Mesophilic-thermophilic temperature-phased microbial aerobic biodegradation of food waste: feasibility study for rapid in-situ disposal strategy

doi:10.21203/rs.3.rs-1560382/v1

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Research Article

Mesophilic-thermophilic temperature-phased microbial aerobic biodegradation of food waste: feasibility study for rapid in-situ disposal strategy

https://doi.org/10.21203/rs.3.rs-1560382/v1

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posted 19 Apr, 2022

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Effective treatment of food waste (FW) is inherently difficult due to heterogeneous composition, high moisture content, and low heating value. Herein, a feasible FW disposal system via microbial biodegradation from vessel to pilot scale was developed. Facilitated by a novel microbial consortium of Bacillus amyloliquefaciens, Bacillus subtilis, and Aspergillus niger, the total solid removal was improved by 3.4-folds (46.6%) compared with negative control. To couple with bacterial growth and biodegradation related enzyme activity and reduce the electrical energy consumption, a mesophilic-thermophilic temperature-phased aerobic digestion processing was performed in a 40 L self-designed biodegradation tank via daily fed-batch mode, and up to 62.88% of protein, 62.22% of starch and 37.75% of waste oil was respectively decomposed, achieving a total mass reduction of 82.15%. Characterization of the final by-product confirmed its potential to be recycled into resources as organic fertilizers or biosolid fuel. The proposed microbial consortium agent and decentralized mass reduction strategy presented in this study offers practical and environmental approaches for FW management in urban areas.

Food waste

Biodegrading microbes

Mesophilic-thermophilic

Temperature-phased

Aerobic digestion

In-situ mass reduction

The ever-expanding urbanization from rapid population growth has accelerated the generation of municipal solid waste worldwide. In China, the main composition of domestic waste in urban and rural areas is food waste (FW), accounting for up to 40%. Due to its exceptionally high content in both moisture and fermentable organic matter, inappropriate disposal of FW will lead to bacteria breeding, odor and toxic gas emissions, groundwater contamination and other problems, causing serious environmental pollutions. The total amount of FW has been increased up to 100 million tons per year with an annually increase of more than 10% in recent decades (Guo et al. 2019), which puts significant pressure on the handling capacity of recycling facilities. At present in China, the dominant disposal strategy of FW is landfill or incineration, accounts for 52% and 45% of the total amounts respectively (Lu et al. 2017). Due to the high moisture content of FW, landfills are prone to produce leachate and lead to water pollution, while incineration need high extra-heat investment and produces dioxin and other harmful gases (Jiang et al. 2015). Therefore, to relieve or distribute the environmental burden, construction of more efficient disposal facilities and strategy for the processing of FW is in urgent need.

Most recently, microbial treatment strategies of FW such as anaerobic digestion, aerobic compost and biological drying have received more attentions and are confirmed to exhibit higher efficiency in both mass reduction and re-utilization (Meng et al. 2022; Lu et al. 2022; Yuan et al. 2019). Anaerobic digestion has a high resource utilization capacity based on the biogas generation, which can be used as a new energy supply. However, this technology has some limitations, such as large project area, long processing period, high equipment investment and high requirements for pretreatment and operation (Pramanik et al. 2019). Traditional composting is a kind of aerobic decomposition process that organic waste is transformed into humus through natural degradation by endogenous microorganisms. This treatment method has good versatility and is now widely applicated in the treatment of American courtyard FW (Dung et al. 2014). However, due to the low degradation efficiency, traditional composing takes a long time, generally as long as one or two months, and the degradation rate can be easily affected by the variations in environmental temperature, dissolved oxygen, FW composition, pH value and endogenous microbial structure (Zhai et al. 2015; Wang et al. 2017). Moreover, the high proportion of land occupation in traditional composting also significantly hampered its application in the growing demand of urban domestic waste mass reduction (Kumar Awasthi et al. 2019; Guidoni et al. 2018; Guo et al. 2018). Bio-drying technology removes water from FW and the remaining materials can be used as fuel. However, the longtime duration in the pretreatment of FW with high moisture (7–15 days) (Mohammed 2017) and the high extra-heat investment (Jiang, et al. 2015) are still the main barriers noticeably for its wide application.

