Bioenergy recovery from food waste through dark fermentation direction

doi:10.21203/rs.3.rs-1965745/v1

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Bioenergy recovery from food waste through dark fermentation direction

https://doi.org/10.21203/rs.3.rs-1965745/v1

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The increase in food waste generation has encouraged the scientific community to convert it into value-added resources. Hydrogen energy provides a sustainable option to fossil fuels due to its purity, high energy content, and reduction of global warming. This study investigates the batch fermentative biohydrogen production from food waste, which takes into account the effects on the process stability and energy recovery. Experiments were carried out at different ranges of pH (5.5, 6.0, and 6.5), FW concentration (1, 5, 10, 15, 20, 25, and 30 g-VS/L) at mesophilic (37˚C) and thermophilic (55˚C) temperature through 24h incubation of batch dark fermentation process. The FW concentration of 15–20 g-VS.L^− 1 was found to be the proper concentration for biohydrogen production at 55˚C fermentation with the highest yield of 80.41 ml-H₂/g-VS.L^− 1, hydrogen content of 76.53%, at pH 6.0. Under mesophilic temperature, a lower hydrogen yield of 49.36 ml-H₂/g-VS.L^− 1and hydrogen content of 60.53% was produced in the same substrate concentration and pH. The COD removal efficiencies at 37 ˚C and 55 ˚C increased from 16.66–21.87%, and 20.83–29.09% at pH 6.0 with an increase in FW concentration from 1 to 20 g-VS.L^− 1. Further increase in FW concentration showed less yield of hydrogen production. From pyrosequencing analysis, hydrogen fermentation at both temperatures was attributed to Lactobacillus and Clostridium sensu stricto 1, which confirms the potential of the microbial community for biohydrogen production.

Biohydrogen

food waste

dark fermentation

microbial community

With fast urban growth, organic solid waste is a primary issue that negatively affects and threatens the sustainable environment by the emission of greenhouse gases (GHGs), groundwater, and soil contamination [1]. In Malaysia, food waste (FW) is one of the most plentiful and challenging organic solid waste. In 2020, the average amount of FW generated in Malaysia has been estimated 31,000 tons. Statistic on the solid waste quantity is essential to manage all dimensions of solid waste generation [2].

Furthermore, the growing ecological perception, the government has directed to the prompt growth of FW management, and the circular economy has been planned to decrease and recycle the FW [3]. Therefore, efficient management and scheme are needed to protect and reduce the catastrophic environmental consequences associated with FW. Fermentation of organic biomass, especially FW is considered as a good option with utmost potential due to a well-balanced content of carbon and nutrient with high-energy content and biodegradability for energy production through anaerobic fermentation [4]. Bio-hydrogen production from FW is a promising method for sustainable source of energy generation, and waste treatment. Dark fermentation process seems to be the most proficient and economical approach, among other biological methods [5, 6]. This treatment includes four steps viz. hydrolysis, acidogenesis, acetogenesis, and methanogenesis, which a group of microorganisms (acidogenic, acetogenic, and methanogens, etc.) produces biogas from organic biomass. Acid forming or acidogenic bacteria utilize and hydrolysis the organic carbons for their growth, and then during this process accumulates the volatile fatty acids (VFAs: acetic acid, propionic acid, butyric acid, and valeric acid) with water (H₂O), hydrogen (H₂), and carbon dioxide (CO₂) which is called acidogenesis. The second group of bacteria (acetogenic bacteria) utilizes the VFAs except for acetic acid, and produce acetic acid and H₂, which is called acetogenesis. At the fourth step, acetic acid, H₂, and H₂O are utilized by the methanogens in the form of methane (CH₄) [7, 8].

Biohydrogen production process is significantly affected by many parameters such as inoculum, pH, temperature, substrate concentration, microbial community structure, and etc. [9]. For biohydrogen production, the activity of methanogens bacteria must be prevented; hence, operation the system at short hydraulic retention time (HRT), low pH, or applying heat treatment of the sludge are the approaches to overwhelm the methanogens bacteria and increase the yield of H₂ production [10, 11]. Among these methodologies, heat treatment has been considered as an alternative to enrich the community of hydrogenase bacteria from anaerobic inoculum culture [12, 13]. The influence of the required fermentation pH of 5.5 to 6.0 as an effective range on the efficiency of biohydrogen production has been demonstrated in several studies [9, 13, 14]. During the bio-H₂ production process, the acids with higher molecular weight (acetic acids, butyric acids) are accumulated in the operation system. Therefore, the microorganisms are restrained from growing and halt the biohydrogen production if not appropriate range of pH provided [11]. Therefore, it is found that the range of pH value mainly affects the enzymatic activity and metabolism of the bacteria population. From the studies, the optimum value for pH was found to be 6.0; however, a wide range of pH was examined on different substrates on the performance of enzyme activity on the fermentation process [15].

