BACKGROUND
In 2000, food waste contributed significantly to municipal solid waste (MSW) across various regions, accounting for 37.78% in Asia, 42.36% in Africa, 28.75% in Europe, and 42.37% in America (IPCC, 2006) Generated from households, convenience stores, restaurants, and food-processing facilities, food waste is prone to putrefaction, emitting unpleasant odors and leachate that pose health risks Historically, food waste was not separated from other MSW, which was typically sent to landfills or incinerators However, with many landfills closing and incinerators under construction, alternative food waste treatment methods are urgently needed Composting is an ideal recycling method due to the nutrient-rich nature of food waste, yet its low pH poses challenges for effective composting The acidity arises from organic acids present in the raw materials and produced during composting (Nakasaki et al., 1993; Reinhart, 2002; Sundberg and Jửnsson, 2005) Therefore, adjusting the pH to a suitable level for microbial activity is crucial, and numerous studies have explored the use of alkaline materials such as NaOH, fly ash, and lime as pH control amendments (Nakasaki et al., 1993; Wong et al., 1995, 1997; Fang et al., 1999; Wong and Fang, 2000) These amendments have proven effective in preventing pH drops during composting, thereby enhancing organic matter degradation.
To prevent pH disruption during composting, a temperature control strategy was implemented during the mesophilic phase (Smar et al., 2002) However, the addition of alkaline materials to food waste compost can negatively impact microbial activity, as elevated pH levels may inhibit these microorganisms.
A study by Sasaki et al (2003) revealed that inoculating the newly isolated yeast, Pichia kudriavzevii RB1, enhances composting by degrading organic acids and eliminating the initial lag phase Interestingly, composting commenced even without RB1, with the presence of mesophilic lactic acid bacteria, Pediococcus acidilactici TM14 and Weissella paramesenteroides TA15, which contributed to favorable environmental conditions for microbial activity These mesophilic organisms are effective only in specific temperature ranges, and the composting process is hindered during the thermophilic phase if organic acid accumulation occurs This research clarifies the role of lactic acid bacteria in accelerating composting and their interactions within the process Additionally, a newly isolated thermophile capable of raising pH levels under thermophilic conditions was introduced to enhance composting efficiency.
OBJECTIVES OF DISSERTATION
The general objective of this study was to elucidate the effects of inoculum on the composting of simulated food waste
To achieve the general objective, the following were the specific objectives:
1 To explain why acid producer Pediococcus acidilactici TM14 (PE) appearing in the early stages of composting can solve the low pH problem, to confirm that QH1 degrades organic acids during composting, and to elucidate the mechanism of composting acceleration by PE
2 To elucidate the effects of another lactic acid bacteria, Weissella paramesenteroides TA15 (WE), in accelerating the composting and to clarify to what extent of the contribution of both lactic acid bacteria (PE) and (WE) in accelerating the composting when they are co-existed
3 To obtain the thermophilic, organic acid-degrading microorganism and to investigate the ability of this thermophilic, organic acid-degrading microorganism in accelerating the simulated food waste with high concentration of carbohydrate
The detailed discussion of each specific objective is consecutively presented in the following chapters 3, 4, and 5
This chapter highlights the critical issue of food waste accumulation in municipal solid waste management and proposes composting as an effective recycling method It identifies low pH levels resulting from organic acid buildup during composting as a significant challenge, and suggests potential strategies to address this issue Additionally, the use of organic acid-degrading microorganisms is recommended as a viable solution to mitigate the low pH problem in composting processes.
Understanding the interactions among coexisting microorganisms is essential for successful composting This study outlines the goals and objectives necessary to address these demands.
PHYSICOCHEMICAL CONDITIONS THAT AFFECT COMPOSTING
Composting is a complex process where diverse microorganisms decompose mixed organic matter The rate and extent of this decomposition are significantly influenced by various environmental physicochemical factors, including temperature, pH, aeration, and moisture content.
Composting is an exothermic process that involves the bio-oxidative degradation by various microorganisms Achieving the ideal temperature for every microbial group is challenging; however, a compromise can be found if the optimal temperature aligns closely with the needs of most microorganisms Therefore, the ideal temperature for composting represents a balance that accommodates the diverse temperature preferences of the microorganisms involved in the process.
Although many studies have put the effort on defining the optimal temperature for composting, the high temperature during the composting of various materials is required for the
Research by Nakasaki et al (1985) investigated the effects of composting temperatures of 50°C, 60°C, and 70°C on the degradation of organic matter The findings indicated that 60°C was optimal for maintaining a high CO2 evolution rate and achieving the greatest carbon conversion While 70°C exhibited a higher specific CO2 evolution rate, the peak CO2 evolution occurred at 60°C Additionally, Lin (2008) demonstrated that total coliforms, used as indicators of pathogens, were effectively eliminated when temperatures reached 65°C.
The optimal pH range for composting is between 5.5 and 8.0, where neutral pH levels support bacterial growth, while slightly acidic conditions favor fungal development Notably, fungi exhibit greater tolerance to varying pH levels within this range compared to bacteria.
The impact of pH on microbial activity and composting varies with the type of material used According to Nakasaki et al (1993), the ideal pH range for maximizing microbial growth and protein degradation activity is around 7.
The study examined the impact of pH on microbial activity during composting, revealing that controlled pH conditions, particularly with lime addition, significantly enhanced the degradation rate of organic matter compared to uncontrolled pH While pH is not a critical factor in composting animal manures, as most materials fall within the optimal range of 5.5 to 8.0, it plays a vital role in managing nitrogen loss due to ammonia volatilization, which increases at higher pH levels To mitigate excessively high pH during composting, sulfur amendments are recommended In contrast, pH levels pose a more serious challenge in composting food waste, necessitating careful management to ensure effective decomposition.
Food waste typically has a low pH value ranging from 4.7 to 6.1, which decreases further during composting due to organic acid accumulation (Sundberg et al., 2011) To adjust the pH levels of raw compost materials during the acid-forming phase, the addition of alkaline substances like lime (Ca(OH)2) is recommended Additionally, introducing microorganisms that degrade organic acids can further accelerate the composting process.