Processing the increased FW via centralized treatment approaches as discussed above require longer time as well as inefficiency, generating more waste collection and transportation (Taşkın et al. 2020). A closer look at the amounting quantities of FW and the related emissions along the food production-consumption chain brings to light both the transportation and terminal disposal stage of FW faces difficulties in high investment (Govindan 2018). Cost of the collection and transportation in urban areas accounts for 34% of the total cost for food waste disposal (Oh et al. 2017), to diminish the transport requirements and related pollutions, in-situ disposal strategy of FW is therefore considered (Sakarika et al. 2019), which has been suggested as one way to relieve the burdens of conventional disposal facillities through mass reduction of FW (Manu et al. 2019). To reduce the considerable volume of FW in urban areas of Korea, a pilot-scale on-site system combing biological treatment and a drying stage was applied to achieve mass reduction (Jeon et al. 2020). In addition, a zero food waste system for sustainable residential buildings in urban areas reduction strategy of food waste was constructed and food waste was pretreated by a complex enzymes degrading in a reactor operating at 30–40°C, achieving a total mass reduction of 94% and producing fertilizer as the final porduct (Oh, et al. 2017). Apart from the weight reduction monitor in the decentralized systems, the entire disposal approach should be inexpensive, require low instrument investiment and easy handling for maintenance. A continuous mode is suggested to perform during digestion process, variation in regular waste composition and changes in physico-chemical parameters such as moisture content, pH value and structure of microorganism should be also monitored (Perin et al. 2020; Waqas et al. 2018).

The synergetic degradation potential of microbial consortium in FW decomposition has attracted more attention on the facilitating mass reduction of organic matters within food waste. Inoculation of a mixture of 71 strains in chicken manure and corn stover compost accelerated the degradation of organic matter (Zhang et al. 2020). Our previous study indicated that a combination of gram-positive and -negative bacteria largely improved lipid degradation owes to the cooperative activity of different microbial isolates (Ke et al. 2021) and high-temperature-resistant oil degrading microbial consortium was further applied in the biodegradation of oily food waste (Ke et al. 2021). Several artificial microbial consortia with excellent characteristics in the degradation of FOG (fats, oils and greases) have been successfully applied for the co-digestion of municipal wastewater sludge (Awasthi et al. 2014) and FW (Deaver et al. 2020), which can potentially improve the efficiency and effectiveness in organic matter decomposition.

In addition, temperature conditions are critical in the biodegradation of food waste. Food waste processed at higher temperatures and longer heating times can break down both organic and inorganic compounds more efficiently (Jin et al. 2016). However, prolonged heating and high temperature conditions mean accelerated consumption of electrical energy, and this amount cannot be ignored. Therefore, to develop a new degradation process that can reduce energy consumption while keeping the degradation efficiency of food waste unchanged is vital for its practical application. Our previous work demonstrated that the electricity cost accounts for the major proportion (above 50%) of total expenditure for equipment maintenance under high temperature (up to 55°C ) (Zhou et al. 2022). Accordingly, heat treatment of food waste may be the most suitable pretreatment method, while its energy consumption was as high as 49.3 MJ/t (Jin et al. 2015). At the same time, food waste has a high moisture content (up to 70%-90%), which is not suitable for direct microbial degradation, and often requires dehydration. Due to its poor dehydration, it takes an energy input of 137MJ/t to dry to 60% water content (Zhang et al. 2021). Therefore, it is in urgent need to develop new processes with lower energy consumption and higher efficiency.

The primary object of this study was to develop a rapid in-situ mass reduction of FW via microbial degradation strategy. Specifically, bacterial isolates with superior biodegradation towards starch, protein, fat and cellulose were screened and further compounded for both lab- and pilot-scale application. Besides, a mesophilic-thermophilic temperature-phased bio-degradation processing were adopted in fed-batch processing and key parameters was respectively monitored to evaluate the effectiveness of microbial facilitated FW decomposition, the practical application prospect was finally confirmed by the byproduct composition analysis.

Physical-chemical characteristics of food waste

The post-consumption FW was collected from the dining hall in Zhejiang University of Technology and was homogenized for physical-chemical analyses. As listed in Table 1, characteristics of FW used in this study were representative of the characteristics of FW in Eastern China, having a high-water content, high ratio of volatile solids (VS) to total solids (TS) (VS/TS) as well as high concentrations of organic acids as reported in a previous study (Zhou et al. 2021).