Moreover, one of the essential parameters affecting the biohydrogen fermentation process is temperature, which influences the production rate, bacterial growth, bacterial community, and substrate degradation. Many studies have been conducted on mesophilic temperature (30–37^ºC), thermophilic temperature (50–60^ºC), or even at room temperature. Furthermore, hydrogen and metabolite production can be affected by the structure of the microbial community. Hawkes et al., [16] studied the structure of the microbial community and revealed that Clostridia species, such as Clostridium butyricum yield butyrate mostly while Clostridium propionicum produce propionate. Thus, from the past studies, the most strong hydrogen producers are identified as Clostridium pasteurianum, Clostridium butyricum, and Clostridium beijerinki [17]. Another important factor that is considered for the fermentation process of biohydrogen production is the substrate concentration, which affects the metabolism of substrate degradation. Hence, the lower substrate concentration follows the slower reaction efficiency due to the lack of food to the microorganism’s ratio (F/M) [11]. Therefore, there should be a balance between the degree of substrate metabolism and the efficiency of the reaction. According to these experiences, this study aimed to assess the different key operational parameters affecting biohydrogen production through dark fermentation process of food waste. Moreover, the structure of the community of microorganisms under two mesophilic and thermophilic conditions were studied at the high amount of hydrogen content, and the results compared with the inoculum before and after the heat-treatment suggesting the microbial activity and the role of heat-treatment in biohydrogen production.

2.1 Inoculum pre-treatment and acclimitization

The inoculum was provided from anaerobic sludge pond of Jugra palm oil mill Sdn. Bhd., Selangor, Malaysia [6]. The anaerobic sludge was stored in 1 L glass bottle, and fed daily by sugar 10 g/l for one week to revive the sludge. From the biogas produced in the bottle could be found that the microorganisms have the high activity of methanogens as indicated from the batch control test. Hence, the anaerobic sludge was heat-treated in waterbath with 80 ºC for 1h to inactive the methanogen bacteria to enrich hydrogen-producing bacteria [13]. Then the sludge was cooled down to ambient temperature and ready for further analysis.

2.1 Food waste feedstock

The food wastes (FW) collected from the Zero Waste Campaign, University of Malaya, Kuala Lumpur, Malaysia, and stored in freezerm (-20 ºC) to avoid the influence of indigenous microorganisms in the FW on the process of hydrogen production. Before use in the batch test studies, the food waste was defrosted a few hours under ambient conditions in laberatory. To prepare the substrate, water was added to the FW to simplify the grinding and blending using an electrical blender and sieved with mesh no. 1 mm. The characteristics of FW such as total solids (TS), volatile solids (VS), chemical oxygen demand, total phosphorus (TP) concentration were measured according to standard methods [18]. The characteristics of FW and inoculum after heat pre-treatment are given in Table 1.

Scanning electron microscopy (SEM) of POME sediments

Food waste was dried in oven at 60 ˚C overnight. The The surface morphology of solids FW particles were characterized by SEM images to provide a clear perspective on the physical characteristic of this substrate. The SEM of FW particles content is shown in Fig. 1. Moreover, Energy Dispersive X-Ray Analyzer (EDX) was used to provide elemental identification and atomic specification information with 5 analysis spots at different points of samples [13].

Table 1

Characteristics of food waste and anaerobic inoculum sludge
Parameters	Food waste	Anaerobic inoculum sludge	Unit
pH	4.2 ± 0.2	7.6 ± 0.1	-
Total solids (TS)	21 ± 1.1	40 ± 2.2	g. L^− 1
VS	19.4 ± 1.4	36 ± 1.2	g. L^− 1
VS/TS	0.92 ± 1.2	0.9 ± 1.7	%
COD	85.5 ± 5.8	-	g COD. L^− 1
Total Carbohydrate	75.6 ± 1.3	-	g COD. L^− 1
Total Kjeldahl nitrogen	4.1 ± 0.2	-	g. L^− 1
Lipids	4.4 ± 0.9	-	g. L^− 1
Alkalinity	0.5	-	g CaCO₃. L^− 1

