Abstract
Co-digestion of biowastes for hydrogen (H2) production using defined mixed cultures can overcome the high risk of failure due to contamination and imbalanced nutrient status. H2 production from biowastes—pea-shells, potato peels (PP), onion peels (OP) and apple pomace, either individually or in various combinations was evaluated by hydrolyzing with defined hydrolytic mixed bacterial culture (MHC5) and subjecting the hydrolysate to mixture of defined H2 producers (MMC6). Co-digestion of OP and PP hydrolysate supplemented at H2 production stage with GM-2 and M-9 media resulted in 95 and 102 l H2/kg of Total solids (TS), respectively compared to 84 l H2/kg of TS in control. Upscaling the process by digesting 4.0 l slurry (16-fold) resulted in 88.5 and 95 l H2/kg of TS, respectively compared to 72 l H2/kg of TS in control. Thus, H2 production by co-digestion of biowastes could be improved through the supplementation with very dilute medium (0.1 ×) and selection of suitable biowastes under unsterile conditions. The overall efficiency can be further enhanced by integrating it with bioprocesses for biopolymers such as polyhydroxyalkanoates and or biofuels like methane production.
Keywords: Biowaste, Bacillus, Hydrogen, Onion peels, Potato peels, Mixed microbial culture
Introduction
The production of biofuels as an alternative to fossil fuels has received substantial attention in the recent few decades [1–5]. Various strategies of microbial production of biofuels, including hydrogen (H2), methane (CH4), and bioalcohols have been demonstrated [6–9]. The production of H2 has been recommended as a clean fuel due its two major characteristics: (1) non-polluting nature—water being the end-product of combustion, and (2) high energy efficiency (122 kJ/g) [10–12]. The production of H2 through a biological process under ambient physiological conditions seems to be an attractive approach over other approaches such as chemical, thermal, nuclear and wind energy sources [10]. Different microbial strains have been established as consistent H2 producers especially Bacillus and Enterobacter [2, 13, 14]. These culture dependent approaches have been supported by genomic approach and have proved effective in identifying novel H2 producers [15, 16]. Broadly, biological H2 production through dark-fermentative process is more efficient than photo-fermentative process. Here, the major limiting factors is the requirement of regular supply of light [2, 10].
A variety of sugars, including glucose, fructose and sucrose have been used for producing H2, by microbes which can easily metabolize them [2, 13, 17, 18]. In order to circumvent the high cost of sugars, the use of biowaste as low-cost feed for large-scale H2 production appears economically feasible [19–24]. However, the complex and imbalanced nutrient status in the biowaste and presence of undesired inherent microbes accompanying the biowaste need special attention. To overcome these limitations, three approaches can be employed (1) hydrolysis of biomass through different methods, including physical, chemical, enzymatic and microbial, (2) co-digestion of biowastes and supplementation of nutrients, and (3) use of defined mixed microbial cultures at each stage of fermentation [21–27]. The microbial hydrolysis of biomass seems effective pretreatment for producing H2 even under unsterile conditions [21, 26]. In the present study, the influence of media supplementation on production of H2 was examined by defined mixed bacterial cultures for hydrolysis of feed and as H2 produces using biowastes, including pea-shells (PS), potato peels (PP), onion peels (OP), and apple pomace (AP), using unsterile conditions.
Materials and Methods
Bacteria and Culture Conditions
Strains selected in present study were reported in our previous works [16, 26]. The hydrolytic mixed culture (designated as MHC5, [26]) consists of Proteus mirabilis strains (EGU30 and EGU32), Bacillus sphaericus strains (EGU385 and EGU542) and Bacillus sp. strains (EGU444 and EGU447). The H2 producing microbial mixed culture (designated as, MMC6 [26]) consists of Enterobacter aerogenes EGU16, Bacillus cereus EGU41, P. mirabilis strains (EGU21 and EGU30), Bacillus megaterium HPC686 and B. pumilus HPC464. Bacteria were grown in nutrient broth to prepare inocula of hydrolytic and H2 producing cultures as reported previously [26].
Biowaste Hydrolysis
Biowastes—PS, OP, PP, and AP were used as feed [2% of total solids (TS)]. In brief, 250 ml of slurry was made using distilled-water in reagent bottles (300 ml). The resulting slurry was hydrolyzed using MHC5 by incubating for two days at 37 °C [21, 28]. Similarly, co-digestion of biowastes was carried out by mixing them in combinations of 2–4 [22].