Table 1

Key physiochemical characteristics of food waste.
Parameters	Unit	Mean
Moisture content	%	72.78 ± 3.25
Volatile solid	%	91.63 ± 2.25
Starch	%	18.89 ± 2.59
Protein	%	20.40 ± 1.69
Lipid	%	25.36 ± 1.25
Cellulose	%	13.43 ± 1.68
pH		5.9 ± 1.2
C/N		29.07 ± 1.25
Note: All values were determined based on the dry weight in addition to moisture and pH.

Screening and identification of biodegrading microorganisms

Samples from the commercial microbial agents and FW-polluted area were collected and serially diluted for screening the microbial isolates on solid medium plates containing starch, protein, fat, and cellulose, respectively. For detail, the starch screening medium was comprised of soluble starch 2%, NaCl 0.5%, peptone 0.5%, and agar 2%; the fat screening medium was comprised of peptone 1%, NaCl 1%, CaCl₂·7H₂O 0.01%, Tween-80 1%, and agar 2%, pH 7.4–7.8; the protein screening medium was comprised of nonfat dried milk 5%, K₂HPO₄ 0.1%, MgSO₄·7H₂O 0.02% and agar 1.8%; the cellulose screening medium was comprised of K₂HPO₄ 0.05%, sodium carboxymethyl cellulose 0.188%, MgSO₄ 0.025%, gelatin 0.2%, congo red 0.02%, and agar 1.6% (w/v). The presence of “clearance zones” surrounding the colonies was taken to indicate degradation activity. Moreover, to increase the color contrast, iodine was added to the starch screening medium and neutral red dye to the fat screening medium. The values of the degradation activity represent the means ± standard deviation for three independent experiments. Strains exhibiting high enzyme activity were collected for further experiments. Subsequently, the genomic DNA of the selected strain was extracted using the FastDNA® SPIN kit. The universal primers 27F and 1492R synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) were used to amplify 16S rDNA. The obtained sequences were submitted and compared with those already available in GenBank at the National Centre for Biotechnology Information BLAST service (http://www.ncbi.nlm.nih.gov/BLAST) to identify each isolate.

Enzyme activity and biomass determination in liquid medium

Each isolate was inoculated into the LB liquid medium and cultured at 30℃ for 48 h. The fermentation broth was centrifuged at 8000 rpm/min for 10 min at 4℃, and the supernatant was collected to determine the enzyme activity (including amylase, protease, lipase and cellulose) according to the calorimetric estimation assay. Amylase activity was determined by the 3,5-dinitrosalicylic acid assay (Awasthi et al. 2018) and protease activity was measured by the foline-phenol method (Li et al. 2005).The p-nitrophenol was used for lipase activity determination (Hernandez-Garcia et al. 2017). The cellulase activity was measured by the content of glucose in its hydrolysate (Awasthi, et al. 2018). In order to study the degradation capacity of four main components within FW by the isolated strain, a simulation medium of FW was prepared according to the composition of FW as listed in Table 1. Each isolated strain was respectively inoculated under 37℃, samples were collected to determine the content of starch and protein after 24 h and the content of oil content was determined at 72 h. Bacterial exhibiting high degradation activity were selected and the cell density was assessed by dried cell weight at indicated time of fermentation.

Microbial compounding agent preparation

Both Bacillus amyloliquefaciens and Bacillus subtilis were activated by LB solid medium for 12 h under 37℃. Well-growing colonies were packed into a 2 L-shake flask containing 700 mL of LB medium and cultured at 37℃, 200 rpm for 24 h. Spores of Aspergillus niger were scraped into PDA liquid medium and fermented at 28℃ for 72 h. Wet bacteria of three selected strains were obtained by centrifugation and then was respectively immobilized with diatomaceous earth at a mixed ratio of 1:1, dried at 37℃ for 72 h. Compounding proportion of the three immobilized strains was optimized by the software of Design Expert 11and the degradation ratio of starch, protein and fat was selected as the key factor. Specific results for immobilized microbial agent preparation were described in supplementary material.

Single batch determination of food waste aerobic digestion

The self-designed FW bioreactor with a capacity of 40 L was equipped with three stainless steel baffles operated at 30 rpm/ min for 25 min, and halted for 10 min which can be controlled by frequency and speed of stirring. Air was provided at 4 L/min from the bottom of the reactors with fine-pore diffusers. External heating was realized by water bath and heat preservation, and the internal temperature as well as other operational parameters in batch mode of FW reduction were presented in Table 2. For temperature-phased biodegradation, the temperature on the run C were modified to a dual-stage of mesophilic (37°C for 12h) and thermophilic (55°C for 12 h) periods as indicated in Table 3. Changes in total mass weight and volatile solids, moisture content, microbial colony number and main organic components content were determined with three independent experiments.