2.3 Preparation of batch serum bottles at different operational parameters

Batch biohydrogen production tests were done in the 180 mL serum bottle. To study the process, the effect of three pH levels 5.5, 6.0, and 6.5; different FW concentration of 1, 5, 10, 15, 20, 25, and 30 g-VS.L^− 1 with inoculum MLSS of 40 ± 2.2 g/l on biohydrogen production through dark fermentation were conducted. Prior to use, to reach a exact chemical oxygen demand these batches of food waste were diluted with water in the feed stock used for fermentation experiments to attain a exact chemical oxygen demand for each FW concentration. In the first set, the effect of the mesophilic (37˚C) condition was studied. Each serum bottle was filled with 40 ml inoculum (MLSS: 40 g/l; MLVSS: 28 g/l) and 40 ml of FW at different COD concentrations. The initial pH value of food waste was 4.2. Each series includes a 3-serum bottle with the same FW concentration, adjusted at different pH of 5.5, 6.0, and 6.5. Hence, 7 various series batch tests (21 batch serum bottles) with different FW concentrations were prepared at three different pH values. The same procedure was done for the second set with the difference that the thermophilic (55˚C) condition was used. The experiments were done on triplicate. In total, 63 series of batch test was prepared. The initial pH was adjusted using NaHCO₃ 2.0 g/l. In this study, no nutrient solution was added to the batch system. The bottles were sealed with a bottle rubber cap stopper and aluminum cap cover. Before incubation of the serum bottles, to remove the oxygen gas (O₂), nitrogen gas (N₂) was purged into the serum bottle by inserting the N₂ gas needle into the rubber to purge the N₂ and another needle to withdraw the O₂ for 5 min at 10 mL. min^− 1 to provide an anaerobic condition in the system. Then, each set were incubated on a rotary shaker incubator at 37 ˚C and 55 ˚C, and rpm of 150, respectively. After each operation run (24 h), the bottles were kept in ambient temperature and were allowed to settle. Then the biogas sampling collected in a small glass laboratory tube by water displacement technique, and a syringe was used to measure the volume of the biogas. The cap of the glass tube contains the biogas sample was sealed by a rubber stopper and wrapped using parafilm tape. Then, the samples were prepared to measure the biogas content. Subsequently, the supernatant was filtered by vacuum pump using filter paper with a pore size of 0.45 µm. The clarified samples were used to determine the soluble chemical oxygen demand (sCOD), and pH.

2.4 Microbial study

Four samples were prepared, including the anaerobic sludge before heat treatment (AnBtre), anaerobic sludge after heat treatment (AnAtre), and acclimatized sludge with food waste (FoodW37) at 37 ºC and (FoodWS55) at 55 ºC. the method for preparation of sample is similar to our previous paper [13]. Samples were preserved in deep freezer at -80 ºC before collection of samples to be qualified for amplicon library construction. Then samples were transported to Apical Scientific Sdn. Bhd. (Selangor, Malaysia) at -78 ºC temperature condition in a box filled by dry ice. All the samples analyzed were done by Apical Scientific Sdn. Bhd. (Selangor, Malaysia). The procedure for 16S rRNA amplicon library construction, sequencing, and software pachage used are provided in our previous paper [13].

2.5 Measurement methods

All the experiments solids, COD, VSS, Kjeldahl nitrogen, ammonia, and alkalinity were quantified according to the American Public Health Association (APHA) standard methods [19]. Phenol–sulfuric method with glucose as a standard was used to measure the total carbohydrate [20]. All the equipment and methods used in this study is same as our previous study [12–14]. Lipids were measured by Soxhlet method [21].

