7.2 mLꞏg-1 CODremoved, respectively. The biohydrogen content fluctuated in the range of between 40.7±2.3% and 48.7±3.4% and COD removal rate presented a slight variation from 33.6±1.7% to 34.9±2.1%. However, a further increase OLR to 61.6 g COD L -1 d -1 resulted in the rapid decrease in both biohydrogen production and content as well as COD removal rate. This phenomenon could be explained by overburden of microorganism's degradation and insufficient hydrolysis of organic compounds at short HRT of 6 h. To prevent the system paralysis, the strategy of decreasing OLR to 41.0 g COD L -1 d -1 was adopted to recover the performance of biohydrogen production. According to Fig.4, the biohydrogen production could raise up to 7082±7.7 mL L -1 d -1 , near to the maximum biohydrogen production. This indicated the effective recovery of microorganism's metabolic activities with decreasing OLR, which was reflected by incremental biohydrogen yield and COD removal rate.it could be seen that the main VFAs were ethanol, acetate, butyrate and propionic rate and acetate was the predominant product. it could be seen that the main VFAs were ethanol, acetate, butyrate and propionic rate and acetate was the predominant product C 6  of 2 moles of hydrogen would be accompanied by the production of 1 mole of acetic acid.it could be found that acetate was produced by acidogenesis in anaerobic digestion for biohydrogen and excess of biohydrogen was produced from other microorganisms' metabolic pathway, including ethanol-type fermentation (Eq.2) and butyrate-type fermentation (Eq.3) as well as other metabolic pathways [13]. There was no homoacetogenesis process involving hydrogen and carbon dioxide consumption in this system, though hydrogen accumulation could favor homoacetogenesis [14]. A little amount of propionate which consumed hydrogen during its production was detected in the VFAs besides OLR of 61.6 g COD L -1 d -1 , but this has little effect on final biohydrogen production. The OLR of 41 g COD L -1 d -1 (HRT= 9 h) was most suitable for biohydrogen production at blend proportion of 15% (MGW/HW).  It could be deduced from above results that the possibility to blend HW and MGW for biohydrogen production was useful for providing data support of anaerobic co-digestion. The synergistic effects obtained by blending substrates deserved particular attention and further investigations need to be carried out for implication of experimental findings about anaerobic co-digestion in actual applications.

Conclusion
This study demonstrated the feasibility of strengthening biological hydrogen production from anaerobic co-digestion by blending herbal wastewater (HW) and monosodium glutamate wastewater (MGW). Results showed that high nitrogen content in feedstock (low C/N) led to low microbial yields and aggravated the reduction of hydrogen production, while the ammonia nitrogen concentration in influent of reactor was not the limiting parameter for the descendent biohydrogen production. The production of hydrogen had a similar trend with VFAs at different operating parameters, and VFAs were mainly acetic acid and butyric acid. The highest biohydrogen production of 7260±7.7 mL L-1 d-1 and the highest biohydrogen yield of 508.9±7.2 mL g COD removed -1 were obtained at optimal operation conditions (Blend volumetric proportion (MGW/HW): 15%, OLR: 41.0 g COD L -1 d -1 ) indicated that it was a promising strategy to achieve the higher bioenergy production by blending wastewaters which were not favorable for anaerobic digestion as sole substrate and established the significant process parameter for future industrial application.