Biodegradation of organic matter can occur with or without oxygen, but composting is primarily an aerobic process This is because aerobic composting is odorless, in contrast to the foul odors produced during anaerobic digestion, primarily due to short-chain fatty acids While some odor can still occur in aerobic composting, proper management helps degrade organic acids quickly Additionally, aerobic composting reaches high temperatures that eliminate most plant and animal pathogens, parasites, and weed seeds, addressing public health and crop production concerns Most importantly, aerobic composting facilitates faster degradation of organic matter, as obligate aerobes thrive in oxygen-rich environments, allowing for more efficient breakdown of refractory compounds compared to anaerobic methods.
Researchers have focused on determining the necessary amount of air to maintain aerobic conditions in composting Theoretically, the oxygen needed is based on the carbon content that must be oxidized However, accurately calculating the oxygen requirement solely from the carbon content of waste is challenging, as a portion of the carbon is transformed into bacterial cellular matter, complicating precise estimations.
9 fraction is so refractory that its carbon remains inaccessible to the microbes The numerical value proposed of 510-620 m 3 /ton of volatile matter per day is useful (Diaz et al., 2007)
The positive-pressure aeration system, which introduces air from the bottom of the reactor to enhance composting efficiency, is commonly used to reduce composting time However, this method can lead to the emission of odorous gases, posing significant environmental concerns In contrast, Lin (2008) proposed a negative-pressure aeration system that forces air downward into the reactor while extracting outlet air from the bottom through perforated pipes connected to a blower This innovative approach has been shown to effectively decrease ammonia (NH3) emissions during the composting process.
Water is essential for all microbial activity and should be present in appropriate amount throughout the composting cycle The optimal moisture content is in the range of 40% to 60%
Microbial decomposition of organic matter primarily occurs in thin liquid films on organic particles, with optimal moisture levels being crucial for microbial activity When moisture drops below 30%, microbial activity declines, leading to dormancy Conversely, excessive moisture over 65% can result in oxygen depletion and nutrient leaching, slowing decomposition and causing odor issues Research by Nakasaki et al (1994) highlights that controlled moisture levels significantly impact the composting rate of grass clippings, showing that the CO2 evolution rate and carbon conversion peak at a moisture level of 50%.
PHYSICOCHEMICAL PARAMETERS IN FOOD WASTE COMPOSTING
Composting food waste presents ongoing challenges for researchers due to insufficient understanding of the key factors that influence its efficiency This section highlights essential knowledge regarding the parameters that determine successful composting, drawing insights from a comprehensive review of various food waste composting processes.
A summary of the 10 conditions and controlled parameters from previous research is presented, as noted by Lee et al (2013) Table 2.1 outlines the characteristics of food waste in relation to the initial conditions of the composting processes.
Table 2.2 presents the initial and peak temperatures recorded during previous food waste composting experiments, highlighting that temperature variations are influenced by the specific characteristics of the microbial population and the decomposition rate This relationship between temperature changes and decomposition rates allows for better regulation of the composting process Research by Yu and Huang (2009) demonstrated that when temperatures exceed 50°C, the degradation of organic matter significantly improves Although many studies have explored the use of rapid external heating methods, they often failed to yield satisfactory outcomes, likely due to the detrimental effects of rapid temperature increases on indigenous microorganisms In contrast, a typical self-heating food waste composting process shows a correlation between rising temperatures and increased microbial activity.
Table 2.1 The feedstock, resources, initial conditions and bulking agents used in the previous experiments a: Municipal solid waste b: Leftovers of raw fruit and vegetables c: Pruning wastes
Feedstock Resource C/N ratio Moisture content (%)
Food waste Collected from eight families N/A 67-90 5-5.4 47-50 Rice straw and grass
Food waste Dining halls, hotels, cafeterias 17-21 61-64 4.2-5.0 25-35 Wood shaving and mulch Food waste School lunch 26-32 61.3-62.2 5.4-5.6 62.8-66.3 Rice hulls and tree cuttings
Food waste Synthetic 12-56.6 55 4 25-30 Rice husks
Food waste Synthetic 21.5-39.2 71.4-79.8 N/A 25-30 Sawdust, rice bran
MSW Bazaar 19-25 N/A 4.9-5.2 N/A Sawdust and peanut shell
Food waste Restaurant 8.85 70-80 3.8-6.5 25-45 Rice husk
MSW Composting plant 26 60 7.13 N/A Dry grass
Table 2.2 Characteristic of temperature variation and composting scale of some food waste composting experiments
The pH levels of food waste composting was varied based on the accumulation of short- chain organic acids and the generation of ammonium compounds, within within a range of 4.9–
8.3 in food waste composting Beck-Friis et al (2001) carried out a series of food waste composting with different temperature regimes, and found that the pH value in the self-heated composting had a larger range of fluctuation than that in the quick external-heating composting
Smørs et al (2002) found that the composting of organic household waste achieves optimal degradation rates when the pH is maintained between 6 and 8 During the initial phase of composting food waste, the accumulation of organic acids can hinder the process To address this, the addition of alkaline additives such as fly ash and lime is essential for maintaining the desired pH level, thereby reducing composting time and improving efficiency Numerous studies have explored the use of alkaline materials like NaOH, fly ash, and lime as pH control amendments to enhance composting outcomes (Nakasaki et al., 1993; Wong et al.).
Research has shown that the addition of pH control amendments during composting effectively prevents pH drops, which in turn enhances the degradation of organic matter (Nakasaki et al., 1993) This strategy has been supported by various studies conducted between 1995 and 2000 (Fang et al., 1999; Wong and Fang, 2000).
Smar et al (2002) proposed a method to stabilize pH levels during food waste composting by maintaining initial temperatures at the mesophilic range until a specific pH is achieved However, the addition of alkaline materials to food waste composting may negatively impact microbial activity due to the inhibitory effects of elevated pH levels, as noted by Sasaki et al.
A previous study demonstrated that the newly isolated yeast, Pichia kudriavzevii RB1, effectively degrades organic acids in raw compost material and accelerates the composting process by eliminating the initial lag phase Additionally, RB1 addresses low pH issues during the early mesophilic stage In cases where waste has a high carbohydrate concentration, pH levels tend to drop during composting Research by Nakaski et al (1996) revealed that Bacillus Licheniformis HA1 can prevent this pH decrease and significantly enhance the rate of organic matter decomposition.