3.1 Effect of pH, temperature, and FW concentration on hydrogen properties

The dataset obtained from the effect of pH on hydrogen content (%), hydrogen yield (ml-H₂/g-VS.L^− 1), and COD removal (%) at different range of FW concentration over 24h fermentation process under both mesophilic (37 ˚C), and thermophilic (55 ˚C) conditions are provided in Table 2. From Figs. 1 and 2, it is clearly seen that a higher amount of biohydrogen content and hydrogen yield achieved at pH 6.0 and thermophilic fermentation than the pH 5.5, and 6.5, and mesophilic fermentation condition. When hydrogen builds up during the process, the pH value attribute to the accumulation of volatile fatty acids (VFAs) is decreased. After 24 h fermentation, the pH from 5.5, 6.0, and 6.5 decreased on average to 4.43, 4.85, and 5.02 at mesophilic temperature; and 4.12, 4.56, 4.78, at thermophilic temperature, respectively. The low value of pH strictly restricts the enzymatic activity and the growth of biomass. The maximum hydrogen content and hydrogen yield obtained 76.53% and 80.41 ml-H₂/g-VS.L^− 1 at pH 6.0 and 55 ºC. However, the results showed that pH 6.0 promoted the prevention of the acid matter inhibition on the microorganism’s activity. As shown in Fig. 1, and Table 2, the volume of produced hydrogen at 55˚C was increased compared to the mesophilic condition. At 37 ˚C at pH 6.0, the volume of hydrogen was 69 ml, and 65.86 ml at FW concentration of 15, and 20 g-VS.L^− 1. With a decrease in temperature, the hydrogen production reduced. Hence, higher temperature enhances the metabolic reactions of biohydrogen production. Moreover, the fermentation process at pH 6.5, and 5.5, showed lower performance efficiency in biohydrogen production; hence, it could be deduced that the adjustment of pH at an intermittent level did increase the hydrolysis process. From Table 2, Fig. 1, and Fig. 2, it was perceived that the increase in hydrogen properties not only depends on the pH, and temperature but also related to the increase in the initial FW concentration. The effect of FW concentration during the mesophilic and thermophilic conditions was studied. The hydrogen constantly produced with an increment concentration of 1, 5, 10, 15, 20, 25, and 30 g-VS.L^− 1 during the fermentation process. An increment of 15-fold in the concentration from 1 to 15 g-VS.L^− 1 caused an increase of 5.1-fold in the hydrogen yield, 2.69-fold in hydrogen content (at pH 5.5, and 37 ˚C). At pH 6.0, and mesophilic temperature, it was increased to 7.31-fold, and 4.14-fold for hydrogen yield, and hydrogen content, respectively; while by increasing the pH to 6.5 at the mesophilic condition, this trend was decreased to 6.94-fold, 3.69-fold, respectively. A greater amount of biohydrogen properties was obtained at intermittent pH 6.0 at 37 ˚C. However, at the optimum pH 6.0, the results obtained at thermophilic conditions 55 ˚C showed a 8.28-fold in hydrogen Yield, 5.01-fold in hydrogen content, respectively. At 37 ˚C, the maximum volume of hydrogen, hydrogen content, and hydrogen yield increased when FW concentration increased from 1 g-VS.L^− 1 to 15 g-VS.L^− 1 and reached from 7.74 to 49.36 ml-H₂/g-VS.L^− 1; 14.6 to 60.53%, respectively. While this upward trend at 55 ˚C increment from 9.71 to 80.41 ml-H₂/g-VS.L^− 1; 15.27 to 76.53%, respectively. This increasing trend might have caused by the appropriate conditions (pH 6.0) and a suitable concentration of FW. At a higher amount of substrate concentration at each pH value, almost the same results with lower hydrogen yield were obtained, which might be due to the lower pH and swift acidification process [3]. Hence, the maximum hydrogen content, and hydrogen yield observed in the FW concentration of 15 g-VS.L^− 1; this might be because of a proper distribution of the biomass and substrate, which reduces the lack of nutrients. Based on this, the higher amount of substrate concentration migh inhibits the high yield of hydrogen [22]. As the concentration increased, the ratio of microorganisms to the substrate was not as suitable as the lower substrate concentration. From the studies, it was shown that the hydrogen yield for food waste could fluctuate from 3 to 196 ml-H₂/g-VS.L^− 1. The finding depends on the inoculum, substrate composition, temperature, and type of fermentation process. A comparison of the yield of biohydrogen using pretreatment technologies on FW at various temperature and pH are given in Table 3. The acid-base pretreatment methods are considered as an easy model and no need for additional requirements for the pretreatment process compared to mechanical methods such as ultrasonic pretreatment. However, ultrasonication has been widely used to increase solubilization and enhance methane production, and a few researches have addressed this method to enhance the yield of biohydrogen production [23]. In this study, heat treatment was used, which involves no need for chemicals (acid-base) or any reactants. Further, this method does not produce any undesired byproducts. The inoculum used to produce biohydrogen was mixed-culture, because the pure culture (strict anaerobes) are exceedingly sensitive to the oxygen. This feature suppresses the capacity of hydrogen production and hydrogen producing bacteria, while facultative bacteria have high activity in micro-oxygen environment. Therefore, mix culture compared to the pure culture perform much better in producing biohydrogen [13]. From Table 3, it is observed that the obtained hydrogen yield of 80 and 47 ml-H₂/g-VS.L^− 1 are similar to the yields of the some previous studies for FW.

3.2 Effect of temperature, and substrate concentration on COD removal

Variations in the soluble COD (sCOD) removal throughout the fermentation process at thermophilic and mesophilic temperature and three different pH values are shown in Fig. 3. The sCOD concentration of FW (1 to 30g-VS.L¹⁻) was increased from 15.6 g/l to 80.0 g/l. After 24h fermentation process, COD removal efficiencies at 37˚C, and 55 ˚C increased from 16–23.49% and 17.94–29.09% at pH 6.0 with an increase in FW concentration from 1 to 15 g-VS. L^− 1; however, with further increase in the FW concentration up to 30 g-VS.L^− 1 less tendency was observed in removal efficiency. This shows that the substrate concentration should be balanced to promote bacterial activity and degradation efficiency [11]. Hence, the digestion of the substrate during the fermentation process might be restricted due to failure in the mass transfer by higher concentration, which resulted in an interrupt in the substrate acclimatization, degradation process, and disparity of cellular osmosis. Thus, it was found that COD removal decreased as the FW concentration increased, demonstrating that the maximum levels of substrate degradation occurred at 10–15 g-VS.L^− 1 was 25–31% at 55 ˚C. The hydrolytic-acidogenic stage through substrate degradation can be the limiting step due to the particulate form of material [24]. Hence, a suitable organic matter conversion process and solubilization is necessary, which is influenced mainly by the concentration of the substrate.