Table 2

Changes in mass reduction of food waste via single batch digestion at 55℃.
Parameter	Processing	Unit	Run A^a	Run B^b	Run C^c
FW weight	Input	kg	5.00	5.00	5.00
	Output	kg	1.49	1.40	0.83
	Reduction	kg	3.51	3.60	4.17
		% of initial	70.25	72.01	82.35
Total solids	Initial	kg	1.52 ± 0.03	1.51 ± 0.08	1.49 ± 0.05
	Final	kg	1.43 ± 0.05	1.35 ± 0.05	0.79 ± 0.04
	Reduction	kg	0.09 ± 0.02	0.16 ± 0.05	0.7 ± 0.09
		% of initial	5.9 ± 0.27	10.59 ± 0.32	46.60 ± 1.50
Moisture content	Initial	kg	3.42 ± 0.30	3.41 ± 0.4	3.42 ± 0.50
	Final	kg	0.24 ± 0.01	0.39 ± 0.05	0.56 ± 0.08
	Reduction	kg	3.18 ± 0.29	3.03 ± 0.35	2.86 ± 0.40
		% of initial	92.98 ± 0.21	88.85 ± 0.40	83.60 ± 0.54
^a Single-batch digestion was maintained for 24 h at 55℃.
^b Extra supplementation of 500 g sawdust at 0 h.
^c Extra supplementation of 500 g sawdust and 50 g of the immobilized microbial agent at 0 h.

Table 3

Comparison of food waste reduction by microbial consortium under different digestion temperature.
Temperature	Total mass reduction (%)	Total solids reduction (%)	Moisture content (%)	Total microbial colony number (cfu/g)	Decomposition of starch (%)	Decomposition of protein (%)	Decomposition of oil (%)
37℃	76.95 ± 1.45	35.95 ± 1.69	61.95 ± 2.69	8.9×10⁸	71.42 ± 1.42	77.29 ± 1.58	13.35 ± 1.24
55℃	82.80 ± 1.24	46.60 ± 1.50	10.66 ± 0.56	7.6×10⁷	80.51 ± 2.11	77.42 ± 1.46	25.51 ± 1.33
37 to 55℃^a	84.65 ± 2.14	53.32 ± 2.13	40.58 ± 3.56	3.5×10⁸	81.69 ± 1.69	81.26 ± 1.25	18.69 ± 1.05
^a Temperature-phased digestion strategy was provided including a mesophilic stage (37℃for 12 h) and a thermophilic stage (55℃ for 12 h) for every single batch treatment.

Mesophilic-thermophilic temperature-phased semi-continuous digestion of food waste

Fed-batch mode as semi-continuous digestion of FW was carried out for 8 days. 1% of constructed microbial agent (50 g) was initially inoculated without any further supplementation. 5 kg of fresh FW was daily loaded into the bioreactor and a dual-stage of mesophilic (37°C for 12h) and thermophilic (55°C for 12 h) period was maintained respectively for one day and lasted for 8 days. Changes in pH, moisture content, and organic matter components of FW were measured every 12 h with three independent experiments. After the entire processing of biodegradation, the FW residue was collected from three different locations and analyzed to investigate the potential of resource recycling.

Microbial analysis

Samples were collected from three different locations inside the digestion tank for further analysis. The total bacterial colony number is determined by the plate dilution coating method. Total cell number of each microbial isolates was further estimated by absolute quantification of gene copy numbers (Bücker et al. 2018). Samples of FW were collected at different degradation times to perform DNA extraction and quantitative real-time PCR performed using the Platinum® SYBR® Green qPCR Super Mix-UDG w/ROX (Invitrogen, USA) in 96-well optical plates (MicroAmp Fast Optical). To quantify the microbial cell number from different species, specific primers that targeting unique genes that was only encoded in one species was designed for quantification: for B. amyloliquefaciens, the forward primer 5'-ATGTTTTTCCGTTCGCCTTT-3' and reverse primer 5'-TTGCGTAAATAATAGCTGCG-3' was used. For B. subtilis, the forward primer 5'- TAAAACCGTCAGAATCAGGT-3', and reverse primer 5'-TCTTCATTTGCCACATTTCC-3' were used. For A. niger, the forward primer 5'-GGAAGAAGTCAATGGCTCGT-3', and reverse primer 5'-CAGGATCAAAGGTGTGGATG-3' were used. To obtain the standard curve, a segment of the DNA target was used at different concentrations (dilutions from 10^− 1 to 10^− 8). The specificity of PCR amplification was determined by melting curve analysis and agarose gel electrophoresis.