Most food waste composting experiments utilized an airflow rate of 0.1–1 L min -1 kg -1 of organic matter Detailed aeration conditions and air supply systems are outlined in Table 2.3 Additionally, both positive and negative pressure aeration methods were occasionally employed in these composting processes.
Table 2.3 Different aeration systems and corresponding final results in the previous food waste composting experiments
Kumar et al (2010) compared the composting with various moisture contents (45–75%) and found that the optimum moisture was 60% for green waste and food waste co-composting
2.2.5 Other consideration for food waste composting
When composting food waste, the salt content (NaCl) must be considered, as it can significantly impact microbial activity Although limited research exists on the effects of salt during food waste composting, studies by Lee et al (2002) and Park (2011) provide valuable insights into salt concentrations in composting materials, particularly from Korean food waste, which is often high in salt due to ingredients like Kimchi, soy sauce, and soybean paste In their findings, Lee et al reported a salt concentration of 12.2 g/kg, while Park noted levels between 0.38% and 0.46% Notably, both studies observed a slight increase in salt concentration throughout the composting process.
Table 2.4 The physicochemical properties of the initial feedstock (IF), intermediate compost (IC) and final compost (FC) during the composting process
PH MC NaCl Total-C Total-N
C/N EC (-) (%, wb) (%, db) (%,db) (%, db) (-) (dS/m)
Table 2.5 Changes in the contents of sodium and organic acids of food waste compost and germination index during the composting process
The NaCl concentration in food waste is crucial due to its impact on osmotic pressure, which influences microbial growth Osmotic pressure refers to the movement of water across a semipermeable membrane, shifting from areas of higher solute concentration to lower concentration Understanding this relationship is essential for managing food waste effectively.
Maintaining osmotic pressure within appropriate ranges is crucial for cellular health, as excessively high osmotic pressure due to elevated solute concentrations, such as sodium chloride (NaCl), can lead to the depletion of essential water from cells For optimal bacterial growth, a NaCl concentration of 0.5-2% is ideal, while concentrations exceeding 3% can negatively impact many microorganisms (Balkose et al., 2014).
While the concentration of NaCl may concern composting engineers, studies by Lee et al (2002) and Park (2011) indicate that the salt levels are within an acceptable range, posing no harm to microorganisms However, if salt concentrations become excessively high, it is essential to implement solutions Simple methods to address high salt levels include washing food waste with water to reduce salt content or blending it with low-salt organic materials Furthermore, research by Oh et al (2008) explored methods to enhance composting processes.
Total Na (g kg -1 ) 12.2 13.2 12.5 13 12.9 14.5 14.3 14.9 15.7 16.4 16.6 Water sol Na (g kg -1 ) 10.8 10.6 10.5 11.2 11 12.1 12.6 13 14.6 15.2 15.5 Organic acids (mg kg -1 )
Anaerobic digestion of food waste with high salt concentrations can enhance methane production, as explored in the study by Oh et al (2008) The research highlights the role of osmoprotectants, which lower the water potential inside cells and help retain intracellular water These osmoprotectants comprise a diverse range of compounds, including amino acids like proline and quaternary ammonium compounds such as glycine betaine, choline, carnitine, and trehalose.
MICROBIOLOGY OF COMPOSTING PROCESS
2.3.1 Microbial succession with increasing in temperature
Composting is a controlled, aerobic biodegradative process that differs from natural rotting, as it involves self-heating and a mixed microbial community that breaks down complex organic matter This microbial activity generates heat, making composting an efficient method for organic waste degradation (Ryckeboer et al., 2003).
The temperature is changed based on the activity of microorganisms If the composting operation is optimal, the composting can be divided into four phases: (i) an initial phase (first
The decomposition process consists of four key phases: (i) an initial mesophilic phase (10-42°C) lasting a few hours, (ii) a thermophilic phase (45-70°C) whose duration varies based on the type of organic matter, (iii) a second mesophilic phase characterized by a temperature decline and the recolonization of substrates by different mesophiles, and (iv) a maturation and stabilization phase that can extend for several weeks or months.
Different microbial consortia dominate each composting phase, adapting to specific environments, as illustrated in Figure 2.2, which shows the temperature ranges of grouped microorganisms Primary degraders establish a suitable physico-chemical environment for secondary organisms that cannot process the initial material, with crossfeeding observed, where metabolites from one group benefit another Initially, a rapid temperature increase signifies a shift from mesophilic to thermophilic microorganisms; however, a temperature plateau often occurs between 42°C and 45°C, inhibiting mesophilic microflora while thermophiles are still developing Once sufficient thermophiles are present, the temperature rises again, peaking above 60°C, which can inhibit organisms due to enzyme inactivation and reduced oxygen supply Effective management, such as regular aeration, can sustain the thermophilic stage until heat production declines as easily degradable substrates are exhausted High temperatures facilitate the breakdown of recalcitrant organics like lignocellulose and eliminate pathogens In the subsequent cooling phase, nutrient limitations lead to reduced microbial activity and heat output, while the maturation phase sees a further decline in substrate quantity and the formation of non-degradable compounds like lignin-humus complexes (Ryckeboer et al., 2003).
The duration of the composting process varies based on several factors, including the breakdown of organic matter, the efficiency of the composting method used, the availability of oxygen, and the moisture levels present in the compost.
Although composting is an ancient art, usually operate itself, better understanding about the microbial community will help to improve the process as well as the quality of end-products
Starting phase-first mesophilic phase
The limited knowledge of indigenous microorganisms is due to the heterogeneous nature of the original material and is documented in only a few studies In the early stages of composting, ambient temperatures and low pH levels create an acidic environment that encourages the growth of fungi and yeasts.
An increase in pH levels favors bacteria, enabling them to out-compete fungi within a short timeframe Bacteria represent the most diverse group in composting, utilizing various enzymes to break down a wide array of organic materials Their average generation time is significantly shorter than that of fungi, providing a competitive edge during rapid changes in substrate availability and environmental conditions such as temperature, moisture, and aeration Consequently, bacterial populations, including actinomycetes, typically exceed those of fungi, making them the primary agents of initial decomposition and heat generation in composting processes.