Energy Dispersive X-Ray Analyzer (EDX) was used to provide elemental identification and atomic specification information with 5 analysis spots at different points of samples. From the EDX data presented in Table 4, the elemental identification and atomic specification information showed that the structure of FW is rich in carbon and some elements, including Potassium, Magnesium, silisium, Phosphorous, Aluminium, Ferrat, and Calcium as basic macronutrients for anaerobes growth, which made this substrate suitable for biohydrogen production [13]. Carbon, Potassium, and Magnesium were the main elements with weight concentration of 56.62%, 42.15%, and 2.95%, respectively, highlighting that food waste comprised of organic material. This approach gives a clear insight into this feedstock, which resulted in maximum hydrogen content of 76.53% through dark fermentation.

Table 2

Experimental dataset at mesophilic and thermophilic temperature
Amout of Food waste used at each batch serum bottle test		1 g-VSL^− 1	5 g-VS L^− 1	10 g-VSL^− 1	15 g-VS L^− 1	20 g-VS L^− 1	25 g-VSL^− 1	30 g-VS L^− 1
Mesophilic condition (37˚C)
pH 5.5	Initial COD, (mg/l)	15600	25900	49200	53592	64642	78900	82000
	COD removal, (%)	13.0	12.12	19.41	16.50	15.43	16.02	12.32
	F/M ratio	0.39	0.64	1.23	1.33	1.61	1.97	2.05
	Hydrogen yield, (ml-H₂/g-VS.L^− 1)	4.85	8.01	19.23	24.76	12.38	9.71	10.12
	Hydrogen content in biogas (%)	12.54	16.69	15.83	33.81	24.29	20.02	18.14
pH 6	Initial COD, (mg/l)	15600	25900	49200	53592	64642	78900	82000
	COD removal, (%)	16.66	22.58	15.56	19.76	21.87	21.26	14.32
	F/M ratio	0.39	0.64	1.23	1.33	1.61	1.97	2.05
	Hydrogen yield, (ml-H₂/g-VS.L^− 1)	7.74	18.21	34.77	49.36	47.56	38.33	36.39
	Hydrogen content in biogas (%)	14.6	33.81	50.10	60.53	53.76	55.61	75.78
pH 6.5	Initial COD, (mg/l)	15600	25900	49200	53592	64642	78900	82000
	COD removal, (%)	16.02	21.62	18.59	25.36	24.62	21.22	14.02
	F/M ratio	0.39	0.64	1.23	1.33	1.61	1.97	2.05
	Hydrogen yield, (ml-H₂/g-VS.L^− 1)	5.47	15.0	34.93	38.0	30.74	29.55	31.27
	Hydrogen content in biogas (%)	12.75	20.28	55.61	47.11	52.96	56.49	61.04
Thermophilic condition (55˚C)
pH 5.5	Initial COD, (mg/l)	15600	25900	49200	53592	64642	78900	82000
	COD removal, (%)	20.46	17.27	31.2	30.55	26.13	22.72	22.38
	F/M ratio	0.39	0.64	1.23	1.33	1.61	1.97	2.05
	Hydrogen yield, (ml-H₂/g-VS.L^− 1)	7.39	12.83	31.81	47.87	29.73	14.93	13.44
	Hydrogen content in biogas (%)	15.64	16.69	40.34	50.99	43.41	31.59	26.51
pH 6	Initial COD, (mg/l)	15600	25900	49200	53592	64642	78900	82000
	COD removal, (%)	20.83	21.62	25.20	29.09	21.91	18.75	16.46
	F/M ratio	0.39	0.64	1.23	1.33	1.61	1.97	2.05
	Hydrogen yield, (ml-H₂/g-VS.L^− 1)	9.71	28.69	44.95	80.41	71.35	38.46	33.36
	Hydrogen content in biogas (%)	15.27	33.16	72.97	76.53	74.25	76.86	78
pH 6.5	Initial COD, (mg/l)	15600	25900	49200	53592	64642	78900	82000
	COD removal, (%)	21.02	26.34	24.55	24.63	22.65	23.06	20.14
	F/M ratio	0.39	0.64	1.23	1.33	1.61	1.97	2.05
	Hydrogen yield, (ml-H₂/g-VS.L^− 1)	9.45	23.97	36.83	58.32	44.60	44.28	40.78
	Hydrogen content in biogas (%)	26	29.54	50.3	73.43	53.05	48.12	77