Chemical analysis

Samples of FW were oven-dried at 105°C for 24 h to determine the moisture content, and the dried samples were ignited at 550°C in a muffle furnace for 16 h to determine the total content of volatile solid. The total mass reduction was evaluated based on the change in the total weight of FW input, and the final product was calculated by dry weight. The fresh FW diluted 6 times with deionized water was used for pH measurement. The starch content of the sample was determined by sequentially digesting the air-dried sample in diethyl ether, ethanol and boiling HCl solution (6 mol/L), and then the solution was titrated with basic copper tartrate (Yuan et al. 2018). Protein content was determined according to the Kjeldahl method and the lipid content was measured using a SOX406 fat analyzer (Zhejiang Nade Scientific Instrument Co., Ltd. China). Lactic acid was quantified using high performance liquid chromatography (HPLC, Young Lin Instrument Co., Korea) with an Aminex HPX-87H organic acids column and UV detector. Heavy metals were measured by an inductively coupled plasma-optical emission spectrometry (ICP-OES, Agilent 720, Agilent Technologies) with a VistaChip II charge coupled device detector.

Data analysis

Experiments were performed in three independent experiments and the average values and standard curves were presented. All the figures in this paper were made by the software OriginPro 2018C 64-bit.

Biodegrading microorganism isolation, characterization and compounding

A total number of 34 microbial isolates including 27 bacteria, 5 actinomycetes and 2 fungus were initially selected as the biodegrading strain according to comparatively larger area of “clearance zones” surrounding the colonies. Extracellular hydrolases secreted by microbial play an important role in biodegradation of organic matters. Enzyme activities towards protein, lipid, starch and cellulose were therefore determined and 6 dominant strains of Bacillus. amyloliquefaciens, Pseudomonas putida, Bacillus subtilis, Aspergillus niger, Streptomyces sp. and Acinetobacter sp. exhibited higher hydrolase activity as shown in Fig. 1. Among which, both the B. amyloliquefaciens (CCTCC No. M2019423) and B. subtilis (CCTCC No. M2017387) exhibited the highest activities of protease, lipase and amylase, and Aspergillus niger (CCTCC No. M2019422) shown the highest activity of cellulase, therefore, all the three isolates were respectively preserved in China Center for Type Culture Collection (CCTCC). Consistently, results of biodegradation capability assayed in the simulative medium of FW further supported the superior roles of the three above strains in digestion of protein, starch and lipids compared with the other strain, as shown in Fig. S1. Furthermore, enzyme activities and growth curve of the three selected strains was further evaluated separately at different temperatures (37, 45, 55℃). As shown in Fig. 2A, 2B and 2C, compared with approximately 1.50 g/L of biomass accumulated in all three strains under 37℃, obvious growth inhibition was observed with the increase of culture temperature when up to 55℃. On the other hand, although notable reduction of bacteria growth was observed at 55°C, the biodegradation ratio of the main organic matters was still remained as high as that under 37°C. Even more, the biodegradation of oil was largely induced under higher temperature especially in B. amyloliquefaciens and B. subtilis, indicating that the specific lipase activity was significantly enhanced under thermophilic condition as confirmed in our previous study (Ke, et al. 2021).

The synergetic effect on the biodegradation of organic matter under the cooperation between different microbial has been widely confirmed. The compounding ratio among the three isolated strain was therefore optimized. Compared to the relatively high decomposition ratio respectively for protein and starch, oil degradation seems to be the main obstacle for the processing of microbial degradation of FW (Table S1). The compounding ratio was further optimized mainly according to the removal efficiency of waste oil. As shown in Fig. 3A, a microbial consortium with optimum mixed ratio of B. amyloliquefaciens: B. subtilis: A. niger = 0.35: 0.45: 0.2 demonstrated the highest waste oil removal (45.36 ± 0.47%). The microbial agent was then prepared and inoculated into the simulated medium of FW with different inoculation (0.1%, 0.5%, 1%, 5%, 10%) and changes in organic matter components (starch, protein, oil) were measured. As shown in Fig. 3B, with the increase of microbial agent content from 0.5 to 1%, a significant improvement of organic degradation was obtained and approximately 90% of protein and starch were degraded at 24 h and 55.21% oil was decomposed at 96 h. Further increase of microbial agent inoculation ratio (more than 1%) could not facilitate FW biodegradation.