Actinomycetes develop more slowly than most bacteria and fungi and are rather ineffective competitors when nutrient levels are high
The thermophilic phase of organic matter degradation exhibits significantly higher activity compared to the initial mesophilic phase, particularly when temperatures rise between 40°C and 60°C During this temperature range, mesophilic microorganisms become inactivated, leading to a decline in their populations, while thermophilic and thermotolerant microorganisms flourish Despite this increase, there is a notable reduction in overall bacterial species diversity during the thermophilic phase Endospore-forming bacteria, such as Bacillus sp., demonstrate high activity at temperatures between 50°C and 60°C, whereas non-spore-forming bacteria like Hydrogenobacter spp and thermos spp thrive at even higher temperatures, exceeding 70°C and reaching up to 82°C (Ryckeboer et al., 2003).
Thermal inactivation of pathogens is essential for ensuring the safety of products, addressing both phytohygiene and human health concerns Typically, higher temperatures lead to greater pathogen destruction However, research indicates that Salmonella spp is suppressed more rapidly during composting at 55°C compared to 70°C.
High-temperature composting during the thermophilic phase differs significantly from normal putrefaction processes While it is commonly believed that elevated temperatures enhance the degradation of organic matter, several factors must be considered High temperatures can reduce gas saturation constants, leading to decreased oxygen availability Furthermore, research indicates that the degradation of organic matter may be hindered at high temperatures due to a reduction in functional diversity Additionally, certain materials require a cooling and maturation phase in the composting process, as high temperatures can inhibit the regrowth of mesophilic microorganisms during the subsequent mesophilic phase (Ryckeboer et al., 2003).
Cooling or second mesophilic phase
As the organic matter becomes exhausted, the activity of thermophilic organisms declines, leading to a decrease in temperature This allows mesophilic fungi to recolonize from protected microniches or external sources While the number of bacteria may decrease by about one order of magnitude, the diversity of microorganisms increases, introducing essential species for composting that were absent during the thermophilic phase (Ryckeboer et al., 2003).
The maturation phase is essential for composting waste with high concentrations of recalcitrant compounds like lignin and lignocellulose, which can be found in materials such as paper, containing about 20% lignin This phase is particularly important for waste types like tree bark and agricultural residues Fungi play a crucial role in breaking down these recalcitrant compounds, making the compost from this phase valuable for isolating cellulose, hemicellulose, and lignin Degrading these compounds facilitates the conversion of waste into useful resources.
The degradation of recalcitrant organic matter into humus compounds during composting can be hindered by low water concentration, which limits enzyme accessibility to the substrate (Ryckeboer et al., 2003).
21 Fig 2 1 Typical process parameters and microbial abundance during composting (Source: Ryckeboer et al., 2003)
Fig 2 2 Temperature range of psychrotolerant, mesophile and thermophile organisms and their generation time
Gray et al (1971) highlighted the controversial benefits of inocula, suggesting that their use is only essential when indigenous microflora cannot proliferate due to environmental constraints However, the validation of this concept was not established at that time.
The effectiveness of inoculation remains a contentious topic, with various studies yielding mixed results Yadav et al (1982) found that fungal inoculation significantly enhanced the decomposition of wheat straw and individual farm waste, but had no impact on mixed materials comprising farm waste, cattle dung, and soil Additionally, Nakasaki et al (1985) investigated the effects of seeding on thermophilic composting of sewage sludge, focusing on CO2 evolution rates and microbial populations, highlighting the complexities in the role of inoculation in composting processes.
23 thermophilic actinomycetes clearly reflected the effect of seeding, no clear difference was observed in the overall rate of composting or quality of the composted product
The positive impact of inoculation was not evident in the two studies mentioned, likely due to the absence of environmental inhibitions in the target waste that could be addressed by inoculation Tang et al (2011) conducted treatments that highlight this aspect.
INTRODUCTION
The urgent need to address food waste arises from its tendency to decompose rapidly, emitting harmful odors that negatively impact environmental quality Composting effectively transforms putrescible organic materials into a stabilized soil conditioner, mitigating phytotoxicity risks (Haug, 1993) Consequently, the use of compost derived from food waste in agriculture has gained significant attention (Lee et al., 2004; Li et al., 2013) However, food waste composting often faces challenges due to low pH levels, which can inhibit microbial activity and delay the composting process (Cheung et al., 2010) Low pH conditions affect the charge interactions of amino acids and alter protein conformations, ultimately hindering the enzymatic functions of microorganisms.
28 damages the microorganisms themselves, causing them to die (Haug, 1993; Srivastava and Srivastava, 2003)
Composting can occur naturally through indigenous microorganisms in raw materials, but its efficiency may be hindered by environmental factors or inadequate microbial populations Research indicates that microbial inoculants can significantly enhance the composting process, as shown in studies where the addition of Thermoactinomyces vulgaris A31 improved compost maturity, and the use of amylolytic and cellulolytic thermophilic bacteria from Geobacillus species increased total bacterial counts and biodegradability.
The use of an inoculant in composting simulated food waste with low pH has shown benefits, as noted by Nakasaki et al (2013) The newly isolated yeast, Pichia kudriavzevii RB1, is capable of degrading organic acids, which raises the pH of composting materials above neutral levels, promoting bacterial proliferation and enhancing organic matter degradation This study aimed to demonstrate the effectiveness of RB1 in accelerating the composting process Interestingly, composting commenced even in the absence of RB1 inoculation, highlighting the resilience of the composting process Additionally, in the absence of RB1, two characteristic lactic acid bacteria were identified.
During the temperature-raising phase of composting, Pediococcus acidilactici, Weissella paramesenteroides, and a mesophilic fungus were identified, indicating their significant role in modifying environmental conditions These lactic acid bacteria facilitate the activity of essential microorganisms in the composting process This study aimed to clarify the mechanisms by which the inoculation of these lactic acid bacteria accelerates aerobic composting.
Fig 3 1 Changes in temperature (a), organic acids (b) with time during the composting period of Run 3-A (With RB1) and Run 3-B (Without RB1) (Nakasaki et al., 2013).