Table 3

Comparison of the hydrogen yield at various condition from different studies
Substrate	Inoculum	pH	Temperature, ˚C	Hydrogen yield, ml-H₂/g-VS.L^− 1	Ref.
Beer lees^a	Pretreated	6.0	36	69	[25]
Jackfruit peel	Pretreated	5.1	35	228	[26]
Corn stalk^a	Pretreated	7.0^c	36	150	[27]
Wheat straw^a	Pretreated	6.5^c	36	68	[28]
Steamed rice	Pretreated	4.5	37	305	[29]
Food waste	Pretreated	5.0–6.0	35	67	[30]
Food waste	Raw	n.c.^b	50	57	[31]
Food waste	Raw	n.c.^b	35	39	[31]
Food waste	Heat-Pretreated	6.0	55	80	This study
Food waste	Heat-Pretreated	6.0	37	49	This study
a Pretreated by acid. b n.c.: noncontrol. c Initial level. d Pretreated by Ultrasonic. e Pretreated by base.

Table 4

Elemental identification of FW structure by EDX
Element Symbol	Atomic Conc.	Weight Conc.	Oxide Symbol	Stoich. wt Conc.
C	75.90	50.62	C	50.62
K	19.42	42.15	K	42.15
Mg	2.19	2.95	Mg	2.95
Si	0.82	1.29	Si	1.29
P	0.74	1.27	P	1.27
Al	0.65	0.98	Al	0.98
Fe	0.15	0.47	Fe	0.47
Ca	0.12	0.27	Ca	0.27

3.3 Fermentation microbiology

3.3.1 Effect of heat-treatment on the structure of the microbial community

The source of anaerobic sludge taken from Jugra palm oil mill Sdn. Bhd used in this study was same as our previous study [13]. The results for microbial analysis before and after heat treatment are provided in Table 4. Anaerobic inoculum fed by FW at both mesophilic (FWS37) and thermophilic (FWS55) conditions was sampled at the highest hydrogen content in batch test study. High throughput pyrosequencing was used to analyse the samples. Different microbial indices was studied such as Shannon, and Simpson which shows the alpha diversity; whereas Chao 1, and Ace indicates the microbial richness shown in Table 4. High-quality sequence tags of 330 with high coverage 99.91%, and 522 with high coverage 99.88% was observed for the FoodWS37, and FoodW55, respectively. The lower value of Simpson index and higher value of Shannon index shows the higher diversity of microorganisms community [13]. The Shannon index showed an increasing trend from 1.87 to 3.05 over the process from mesophilic to thermophilic condition. The values of Chao1 and Ace shows an increase from 376 to 609 at 37˚C, and 393 to 596, respectively, which further confirmed the microbial productivity and diversity in the sludge. In our previous work [13] which is start-up of the UASFF reactor operated for about 70 days, alpha diversity was measured for the control (anaerobic sludge before heat-treatment), pre-treated sample (anaerobic sludge after heat-treatment) used as inoculum in this study as well. The results are provided in Table 5, which shows a change in diversity of the microbes through the process. FoodW55 showed the highest indices of Chao, ACE, Shannon, and the lowest Simpson index, demonstrating the major bacterial species diversity and richness. The results indicated that inoculum at thermophilic condition rather than mesophilic condition was more diverse in the bacterial community. This might be due to the shifting metabolic pathways of microorganisms and variation in the inoculum sludge structure, which also lead to different hydrogen production at two different temperatures of 37˚C and 55˚C.