Microbial consortium facilitated food waste biodegradation

The constructed microbial consortium of B. amyloliquefacien, B. subtilis and Aspergillus niger was directly applicated in batch digestion of FW performed in a self-designed bioreactor. Physical parameters including the initial moisture content of FW, air flow rate and stirring speed were optimized and the optimum running condition were performed as indicated in Table 2. Our data demonstrated that a mass reduction ratio of 82.35% in FW was achieved with supplementation of 1% microbial agents after 24 h of biodegradation in Run C, compared with only 70.25% and 72.01% respectively observed in the negative control of Run A and Run B, which was dominantly depending on the volatilization of moisture. Moreover, 46.6 ± 1.50% of the total solids within FW was decomposed compared with 5.9 ± 0.27% and 10.59 ± 0.32% respectively achieved in the negative control. Variation of pH and total microbial cell number were also monitored as indicated in Fig. 4. At the initial stage of digestion, the pH value in run C was dropped more obviously compared with the control group, probably due to the organic acids produced by decomposition of organic matter; During 20–24 h, the pH value within FW showed an upward trend, indicating that the organic acid produced in early stage began to decomposition. The total microbial cell number within FW in three groups was respectively increased and reached its highest peak at 9 h, and the maximum value achieved to 1.5×10⁸ cfu/g after external inoculation compared with approximately 7.5×10⁶ cfu/g in the negative control. All the above data strongly confirmed the FW biodegradation was efficiently enhanced by the external inoculation of the constructed microbial consortium, and thereby significantly improved mass reduction within a short period of processing.

Mesophilic-thermophilic temperature-phased processing enhanced food waste biodegradation

Temperature is a key parameter for the bacterial growth and enzyme production in most microbials (Liu et al. 2020). As indicated in Fig. 2, all three selected strain of B. amyloliquefaciens: B. subtilis: A. niger exhibited an obvious preference to grow under mesophilic condition (37°C), while were tended to produce more hydrolases of protease, lipase, amylase and cellulase under thermophilic condition (55°C). To couple or combine the two stages for rapid mass reduction, a dual-stage of mesophilic-thermophilic digestion processing was performed. As shown in Table 3, compared with single mesophilic (37°C) or thermophilic (55°C) condition, the highest value in mass reduction (84.65 ± 2.14%) and total solids removal (53.32 ± 2.13%) was achieved in the group of dual-stage processing, and the main component including starch (81.69 ± 1.69%), protein (81.26 ± 1.25%) was most degraded correspondingly. In comparison, oil trend to be degraded under thermophilic condition. Besides, a total bacterial colony number of 3.5×10⁸ cfu/g was detected under the dual-stage processing, which was a bit lower than that achieved under 37°C with a value of 8.9×10⁸ cfu/g.

Rapid mass reduction of food waste by fed-batch microbial degradation

A fed-batch model of semi-continuous digestion strategy of FW was performed and changes in key parameters involved in aerobic degradation were monitored. As shown in Fig. 5A, during the first batch of degradation, the moisture content was gradually decreased but remained above 40% after 24 h. Previous reports confirmed a moisture content between 55% and 60% could maintain high degradation activity and well mineralization of organic matter in traditional aerobic composting process (Guo et al. 2012) (Said-Pullicino et al. 2007; Zhao et al. 2017). After loading of 5 kg fresh FW into the digestion tank, the total moisture content raised back to above 50%, providing a constant moisture content at approximately 50% during the entire stage of processing. The fluctuation of pH value was indicated in Fig. 5B. Consistent to single batch model, the pH value dropped initially and then increased slightly at 24 h after one batch. It was proved that lactic acid, acetic acid, propionic acid and butyric acid were four characteristic organic acids in food waste (Jurado et al. 2014). Changes in the total number of colonies was also monitored as shown in Fig. 5C. It reached to highest level of 1.52×10⁹ cfu/g at 20 h after 1% inoculation. With a few cycles of processing, the total number of colonies was gradually decreased but still remained above 1×10⁸ cfu/g without any further external inoculation. Moreover, to monitor the variation in total colony number of each strain in the constructed microbial consortium, primers specially directing the protein that was uniquely expressed in the individual strain was designed and the quantitative real-time PCR was performed as indicated in material and methods. Each strain showed distinct characters in growth behavior and environmental tolerance during the degradation of FW as shown in Fig. 5D. The total number of both B. subtilis and B. amyloliquefaciens colonies achieved to above 1× 10⁸ cfu/g, and trend to be consistently stable during the entire period of processing. In comparison, notable fluctuation in total number of A. niger colonies was found and it was gradually dropped below 1× 10⁷ cfu/g at late stage. All the above data supported that B. amyloliquefaciens and B. subtilis were the predominant flora within the FW along the fed-batch processing. Besides, changes in total colony number of other originated bacteria especially for the food waste endogenous microbials needs further exploration. The total degradation ratio and mass reduction of the three main components within FW were concluded in Table 4. Compared to the high decomposition ratio achieved in both starch (62.22 ± 0.41%) and protein (62.88 ± 0.51%), comparatively lower ratio of oil was removed (37.75 ± 0.33%), but still much higher than that achieved in flask-vessel experiment (18.69 ± 1.05%), and a total mass reduction of 82.15% was achieved, correspondingly.