16 acetic acid propionic acid butyric acid lactic acid
0 2 4 6 8 10 acetic acid propionic acid butyric acid lactic acid
Lactic acid bacteria play a crucial role in anaerobic composting by adhering to food waste and dominating microbial communities during storage Research by Hemmi et al (2004) and Asano et al (2010) highlighted their effectiveness in maintaining low pH and high temperatures in anaerobic conditions, which aids in stabilizing compost by suppressing unwanted microorganisms and preventing garbage putrefaction However, there is a lack of studies investigating the mechanisms through which lactic acid bacteria may accelerate aerobic composting.
MATERIALS AND METHODS
Commercial rabbit food, known as Rabbit Food Timothy TM from Easter Co Ltd in Tatsuno, Japan, was utilized as a model for food waste (Nakasaki et al., 2013) Elemental analysis revealed that the carbon and nitrogen content of the dry weight of the rabbit food was 44.0% and 2.43%, respectively, resulting in a C/N ratio of 18.1 To measure the pH of the rabbit food, a suspension was created by homogenizing the food in water at a 1:9 (w/w) ratio using an ACE homogenizer from Nihonseiki Kaisha Ltd in Tokyo, Japan.
The pH of the compost raw material was determined to be 5.7 using a pH meter (Model D-51; Horiba Co., Ltd., Tokyo, Japan) To create the compost, rabbit food was combined with sawdust and commercial seeding material (Alles G TM; Matsumoto Laboratory of Microorganism Co Ltd., Matsumoto, Japan) in a ratio of 10:9:1 The cell densities of mesophilic fungi, mesophilic bacteria, and thermophilic bacteria in the seeding material were measured at 4.12 × 10^4, 1.56 × 10^6, and 7.18 × 10^5 colony-forming units (CFU) per gram of dry solid material, respectively, while the seeding material's pH was recorded at 7.8 Food waste is typically acidic due to organic acids produced during storage, including lactic acid, acetic acid, propionic acid, and butyric acid, as identified by Sundberg et al (2011) These organic acids were incorporated into the compost raw material at concentrations of 12.45, 2.90, 3.02, and 2.43 mg, respectively.
After the addition of organic acids, the pH of the raw compost mixture reached approximately 5.2, while the initial moisture content of the compost material was adjusted to 60% (w/w) (Nakasaki et al., 2013).
Fig 3 2 Changes in temperature (a) and growth of mesophilic fungi (MF) and mesophilic bacteria (MB) (b) with time during the composting of Run 3-B (Without RB1) (Nakasaki et al.,
Two characteristic lactic acid bacteria closely related to Pediococcus acidilactici and
Weissella paramesenteroides were isolated from the compost produced without inoculation of
In a study by Nakasaki et al (2013), two strains were isolated from a deMan-Rogosa-Sharpe (MRS) agar plate, identified as P acidilactici TM14 and W paramesenteroides TA15, and preserved at -80°C for future use These strains were subsequently referred to as PE and WE, with PE being selected as the sole inoculant to investigate the role of lactic acid bacteria in accelerating the composting process.
Fig 3 3 The colony appearance of Pediococcus acidilactici TM14 (a) and Weissella paramesenteroides TA15 (b)
Two composting experiments were conducted: Run 3-A served as the control without inoculation, while Run 3-B was inoculated with PE at a cell density of 10^8 CFU g^-1 DS The inoculum preparation involved preculturing PE in MRS medium at 37°C for two days with shaking at 150 rpm, followed by washing the PE in distilled water and suspending it in distilled water.
The composting experiments utilized a bench-scale reactor measuring 300 mm in diameter and 400 mm in depth Constructed from thermoresistant polyvinyl chloride resin and insulated with polystyrene Styrofoam, the reactor was designed to minimize heat loss during the composting process.
The composting was started by introducing approximately 3,000 g of compost raw material into the reactor Air was supplied from the bottom of the reactor at a rate of 45 l h -1 Temperature
The composting process reached the optimal temperature of 60°C due to self-heating from microbial activity, as noted by Nakasaki et al (1985) Once this temperature was achieved, it was maintained by adjusting the air feed rate However, in the later stages, heat generation decreased as easily degradable organic matter was depleted, necessitating the use of an electrical ribbon heater to sustain the temperature The exhaust gas was treated with an H2SO4 solution to remove ammonia before measuring the CO2 percentage with an infrared analyzer, as detailed by Ohtaki et al (1998) A schematic diagram of the experimental system is illustrated in Fig 3.4.
The organic matter degradation was expressed as the CO2 evolution rate and the conversion of carbon, calculations for which were detailed in a previous paper (Nakasaki et al., 1998) The
The CO2 evolution rate reflects the degradation of organic matter, while the conversion of carbon indicates the mineralization degree of that matter In the composting process, materials were turned and sampled daily, with moisture content consistently maintained at 60% by adding distilled water as needed The composting duration lasted for 15 days, and previous studies have shown that this composting system yields highly reproducible results (Ohtaki et al.).
The compost samples were analyzed for moisture content, pH, and organic acid concentration Moisture content was assessed by measuring weight loss after drying at 105°C for 24 hours in a dry oven The pH levels of the compost samples were determined as outlined in section 2.1 Additionally, organic acid concentration was quantified using high-pressure liquid chromatography.
(HPLC) system equipped with an UV-2075 detector (JASCO Corp., Tokyo, Japan) and a
SUGAR SH 1011 column (Shodex, Tokyo, Japan)
34 Fig 3 4 The schematic diagram of the experimental system (Bench-scale reactor)
For HPLC measurement of the liquid sample, the compost suspension was filtered using a 0.2-μm cellulose acetate membrane filter The HPLC conditions followed the manufacturer's guidelines, utilizing a mobile phase of 5 mM sulfuric acid at a flow rate of 1 ml/min, with the temperature maintained at 50°C.