The microbial community for FoodW37 and FoodW55 samples are shown in Fig. 4. The results obtained from the phylum level showed that microbial community in inoculum fed by FW at 37˚C was different with phyla sequences, including Firmicutes (97.89%), Bacteroidetes (0.88%), Proteobacteria (0.26%), Chloroflexi (0.23%), Caldatribacteriota (0.25%), Actinobacteria (0.071%), Desulfobacterota (0.16%), Thermotogota (0.144%) and Cloacimonadota (0.04%) (Fig. 4a). By operating at 55˚C, the sequences related to Firmicutes was 83.1%, Bacteroidetes (2.04%), Proteobacteria (10.66%), Chloroflexi (0.35%), and Actinobacteria (3.15%). It has been supposed that phylum Firmicutes included by the most HPBs could make endospores to tough extreme environmental conditions [23, 24]. Further genus level analysis specified that microbial community altered moderately at thermophilic condition. Lactobacillus, Clostridium sensu stricto 1, Trichococcus, Bacterioidetes_vadinHA17, and Clostridium_sensu_stricto_12 suited prime types with an entire abundancy of 71%, 10%, 12.5%, and 12% in the microbial community, respectively (Figure. 4b). Clostridium with producing enzymes potential is a distinct hydrogen-producing genus in consumption and degradation of the simple sugars and complex organic wastes over dark fermentation process [25–27]. The lactic acid bacteria abundancy at 37˚C was 28%, their presence have already been reported in biohydrogen system [25]. It was observed the main species altered from Clostridium intestinale and Klebsiella pneumoniae to Clostridium sulfatireducens and Bacillus sp., in dark fermentation process by increasing the temperature from 35˚C to 40˚C.

Table 5

Alpha diversity measures for acclimatized sample (FoodW37) and (FoodW55)
Observed	Chao 1	ACE	Shannon	Simpson	Ref.
767	804	809	3.17	0.85	Previous study
791	863	846	2.99	0.79	[13]
330	376	393	1.87	0.952	This study
522	609	596	3.05	0.657	This study

The effect of key operational parameters on biohydrogen production from food waste during batch dark fermentation in serum bottle was studied under mesophilic and thermophilic conditions using the anaerobic sludge taken from anaerobic pond as the inoculum. Controlling the pH, and food waste concentration at a proper level was found to be a feasible approach to enhance hydrogen production. Under thermophilic conditions, a maximum HY yield of 80.41 ml H₂/g-VS.L^− 1 could be obtained at pH 6 and FW15-20g-VS.L^− 1 after 24 h of fermentation, while under mesophilic conditions the highest HY was 49.36 ml H₂/g-VS.L^− 1 at at same pH and FW concentration. The results obtained in this study are useful for designing a batch reactor for hydrogen production from food waste feedstock. From pyrosequencing analysis, hydrogen fermentation at both temperatures was attributed to Lactobacillus and Clostridium sensu stricto 1, which confirms the potential of the microbial community for biohydrogen production.

Acknowledgements

This work was supported by the University of Malaysia (UM), Project No: RU003-2016. Authors would like to thank Mr. Mohd Faiz bin Ibrahim from University Malaya, Faculty of engineering, his help during the research.

Declaration

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.