H2 Production
The biowaste slurries hydrolyzed by MHC5 were used as feed-stock. Production of H2 was carried out by digesting biowaste hydrolysates with MMC6 under batch culture conditions. After, adjusting pH of the slurries to 7.0, argon was flushed to maintain anaerobic conditions. The reactor bottles were incubated at 37 °C. The daily production of resulting biogas was calculated by water-displacement procedure [13].
Effect of Medium Supplementation
The influence of medium supplementation on H2 producing abilities of MMC6 were checked by adding 0.1 × of M-9 or GM-2 at the following stages: (1) hydrolysis and (2) H2 production from individual and mixed biowastes.
Effect of Feed Concentration
The influence of feed concentration on H2 production was assessed by mixing biowastes (OP and PP) at 1, 2, 3, 5 and 7% TS.
Up-Scaling of H2 Production
The batch culture up-scaling of H2 production was tested using 0.75, 1.5 and 4.0 l of mixed biowaste (OP and PP) as a feed in the reactors of 1, 2 and 5 l capacities, respectively.
Analytical Methods
Gas Analysis
The evolved biogas gas contents were measured by gas chromatography system (Nucon GC5765, India) as reported previously [13, 29].
Results
Microbial fermentation of biowastes is limited by their initial hydrolysis into simpler forms and their biotransformation into useful bioproducts including H2. Well-defined mixture of bacteria with ability to produce hydrolytic enzymes were used to provide feed for H2 producing bacteria.
Production of H2 from Biowaste
Hydrolysis of different biowastes for 2 days with MHC5 followed by incubation with MMC6 resulted in H2 production of 225–350 ml/250 ml of slurry (Table 1). Biogas produced had a H2 content of 58.9–63.0%, such that the H2 yield (l/kg of TS) ranged from 45 with PS, to 70 with PP. It showed that the bacterial culture had a very high potential to produce H2, and it is greatly influenced by the type feed material. H2 yield from PP was 56% higher than that recorded with PS as feed. In view of the increase in H2 yields on switching the feed, it was envisaged that supplementation of nutrients may help to further enhance the H2 producing ability of the bacterial cultures. A comparative study was carried out by adding nutrients at two stages during biowaste fermentation process: (1) hydrolysis, and (2) H2 production. Addition of nutrients as M-9 and GM-2 media at the hydrolytic stage, lead to H2 yields of 36–61 l/kg of TS depending up on the biowaste. However, these yields were 5–21% lower than their respective controls (Table 1). Addition of GM-2 to different biowastes being subjected to hydrolytic bacterial culture resulted in further decline of 18–36% in H2 yields in all the cases. In each of these cases, maximum loss in H2 was recorded with PS as feed.
Table 1.
Hydrogen production by defined bacteria from biowastes
| Feeda | Hydrogen (H2) | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | Supplementation of medium (0.1x) | ||||||||||||||
| At hydrolysis stage | At H2 production stage | ||||||||||||||
| M-9 | GM-2 | M-9 | GM-2 | ||||||||||||
| Volb | % | Yieldc | Vol | % | Yield | Vol | % | Yield | Vol | % | Yield | Vol | % | Yield | |
| Pea-shells | 225 | 58.9 | 45 | 180 | 43.2 | 36 | 145 | 41.9 | 29 | 310 | 61.7 | 62 | 285 | 56.1 | 57 |
| Apple pomace | 300 | 61.9 | 60 | 285 | 57.2 | 57 | 245 | 44.8 | 49 | 415 | 65.4 | 83 | 405 | 59.7 | 81 |
| Onion peels | 335 | 63.0 | 67 | 265 | 49.7 | 53 | 270 | 46.5 | 54 | 425 | 63.8 | 85 | 430 | 61.3 | 86 |
| Potato peels | 350 | 62.9 | 70 | 305 | 51.2 | 61 | 285 | 51.3 | 57 | 460 | 64.2 | 92 | 435 | 60.5 | 87 |
Values are mean of three experiments and SD was < 10%
aTotal volume of feed: 250 ml (2%, TS) hydrolysed with MHC5 (2 days) followed with H2 production by MMC6 (up to 5 days)
bObserved volume (ml) of H2 in the biogas (H2 + CO2)
cl/kg of TS fed
In the next set of experiments, the two media were added at the H2 production stage. Here, addition of M-9, led to a H2 yield of 62–92 l/kg of TS. These yields were 27–38% higher than their controls (Table 1). An interesting feature of this fermentation process was that the H2 yields were influenced positively by the biowaste in the same order (PP > OP > AP > PS) as in the control. The increase in H2 yield was observed to be due to better H2 metabolism i.e. biogas had higher H2 content. Subsequent experiments, where GM-2 media was added at the H2 production stage, a 24–35% increase in H2 yield with respect to the controls was recorded. These H2 yields of 57–87 l/kg of TS were almost similar to those recorded with M-9 supplementation. Hence, the overall improvement in H2 production was recorded on addition of M-9 to AP or PS and GM-2 to AP (Table 1).