The dilution plating technique was utilized to assess the cell density of three microorganism types: mesophilic fungi, mesophilic bacteria (including lactic acid bacteria), and thermophilic bacteria Mesophilic and thermophilic bacteria grew on trypticase soy agar medium (pH 7.3), which contained 17 g/L trypticase peptone, 3 g/L phytone peptone, 5 g/L NaCl, 2.5 g/L K2HPO4, 2.5 g/L glucose, and 20 g/L agar Meanwhile, mesophilic fungi were cultivated on potato dextrose agar from Eiken Chemical Co., Ltd Additionally, the trypticase soy agar medium was enhanced with 100 μL of amphotericin B solution to support microbial growth.
To inhibit fungal growth, 25 mg of amphotericin B was combined with 1 ml of dimethyl sulfoxide, while 1 ml of chloramphenicol solution (100 mg chloramphenicol in 1 ml ethanol) was used to suppress bacterial growth on potato dextrose agar The incubation temperatures were set at 30°C for mesophiles and 60°C for thermophiles, with a 3-day incubation period, except for mesophilic bacteria, which required 5 days to form clear colonies Cell density measurements for each sample were conducted in triplicate, and the average values along with a 95% confidence interval were calculated for accuracy.
3.2.5 The influence of lactic and acetic acids on the growth of fungus with the ability to degrade organic acids
The study examined the effects of lactic acid and acetic acid on the growth of the fungus QH1, which has the capability to degrade organic acids isolated from compost The fungus QH1 was isolated from a compost sample collected on the seventh day of Run B and preserved in a 20% glycerol solution at -80°C To create the inoculum, QH1 was precultured twice in potato dextrose broth from the glycerol stock.
The QH1 sample was incubated at 30°C for three days while being shaken at 150 rpm Afterward, it was washed with distilled water and homogenized using an Ultra-Turrax homogenizer from IKA Works, Inc The solid substrate utilized was identical to the compost raw material.
In this study, two experimental runs, Run 3-C and Run 3-D, were performed using sterilized compost raw material, which was autoclaved at 121°C for 90 minutes Run 3-C involved adjusting the initial concentration of acetic acid to 18.3 mg g -1 DS, while Run 3-D increased the initial concentration of lactic acid to 21.7 mg g -1 DS The concentrations of other organic acids in both runs were maintained at levels consistent with those found in the compost raw material.
Table 3.1 The initial concentrations of organic acids in Run 3-C and Run 3-D
Composting procedure
Rabbit Food Timothy TM, produced by Easter Co Ltd in Tatsuno, Japan, serves as a model for studying food waste (Nakasaki et al., 2013) Elemental analysis revealed that the dry weight of the rabbit food contains 44.0% carbon and 2.43% nitrogen, yielding a carbon-to-nitrogen (C/N) ratio of 18.1 To enhance the composting process, the rabbit food was combined with sawdust as a bulking agent and supplemented with commercial seeding material from Alles G TM, developed by the Matsumoto Laboratory of Microorganisms.
In Matsumoto, Japan, a compost raw material was created using a ratio of 10:9:1, incorporating commercial seeding material with cell densities of mesophilic fungi, mesophilic bacteria, and thermophilic bacteria at 4.12 × 10^4, 1.56 × 10^6, and 7.18 × 10^5 CFU g^-1 dry solid, respectively Food waste typically exhibits acidity due to organic acids produced during storage, including lactic, acetic, propionic, and butyric acids, as identified by Sundberg et al (2011) These acids were added to the raw material, achieving concentrations of 12.45, 2.90, 3.02, and 2.43 mg g^-1 dry solid, leading to a final pH of approximately 5.2 in the mixture (Nakasaki et al., 2013).
Lactic acid bacteria, PE, and WE were preserved at -80°C until needed as inoculum Prior to inoculation, the bacteria were pre-cultured in deMan-Rogosa-Sharpe (MRS) medium at 37°C for 48 hours under shaking conditions at 150 rpm The culture was then centrifuged, washed, and re-suspended in a physiological saline solution.
Compost raw mixtures were inoculated with P and W, individually and in varying combinations of cell density, as shown in Table 4 1
Table 4 1 The initial cell density of P, W and P/W ratio for all experimental runs
Composting was carried out in a mini-reactor comprised of a Pyrex glass cylinder (diameter:
The composting reactors, measuring 45 mm in diameter and 100 mm in depth, were equipped with silicone rubber stoppers and glass pipes for effective aeration Each reactor held around 15 grams of a compost raw mixture, with air introduced from the bottom at a flow rate of 5.5 mL/min throughout the composting process Prior to entering the reactor, the air was filtered through a flask to ensure optimal conditions for composting.
NaOH solution to eliminate CO2 gas and thereafter through a flask with water to saturate the air with moisture The exhaust gas was collected in a 10-L polyvinyl fluoride plastic bag (Tedlar
Bag TM ; OMI Odoair Service Co Ltd., Omihachiman, Japan) for 24 h The plastic bag was
Run No. log.cell density (CFU/g -1 dry solid)
*3: Temperature increased to simulate autothermal composting
The CO2 percentage and total gas volume were measured daily to assess CO2 emissions, while the degradation of organic matter was quantified through the CO2 evolution rate, reflecting the degree of mineralization of organic matter (Tran et al., 2015).
The mini-reactor was incubated at a controlled mesophilic temperature of 30°C using a Model IS 800 incubator from Yamato Scientific Co., Ltd., Tokyo, Japan, to simulate the initial stages of composting This temperature was consistently maintained throughout composting Runs 4-A to 4-F, and during Run 4-G, the temperature remained at 30°C for the initial phase.
To simulate autothermal composting, the temperature was raised to 60°C over a period of 5 days at a rate of 2.5°C per hour, and then maintained at this temperature for the subsequent days The operation of the mini-reactor has been detailed in prior research (Kuok et al., 2012).
Physicochemical analysis was conducted to assess moisture content, pH, and organic acid concentration, along with estimating the cell density of mesophilic fungi (MF) and mesophilic bacteria (MB) Moisture content was measured as a percentage of weight loss after drying samples at 105°C for 24 hours using a dry oven The pH value was obtained with a pH meter after preparing a compost suspension by homogenizing the sample in water at a 1:9 ratio The cell density of mesophilic fungi and bacteria, including lactic acid bacteria, was determined through dilution plating on trypticase soy agar medium, which was supplemented with amphotericin B to support bacterial growth.