Table 4

Changes in total starch, protein and oil content of food waste before and after the microbial digestion processing via a temperature-phased and fed-batch mode.
Main component	Input^a (kg)	Final^b (kg)	Reduction ratio (%)
Oil	3.12 ± 0.31	1.94 ± 0.21	37.75 ± 0.33
Protein	3.07 ± 0.38	1.14 ± 0.44	62.88 ± 0.51
Starch	3.2 ± 0.27	1.21 ± 0.35	62.22 ± 0.41
Total mass	40	7.14	82.15%
^a Components of the fresh food waste was selected and calculated from triplicate measurements.
^b Components of the remaining samples after 8 days of digestion was selected and calculated from triplicate measurements.

Characterization FW by-products for recycled utilization

After 192 h of digestion, the phenotype of FW was changed into dark-brown soft claylike solid fractions after the entire processing. Composition of main organic acids and poisonous metal of FW undergoing biological digestion were summarized in Table 5. Lactic acid was represented as main component for 14.76 g/L, and oxalic acid (2.03 g/L) and acetic acid (1.48 g/L) as the second and third by-product. Levers of heavy metals in the FW were all below permissible limits of fertilizer. Since the moisture content was less than 5%, the by-product can also be considered as biosolid fuel and the final calorific value is expected to meet the requirements, suggesting the feasibility of its re-cycling usage as fertilizer. However, little amounts of fumaric acid, humic acid and maleic acid were detected in the solid fraction, probably due to the short time of the entire processing.

Table 5

Compositions of the main organic acids and poisonous metal in the by-product of food waste after microbial biodegradation.
Parameters	Final solid (mg/ kg)
Oxalic acid	2026.1
Malic acid	195.89
Citrate acid	—
Succinic acid	—
Lactic acid	14760.71
Acetic acid	1102.23
Fumaric acid	1.26
Humic acid	—
Maleic acid	—
Cu	3.37
Pb	0.576
As	0.245
Cd	0.194
Cr	9.14
Ni	3.1
Hg	—
Zn	65.5
Note: “—” represents not detected.

A 24-hour processing cycle of in-situ mass reduction strategy was established in the present study and the processing efficiency was greatly improved by the external inoculation of 1% constructed microbial consortium. Via the mesophilic-thermophilic temperature-phased digestion strategy, both the bacterial growth and enzyme production were perfectly coupled in the simple bio-stirring degradation equipment, offering more cost-effective results that supports the feasibility for rapid FW mass reduction. The degradation rate of organic matter in present study is tremendously improved compared with the traditional aerobic composting that depended on the environment indigenous microbial degradation (Arrigoni et al. 2018; Manyapu et al. 2018) and was also more efficient in mass reduction compared to a Bio-SRF system as previously indicated (Yeo et al. 2019).