H2 Production by Co-digestion of Biowastes
One of the major issues, on the usage of municipal market biowastes is their availability in mixed form. Hence, it becomes important to analyze, which wastes should be co-digested. The four biowastes were co-digested in different combinations (Table 2). Mixing all the four kinds of biowastes in equal proportion resulted in 320 ml of H2, which was 5% higher than the expected value of 305 ml/250 ml slurry. In the next stage, various combinations of three biowastes resulted in 5–11% improvement over their expected values. In the cases, where AP or OP were missing from the combinations of three biowastes, a 15% enhancement in H2 yields was observed, it indicated that these wastes are not compatible with other biowastes. In the third group, co-digestion of two biowastes at a time resulted in 2–23% increase in H2 yields over expected values. In these cases, co-digestion of OP and PP proved to be among the most effective, with an enhancement in H2 from 340 to 420 ml/250 ml slurry. It implies that the presence of PS is more deleterious than addition of AP to the co-digestion of OP and PP (Table 2).
Table 2.
Hydrogen production by defined mixed microbial culture from co-digestion of biowastes
| Biowastesa | Hydrogen (H2) | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control | Medium (0.1 ×) | ||||||||||||||
| M-9 | GM-2 | ||||||||||||||
| Apple pomace | Onion peels | Potato peels | Pea shells | Volb | % | Yieldd | Vol | % | Yield | Vol | % | Yield | |||
| Expc | Obsb | Exp | Obs | Exp | Obs | ||||||||||
| +e | + | + | + | 305 | 320 | 59.7 | 64 | 400 | 365 | 63.2 | 73 | 390 | 345 | 58.7 | 69 |
| + | + | + | −f | 325 | 360 | 62.5 | 72 | 435 | 430 | 61.9 | 86 | 425 | 395 | 63.0 | 79 |
| + | + | − | + | 285 | 300 | 57.8 | 60 | 385 | 375 | 63.6 | 75 | 375 | 365 | 61.2 | 73 |
| + | − | + | + | 290 | 335 | 61.2 | 67 | 400 | 390 | 64.8 | 78 | 375 | 310 | 59.3 | 62 |
| − | + | + | + | 300 | 345 | 62.0 | 69 | 400 | 425 | 64.7 | 85 | 385 | 405 | 59.2 | 81 |
| + | + | − | − | 315 | 345 | 63.3 | 69 | 420 | 460 | 65.3 | 92 | 415 | 410 | 57.7 | 82 |
| + | − | + | − | 325 | 330 | 60.4 | 66 | 440 | 405 | 62.7 | 81 | 415 | 420 | 61.8 | 84 |
| + | − | − | + | 265 | 290 | 58.8 | 58 | 365 | 390 | 61.4 | 78 | 340 | 370 | 58.3 | 74 |
| − | + | + | − | 340 | 420 | 64.2 | 84 | 440 | 510 | 66.7 | 102 | 430 | 475 | 63.2 | 95 |
| − | + | − | + | 280 | 305 | 56.3 | 61 | 365 | 415 | 64.2 | 83 | 355 | 360 | 60.6 | 72 |
| − | − | + | + | 290 | 295 | 58.6 | 59 | 385 | 385 | 61.8 | 79 | 355 | 310 | 58.4 | 62 |
Values are mean of three experiments and SD was < 10%
aFeed in equal ratio: 250 ml (2%, TS) hydrolysed with MHC5 (2 days) followed with H2 production by MMC6 (up to 5 days)
bObserved volume (ml) of H2 in the biogas (H2 + CO2)
cExpected volume of H2, based on the average of H2 producing capacities of mixed microbial culture MMC6 from individual biowaste
dl/kg of TS fed
ePresent
fAbsent
Supplementation of biowaste hydrolysate with medium M-9, resulted in: (1) 10% loss on co-digesting the four biowastes, (2) 1–3% loss on co-digesting three biowastes and 6% enhancement on combining OP, PP and PS, and (3) 7–16% enhancement in most of the co-digestions involving only two biowastes. Here, the maximum benefit in H2 yield was recorded in the co-digestion of OP and PP with M-9, a trend that is similar to one seen in control. Overall, the maximum H2 production of 102 l/kg of TS was recorded by using OP and PP combination, which was hydrolysed by MHC5 and fermented with MMC6 as mixed H2 producers. Supplementation of different combinations of biowastes with GM-2 medium was observed to be quite similar to that observed with the addition of M-9 medium. With GM-2, observed H2 yield was 11% higher than the expected values. Here, the maximum yield of 95 l H2/kg of TS was recorded with the co-digestion of OP and PP.