To inhibit bacterial growth, 1 mL of chloramphenicol solution (100 mg in 1 mL ethanol) was added to potato dextrose agar medium (Eiken Chemical Co., Ltd., Tokyo, Japan), allowing for the observation of mesophilic fungi Both fungi and bacteria were incubated at a temperature of 30°C for a specified duration.
5 days Cell density measurements for each sample were performed in triplicate and the average
57 of 3 measurements at 95% confidence interval for each average value was calculated, as previously described (Tran et al., 2015).
Quantification of individual lactic acid bacterium by real-time PCR
The DNA extraction was conducted using the ISOIL for Beads Beating kit from Nippon Gene Co., Ltd in Toyama, Japan, on the compost sample To quantify the microbial DNA in the compost, real-time PCR was utilized for both the P and W samples, employing specific primer sets for accurate measurement.
For the amplification of the respective 16S rRNA gene regions, the primers used are Pediortf (5’-TTC TGC CAA CCT AAG AG-3’) and Pediortr (5’-GTG CCC AAC TGA ATG CTG GCA-3’) for P, as well as Weisrtf (5’-CCT TGC TAA TCC TAG AAA TAG-3’) and Weisrtr (5’-GTC TCA CTA GAG TGC CCA ACT-3’) for W.
Real-time PCR was conducted using the Smart Cycler system from Cepheid and SYBR Premix Ex Taq from Takara Biomedicals The 25 µL reaction mixture was prepared according to the manufacturer's instructions, incorporating 1 µL of SYBR Premix Ex Taq, 0.2 µM of both forward and reverse primers, 2 µL of extracted DNA, and super-pure water.
A standard curve for quantification was established using a 10-fold dilution series of P and W cell suspensions, ranging from 10³ to 10⁸ cells All measurements were performed in triplicate, and the average values were calculated to ensure accuracy.
4 3.1 Time course of composting with inoculation of individual microorganism
The comparison of pH, CO2 evolution rate, and carbon conversion for Runs 4-A and 4-B reveals significant differences in composting behavior In both runs, the pH initially dropped below 5, but Run 4-A experienced a marked increase in pH, reaching approximately 7.4 by day 7, while Run 4-B showed no such increase The CO2 evolution rate and carbon conversion data indicate that vigorous organic matter degradation commenced on day 4 in Run 4-A, contrasting with the lack of significant degradation in Run 4-B This correlation suggests that the patterns of pH change are closely linked to CO2 evolution rates, with an increase in pH corresponding to heightened CO2 production in Run 4-A.
A, while it was maintained at a low level throughout in Run 4-B, where no pH value increase was observed It is well known that low pH inhibits the activity of bacteria that are main
The study identified 58 contributors to organic material degradation during composting, with carbon conversion increasing in Run 4-A but remaining low in Run 4-B The results suggest that the inoculation of P enhanced pH levels, which activated microorganisms, leading to increased CO2 evolution and carbon conversion in Run 4-A Figure 2 illustrates the changes in organic acid concentrations, showing that lactic acid peaked at 37.1 mg g-1 DS during Run 4-A before significantly decreasing and disappearing by day 7, coinciding with a pH increase from day 4 Conversely, in Run 4-B, acetic acid concentration rose and stabilized around 20 mg g-1 DS throughout the composting process.
The concentration of other organic acids showed little variation, while excessive accumulation of acetic acid inhibited microbial activity, leading to minimal degradation of organic acids during composting in Run B.
During the initial stages of composting in Run 4-A, mesophilic bacteria, primarily composed of PE, reached a high cell density, which correlated with an increase in lactic acid levels This rise in PE facilitated the growth of Paecilomyces sp., a species known for degrading organic acids Conversely, in Run B, mesophilic bacteria dominated by W also increased early in the composting process, aligning with the accumulation of acetic acid.
The study examined the effects of two types of lactic acid bacteria, PE and WE, on organic acid production during composting Inoculating with PE significantly accelerated the composting process, while WE inhibited the degradation of organic materials These contrasting effects highlight the different roles of these bacteria in composting Previous research using an autothermal composting system has shown similar results for PE, reinforcing its effectiveness even under low constant temperature conditions.
The onset of vigorous organic matter degradation occurs more rapidly in low constant temperature composting compared to autothermal composting This difference is likely attributed to the initial temperature of 30°C in the low constant temperature process.
59 composting, was higher than that in the autothermal composting procedure, which subsequently enhanced the activity of PE
Fig 4 1 The change in pH, CO 2 evolution rate, rCO 2 , and conversion of carbon, X C during composting of Runs 4-A and 4-B
C onv er s ion of C ( % ) CO 2 ev ol ut ion r at e x1 0 3 (m ol /d) p H ( -)
During the composting process of Runs 4-A and 4-B, the concentration levels of organic acids, specifically lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA), were measured The data includes error bars that indicate the 95% confidence intervals for the mean values, based on three samples (n=3).
Time course of composting with inoculation of 2 microorganisms
Figure 4 3 shows the time courses of pH, CO 2 evolution rate, and conversion of carbon in
Runs C, D, and E A significant increase was observed in CO 2 evolution rate during Run C with the P/W ratio of 10 1.5 , while CO 2 emission was not remarkable in Run D and E with the P/W
In the study, Run C showed a significant increase in carbon conversion, reaching 34.5% by the end of the experiment, while pH levels drastically rose, with CO2 evolution peaking at 8.5 In contrast, Runs D and E exhibited a slight initial increase in CO2 emissions, linked to organic acid production and a decrease in pH, which subsequently inhibited microbial activity and organic matter degradation These findings indicate that a high PE/WE ratio of 10:1.5 effectively accelerated organic matter degradation, whereas a low PE/WE ratio of 1:10 failed to achieve similar results.
Figure 4.4 demonstrates the variations in organic acid concentrations during composting for Runs 4-C, 4-D, and 4-E In Run 4-C, lactic, acetic, and propionic acid levels initially rose but significantly degraded by day 4, coinciding with a pH increase to approximately 6.9, indicating a consistent relationship between organic acid concentration and pH (see Fig 3) Runs D and E, with PE/WE ratios of 1 and 10^-1 respectively, exhibited similar trends; acetic acid levels peaked at around 20 mg g^-1 DS during the early composting stages and remained stable until completion The elevated acetic acid concentration likely contributed to a reduced degradation rate of organic material in these runs.