Several limitation factors were also confirmed to be essential for efficient microbial degradation during the semi-continuous processing. Accumulation of organic acids with an obvious drop of pH value in solid waste was found at the late stage of digestion, which were toxic to microorganisms because they were permeable to cell membranes(Song et al. 2018). Studies have confirmed the dominant role of Bacillus subtilis during the aerobic composting of food waste (Nakasaki et al. 2019; Zhou et al. 2021), and suggested that strain of Bacillus sp. exhibited more resistant to the environmental change. Anti-acidification microbial, such as lactobacillus sp. may also be existed during the later stage of processing as the accumulation of organic acids was detected according to a previous study (Song et al. 2018). Therefore, further genome sequencing of the existing microbial consortium during processing is suggested to provide more detail information of the microbial community. Additionally, parameters including moisture content as well as the changes in main components within FW during the digestion process were also verified to be critical for the growth and reproduction ability of the strains (Nakasaki, et al. 2019). In present study, via a fed-batch and temperature-phased composting strategy, the total number of microbial colonies could be recovered and maintained at 10⁸ levels with a small fraction of fluctuations in the later period, which supported the system-stability. The breakdown of waste oil in FW was difficult compared to carbohydrates and proteins due to their low bioavailability and the hydrophobicity of fatty acids they contain (Li et al. 2018; Chen et al. 2021). In addition, the accumulated oil on the surface of the material in the compost would increase the viscosity of the material, interfered with the distribution of water and heat, and adversely affected the decomposition of organic matter (Chang et al. 2008; Cerda et al. 2018). High temperature seems to be beneficial for oil degradation as indicated in our study and a previous report (Awasthi et al. 2018). Most recently, our study confirmed that extra addition of Pseudomonas sp. with Bacillus sp. could further enhance oil decomposition (Ke, et al. 2021), which is also convinced in other related studies (Zhao et al. 2018; Tzirita et al. 2019). Moreover, degradation of cellulose is still a challenging mission in present study, although A. niger exhibited highest cellulase activity among the isolated strain, its total colony number was greatly reduced at the later stage of processing. Therefore, strains with high cellulase as well as waste cooking oil degrading capability combined with stronger stress tolerance would be definitely explored in future study.

The characteristics of the total solids from FW in our study satisfied most of the standard requirement for a Bio-SRF in references despite the concentration of chorine and sulfur was not monitored in present study (Yeo, et al. 2019). And the moisture content was comparatively higher (35%) as they suggested that recycling the solids by-products as Bio-SRF is possible if the moisture content in pellets manufacture were less than 5%, a final drying stage was therefore suggested to low down the moisture content at the end of digestion.

The present study developed a novel microbial consortium with excellent biodegradation capacity towards FW and 1% inoculation strongly improved organic matter decomposition within 24 h. A mesophilic-thermophilic temperature-phased digestion processing performed in a fed-batch mode for 8 days was further performed, and the solid residue fraction of the by-products have the potential to be converted into external carbon sources as fertilizers in resource recycling. All the above evidences offer a microbial facilitated rapid mass reduction approach feasible for the cost-effective disposal of the ever-increasing amounts of FW.

Authorship contribution XK: Data curation, Formal analysis, Methodology, Writing -review & editing, JM Y: Methodology and Investigation, Data curation, Original draft and Writing. YX L: Methodology and Investigation. H Y: Data curation and Writing. JC S: Methdology and Investigation. YP X: Conceptualization, Funding acquisition, Methodology, Resources, Project administration, Supervision. YG Z: Funding acquisition, Supervision.

Acknowledgements We would like to thank to Zhejiang Guoyue Biotech Company for providing the stirred-biodegradation equipment for in-situ treatment of kitchen waste.

Data availability statement The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files.

Consent to participate All authors agree to participate.

Consent for publication All authors agree to publicate.

Conflict of Interest The authors declare that there are no conflict of interests.

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Mesophilic-thermophilic temperature-phased microbial aerobic biodegradation of food waste: feasibility study for rapid in-situ disposal strategy

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Abstract

Figures

Introduction

Materials And Methods

Physical-chemical characteristics of food waste

Screening and identification of biodegrading microorganisms

Enzyme activity and biomass determination in liquid medium

Microbial compounding agent preparation

Single batch determination of food waste aerobic digestion

Mesophilic-thermophilic temperature-phased semi-continuous digestion of food waste

Microbial analysis

Chemical analysis

Data analysis

Results

Biodegrading microorganism isolation, characterization and compounding

Microbial consortium facilitated food waste biodegradation

Mesophilic-thermophilic temperature-phased processing enhanced food waste biodegradation

Rapid mass reduction of food waste by fed-batch microbial degradation

Characterization FW by-products for recycled utilization

Discussion

Declarations

References

Competing Interests

Supplementary Files

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