Effect of Feed Concentration on H2 Production
To improve the efficiency of the bioprocess, it is desirable to use higher concentration of feed in the reactor. Based on the experiments described above, different feed concentration (1–7% TS) were tested only for co-digestion of OP and PP (Table 3). With increase in the feed concentration from 1 to 7% TS, the volumetric H2 production was observed to increase from 220 to 1075 ml, equivalent to 61.4–88.0 l/kg of TS. Here, H2 constituted 54.4–64.2% of total biogas produced. Further, the supplementation of M-9 and GM-2 showed higher volumetric production in the ranges of 290–1590 and 275–1410 ml H2/250 ml of slurries, respectively. Here, H2 yields (l/kg of TS) were equivalent to 90.9–116 with M-9 and 80.6-110 with GM-2 supplements. The maximum yields of 116 and 110 l/kg of TS were observed with supplementation of M-9 and GM-2 as compared with control 88 l/kg of TS, respectively. It may be remarked that increasing the TS from 1 to 7% resulted in only marginal reduction in H2 yields: 30%, 22% and 27% in the case of control, M-9 and GM-2, respectively.
Table 3.
Effect of biowaste concentration on hydrogen production by defined mixed bacteria
| Feeda (%) | Hydrogen (H2) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Control | Medium (0.1 ×) | ||||||||
| M-9 | GM-2 | ||||||||
| Volb | % | Yieldc | Vol | % | Yield | Vol | % | Yield | |
| 1 | 220 | 61.0 | 88.0 | 290 | 65.4 | 116 | 275 | 59.5 | 110 |
| 2 | 420 | 64.2 | 84.0 | 510 | 66.7 | 102 | 475 | 63.2 | 95.0 |
| 3 | 580 | 63.3 | 77.3 | 750 | 61.5 | 100 | 710 | 61.7 | 94.7 |
| 5 | 905 | 58.7 | 72.4 | 1230 | 60.2 | 98.4 | 1165 | 58.7 | 93.2 |
| 7 | 1075 | 54.4 | 61.4 | 1590 | 58.9 | 90.9 | 1410 | 55.6 | 80.6 |
Values are mean of three experiments and SD was < 10%
aFeed (OP and PP): 250 ml (2%, TS) hydrolysed with MHC5 (2 days) followed with H2 production by MMC6 (up to 5 days)
bObserved volume (ml) of H2 in the biogas (H2 + CO2)
cl/kg of TS fed
Up-Scaling
Up-scaling of H2 evolution by MMC6 from co-digestion of biowastes (OP and PP) has been presented in Table 4. In control, the volumetric production H2 increased from 0.42 to 5.75 l with an increase in the working volume from 0.25 to 4.0 l. Here, the yield was observed in the range of 71.9–84.0 l H2/kg of TS with H2 contents of 52.8–64.2% of total evolved biogas. These results suggest that the up-scaling of H2 production from biowaste is quite stable, with an over variation of 14% on upscaling to 4.0 l. Volumetric production of H2 from biowastes improved to 7.6 and 7.08 l on supplementation with M-9 and GM-2 medium, respectively. In contrast, to 14% variation recorded in the case of control, the variation in H2 yield was only 7% in the cases where biowastes supplemented with nutrient media were used. Overall, these results demonstrate that 16-fold up-scaling in the working volume of feed was quite consistent.
Table 4.