Comparative analysis of PE and WE inocula, both individually and in a 10:1.5 ratio, revealed significant reductions in lactic acid and acetic acid concentrations Specifically, lactic acid production decreased in Run 4-C compared to Run 4-A, while acetic acid levels were lower in Run 4-C than in Run 4-B.
Fig 4 3 The change in pH, CO 2 evolution rate, rCO 2 , and conversion of carbon, X C during composting of Runs 4-C, 4-D, and 4-E
Fig 4 4 Concentration of organic acids during composting of Runs 4-C,4- D, and 4-E LA, AA,
PA, and BA represent lactic acid, acetic acid, propionic acid and butyric acid, respectively Error bars show 95% confidence intervals for the mean values (n=3)
C o n c e n tr at ion of o rgan ic a c id (m g/ g -d s ) f
Microbial succession during composting with inoculation of 2 microorganisms
Figure 4 5 shows the growth of microorganisms during composting Runs 4-C, 4-D, and 4-E
In the initial phase of Run C, there was a notable increase in mesophilic bacteria, which was subsequently followed by the growth of mesophilic fungi starting from day 2 The predominant mesophilic fungus identified in Run C was Paecilomyces sp., known for its ability to raise pH levels, thereby creating a more favorable environment for the activity of other indigenous composting microorganisms (Tran et al.).
In the composting process, mesophilic fungi, particularly Paecilomyces sp., showed a notable increase after day 2 when PE was dominant, leading to the degradation of organic acids and their maintenance at low concentrations until the end of composting In Runs 4-D and 4-E, although mesophilic bacteria increased, they did not support the growth of Paecilomyces sp., suggesting a difference in the dominant mesophilic bacteria compared to Run 4-C As composting progressed in Runs D and E, mesophilic bacteria experienced a significant decline in cell density, potentially due to the high levels of acetic acid they produced, which may have inhibited their own growth The inoculum with a PE to WE ratio of 10:1.5 effectively facilitated the transition from mesophilic bacteria to mesophilic fungi, thereby enhancing the degradation of organic matter, a phenomenon not observed in runs with lower PE/WE ratios of 1 and 10:1.
Figure 4.6 illustrates the cell density dynamics of PE and WE during Runs 4-C, 4-D, and 4-E, as measured by real-time PCR Initially, in Run 4-C, PE exhibited a higher cell density than WE; however, WE rapidly surpassed PE, indicating a greater growth rate of WE under composting conditions The trends in cell density for WE during Runs 4-D and 4-E, with initial PE/WE ratios of 1 and 10^-1 respectively, showed similar patterns.
Fig 4 5 Cell density of microorganisms during composting of Runs 4-C,4- D, and 4-E MF, and
MB represent mesophilic fungi, and mesophilic bacteria, respectively Error bars show 95% confidence intervals for the mean values (n=3)
L og c e ll de n s it y o f m icr oo rg an ism ( C F U /g -ds )
Fig 4 6 Cell density of Pediococcus acidilactici TM14 (P) and Weissella paramesenteroides
TA15 (W) during composting of Runs 4-C, 4-D, and 4-E Error bars show 95% confidence intervals for the mean values (n=3)
Throughout the composting process, the cell density of WE consistently surpassed that of PE, which correlated with the production of acetic acid Notably, after day 2, the cell densities of W experienced a slight decline until the composting concluded in both Runs 4-D and 4-E The discrepancy in cell densities observed through the dilution plating method and the real-time PCR method may be attributed to the latter's inability to differentiate between viable and non-viable cells.
The real-time PCR method measures both viable and non-viable cells, which may explain why the cell density results from this technique were higher than those obtained through the plating method at later stages.
In Run 4-C, the concentrations of lactic acid and acetic acid were significantly lower than those observed in Run 4-A and Run 4-B, respectively, as illustrated in Figures 2 and 4.
The individual addition of PE and WE led to significantly higher production of lactic and acetic acids, respectively, compared to their combined addition, indicating an interaction between these two lactic acid bacteria According to Freilich et al (2011), interactions can be classified as competition, cooperation, or neutral, with competition resulting in a loss-loss scenario where the activity of each microorganism is suppressed due to nutrient competition The reduced organic acid production observed in Run 4-C can be attributed to this loss-loss interaction, particularly significant when the PE/WE ratio was 10:1.5, potentially due to the higher growth rate of WE compared to PE, suggesting that a higher initial cell density of PE may be necessary to foster competition for nutrient consumption.
Effects of cell density of 2 microorganisms and composting temperature
The study confirmed that maintaining a PE/WE ratio of 10:1.5 with high cell density of both bacteria effectively prevents acetic acid production and promotes the growth of indigenous microorganisms, enhancing organic matter degradation Additionally, it was established that a PE/WE ratio around 10:1.5 significantly accelerates the degradation process In Run F, the PE/WE ratio was similar to that of Run 4-C, but the cell densities of both PE and WE were reduced by an order of magnitude compared to Run 4-C (refer to Table 4.1).
The carbon conversion in Run 4-F began to rise from day 2, displaying a trend similar to that of Run 4-C, despite differing conversion values between the two composting processes The findings from Run 4-F indicate that the P/W ratio plays a more crucial role than the individual cell densities of P and W within the range examined in this study.
The study confirmed that the relationship between the PE/WE ratio and composting acceleration observed at low constant temperatures also applies to increasing temperatures over time, simulating autothermal composting in Run 4-G Significant organic matter degradation began on day 2, with ongoing mineralization observed even after day 5 when the temperature rose to 60°C.
The inoculation of a P/W ratio of 10 1.5 significantly accelerated organic matter degradation during composting, mimicking autothermal conditions Furthermore, the carbon conversion curve's steep slope after day 5 in Run 4-G indicates that elevated temperatures further enhanced the degradation process of organic matter.
Fig 4 7 The change in temperature and conversion of carbon, XC during the composting of
This study highlights the significant role of lactic acid bacteria during the early stages of composting, revealing their interactions Understanding these dynamics can enhance our knowledge of composting processes and improve the efficiency of high-rate composting operations.