Obulisamy PK, Eric T, Sanjeet M, Nicolas B, Jonathan WC (2018) Pretreatment of food waste for methane and hydrogen recovery: a review. Bioresour Technol 249:1025–1039
Abd Manaf L, Samah MAA, Zukki NI (2009) M.J.W.m., Municipal solid waste management in Malaysia: Practices and challenges. Waste Manag 29(11):2902–2906
Yunhui J, Xiao T (2019) C. Hydrogen production from acidogenic food waste fermentation using untreated inoculum: Effect of substrate concentrations. International Journal of Hydrogen Energy. 44(50): p. 27272–27284
Ruihong Z, Hamed MEl, Karl H, Fengyu W, Guangqing L, Chris C, Paul G (2007) Characterization of food waste as feedstock for anaerobic digestion. Bioresour Technol 98(4):929–935
Gorge FK, Karapinar I (2019) Biohydrogen production from hydrolyzed waste wheat by dark fermentation in a continuously operated packed bed reactor: The effect of hydraulic retention time. Int J Hydrog Energy 44(1):136–143
Akhbari A, Zinatizadeh A, Mohammadi P Effect of operational variables on biological hydrogen production from palm oil mill effluent by dark fermentation using response surface methodology.Desalination and Water Treatment, doi: 10.5004/dwt.2018.23169
Kothari R, Pandey AK, Kumar S, Tyagi VV, Tyagi SK (2014) Different aspects of dry anaerobic digestion for bio-energy: An overview. Renew Sustain Energy Rev 39:174–195
Noblecourt A, Gwendoline C, Christian L, Pierre F (2018) Hydrogen production by dark fermentation from pre-fermented depackaging food wastes. Bioresour Technol 247:864–870
Mu Y, Xian JZ, Han QY, Ren FZ (2006) Biological hydrogen production by anaerobic sludge at various temperatures. Int J Hydrog Energy 31(6):780–785
Mu Y, Yu H-Q, Wang G (2007) Evaluation of three methods for enriching H₂-producing cultures from anaerobic sludge. Enzym Microb Technol 40(4):947–953
Li Z, Hui w, Zongxun T, Xiaofang W, Jinob B (2008) Effects of pH value and substrate concentration on hydrogen production from the anaerobic fermentation of glucose. Int J Hydrog Energy 33(24):7413–7418
Akhbari A, Chuen OC, Zinatizadeh A, Ibrahim S, Air (2020) Water. Start-up Study on Biohydrogen from Palm Oil Mill Effluent in a Pilot‐Scale Reactor. Int J Hydrog Energy. https://doi.org/10.1002/clen.202000192
Akhbari A, Chuen OC, Ibrahim S (2020) Start-up study of biohydrogen production from palm oil mill effluent in a lab-scale up-flow anaerobic sludge blanket fixed-film reactor. Int J Hydrog Energy 12:125. https://doi.org/10.1016/j.ijhydene
Evolutionary prediction of biohydrogen production by dark fermentation, Akhbari A, Ibrahim S, Zinatizadeh A, Bonakdari H, Ebtehaj I, Sheikh Khozani Z, Gharabaghi BClean Soil Air Water, https://doi.org/10.1002/clen.201700494
Wang D, Guangming Z, Yinguang C, Xiaoming L (2015) Effect of polyhydroxyalkanoates on dark fermentative hydrogen production from waste activated sludge. Water Res 73:311–322
Hawkes F, Dinsdale R, Hawkes DL, Hussy I (2002) Sustainable fermentative hydrogen production: challenges for process optimization. Int J Hydrog Energy 27(11–12):1339–1347
Seongwon I, Mo-Kwon L, Yeo-Myeong Y, Si-Kyung C, Dong-Hoon K (2020) Effect of storage time and temperature on hydrogen fermentation of food waste. Int J Hydrog Energy 45(6):3769–3775
Rice E, APHA (American Public Health Association) (2012) : Standard method for the examination of water and wastewater.
Herbert D, Philips PJ, Strange RE, Norris JR, Ribbons DW (1971) Chemical análisis of Microbial cells. Methods in Microbiology. : p. 209–336
Fang HHP, Liu H (2002) Effect of pH on hydrogen production from glucose by a mixed culture. Bioresour Technol 82:87–93
Ruibal-Mendieta NL, Delacroix DL, Meurens M (2002) A comparative analysis of free, bound and total lipid content on spelt and winter wheat wholemeal. J Cereal Sci 35:337–342
Ramos C, Germán B, Iván M (2012) Rolando. Effect of the initial total solids concentration and initial pH on the bio-hydrogen production from cafeteria food waste. Int J Hydrog Energy 37(18):13288–13295
Rajesh Banu J, Merrylin J, Mohamed Usman TM, Yukesh Kannah R, Gunasekaran M, Sang-Hyoun K, Gopalakrishnan K (2019) Impact of pretreatment on food waste for biohydrogen production: A review. Int J Hydrog Energy 45(36):18211–18225
Hussy I et al (2003) Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora. 84:619–6266
Fan Y, Gao-Sheng Z, Xin-Yong G, Yan X, Mao-Hong F (2006) Biohydrogen-production from beer lees biomass by cow dung compost. Biomass Bioenergy 30(5):493–496
Vijayaraghavan K, Ahmad D, Ibrahim M (2006) Biohydrogen generation from jackfruit peel using anaerobic contact filter. Int J Hydrog Energy 31(5):569–579
Zhang ML, Yao-Ting F, Yan X, Chun P, Gao-Sheng Z, Jiunn-Jyi L (2007) Enhanced biohydrogen production from cornstalk wastes with acidification pretreatment by mixed anaerobic cultures. Biomass Bioenergy 31(4):250–254
Fang HH, Li C, Zhang T (2006) Acidophilic biohydrogen production from rice slurry. Int J Hydrog Energy 31(6):683–692
Kim SH, Han SK, Shin HS (2004) Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge. Int J Hydrog Energy 29(15):1607–1616
Pan J, Ruihong Z, Hamed M, Huawei S (2008) Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation. Int J Hydrog Energy 33(23):6968–6975
Elbeshbishy E, Hisham H, Bipro Ranjan D, George N (2011) Single and combined effect of various pretreatment methods for biohydrogen production from food waste. Int J Hydrog Energy 36(17):11379–11387

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Bioenergy recovery from food waste through dark fermentation direction

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Abstract

Figures

1. Introduction

2. Material And Method

2.1 Inoculum pre-treatment and acclimitization

2.1 Food waste feedstock

2.3 Preparation of batch serum bottles at different operational parameters

2.4 Microbial study

2.5 Measurement methods

3. Results And Discussions

3.1 Effect of pH, temperature, and FW concentration on hydrogen properties

3.2 Effect of temperature, and substrate concentration on COD removal

3.3 Fermentation microbiology

3.3.1 Effect of heat-treatment on the structure of the microbial community

4. Conclusion

Declarations

References

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