Up-scaling of hydrogen production from biowaste
| Feeda | Hydrogen (H2) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Control | Medium (0.1 ×) | ||||||||
| M-9 | GM-2 | ||||||||
| Volb | % | Yieldc | Vol | % | Yield | Vol | % | Yield | |
| 0.25 | 0.420 | 64.2 | 84.0 | 0.510 | 66.7 | 102 | 0.475 | 63.2 | 95.0 |
| 0.75 | 1.135 | 59.4 | 75.7 | 1.495 | 61.7 | 99.7 | 1.375 | 57.3 | 90.0 |
| 1.50 | 2.175 | 55.5 | 72.5 | 2.735 | 63.2 | 91.2 | 2.510 | 61.5 | 83.7 |
| 4.00 | 5.750 | 52.8 | 71.9 | 7.600 | 59.4 | 95.0 | 7.080 | 57.9 | 88.5 |
Values are mean of three experiments and SD was < 10%
aFeed (OP and PP): 250 ml (2%, TS) hydrolysed with MHC5 (2 days) followed with H2 production by MMC6 (up to 5 days)
bObserved volume (ml) of H2 in the biogas (H2 + CO2)
cl/kg of TS fed
Discussion
Production of H2 from the pure sugars and biowaste as a primary feed is widely evaluated using pure and mixed cultures [2, 5, 13, 26]. Since, biowaste are highly complex in nature, they need pretreatment for their effective metabolization to produce at higher H2 yield [22, 26]. Microbial activity seems to be a viable and cost-effective approach to improve biowaste hydrolysis [21, 22]. In our previous studies, we have effectively demonstrated the use of defined sets of MHCs and MMCs combinations to improve the H2 production from biowaste as feed [26]. Another major limitation which has not been paid much attention is the imbalanced nutrient status of the biowastes. In this study, we have checked the influence of media (M-9 and GM-2) by using them as supplements at two stages of overall fermentation: (1) at hydrolytic, and (2) at H2 production. Secondly, we opted for co-digestion of biowastes to achieve the desired nutritional status.
Using combination of MHC5 and MMC6, H2 production from biowastes—AP, OP, PP and PS (2% TS) was shown to be 60, 67, 70 and 45 l/kg of TS respectively. Here, significant variation in the H2 yield might be associated with the variation in the composition of these biowastes. Interestingly, the supplementation of both M-9 and GM-2 media at H2 production stage showed positive influence on H2 production. An enhancement up to 1.3 to 1.4-fold in H2 yield was recorded using media. In contrast, the supplementation of media at hydrolysis stage did not prove beneficial. Among the various combinations of co-digestions evaluated, the combination of PP and OP resulted in the production of 84 l H2/kg of TS, which was more than the yield of 67 and 70 l H2/kg of TS, recorded with these wastes individually. Further, supplementation of media to different co-digestions of biowastes, once again provided to be most effective in the case of OP and PP combination. Here, the H2 yield was found to get enhanced to 95 l/kg of TS with GM2 and to 102 l/kg of TS with M-9.
Another very interesting feature, which was observed in co-digestion and supplementation was the potential to further improve the H2 production process. It was found that lower feed concentration of 1% TS (OP + PP) supplemented with M-9 and GM-2 media could enhance the H2 yield (l/kg of TS) from 102 to 116 and from 95 to 110, respectively. Since, the process efficiency can be improved by increasing the loading rate, we found that there was a marginal decline in H2 yield on increasing the TS concentration up to 7%. Thus, a 7-fold improvement in reactor size can be achieved at the cost of 22–27% loss in H2 yield. However, at 7% TS level, there was a 1.48-fold enhancement in H2 yield with M-9 in comparison to control.
For all bioprocesses, the ultimate goal is to produce the bioproduct on a large scale [26]. We thus evaluated the efficiency of the process by upscaling the process by 16 times. On up-scaling of process at a working volume of 4.0 l, it exhibited high volumetric production up to 7600 ml of H2 using M-9 medium. Here, 1.3-fold improvement in H2 production was recorded as compared with control. Overall, these results suggest that supplementation of medium at H2 production stage is very effective to enhance the production yield using biowastes under unsterile conditions. Here, H2 production was significantly higher than the previously reported yield of 60.2 l H2/kg of feed with enzymatically pre-treated oil palm [4]. Similarly, undefined mixed culture had shown lower H2 production yield 55.3 l/kg of volatile solids using mixed biowaste consisting of macro-algae (Laminaria digitata) and micro-algae (Arthrospira platensis) [6]. However, the H2 production has been shown to be stabilized using defined mixed cultures [2, 5, 11, 26]. Further, robustness of defined mixed culture can be significantly improved by designing selective microbes with unique feature such as hydrolytic, H2 production, quorum sensing mediated biofilm formation and anti-microbial properties to improve yield under unsterilized conditions [1, 10, 16, 30, 31]. Further economic improvement in this process can be achieved through its integration with processes leading to the production of CH4, PHA or biomethanol, through biorefinery approach [5, 28, 32–38].
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2018H1D3A2001746, 2013M3A6A8073184). This research was also supported by KU Research Professor Program of Konkuk University.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflict of interest.
Footnotes
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Contributor Information
Jung-Kul Lee, Email: jkrhee@konkuk.ac.kr.
Vipin Chandra Kalia, Phone: +82-2-2049-6179, Email: vckaliaku@gmail.com.
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