Studies on Composting Spent Coffee Grounds by Aspergillus sp and Penicillium sp in Aerobic Static Batch Temperature Control

Spent Coffee Ground (SCG) is characterized by high organic content, in the form of insoluble polysaccharides bound and phenol compounds. Phenol compounds are toxic to nature and are a cause of environmental pollution. Composting method of this study is aerobic static batch composting with temperature control with adding activators of some fungi such as Aspergillus sp, and Penicillium sp. The purpose of the research is to fill the research gap from previous studies of spent coffee grounds compost, which requires a long time in composting, so that if it is used directly on the soil and plants, the positive effect also requires a long time. The result of composting for 28 days with this method is that mature compost has black crumb and normal pH, with characteristics of C/N ratio below 10: C1 (7.06), C2 (6.99). This value is far from the control with a C/N ratio of 8.33. Decompose rate of macromolecule are above 40% for lignin and 70% for cellulose. Implementation of compost in radish plants, resulting Germination Index above 80% which indicates that the compost is ripe: control (92.39%), C1 (183.88%), C2 (191.86%). The results of the analysis with FTIR also showed that the compost was mature and stable, and rich in minerals. So, it can be concluded that this composting method can speed up composting time and optimize the results of compost produced.

The addition of activator in the composting process has been widely studied by researchers. However, the use of specific combination types of fungi activators with temperature control for SCG composting is still very little studied.
Yamane et al. (2014) studied the use of coffee grounds directly on plant growth, the results of which took a long time for the benefits of coffee grounds to function positively for the soil and plants [22]. The difference in the use of fresh and composted spent coffee ground is also studied and composted SCG is better for plants [23]. SCG compost using solid state fermentation was also studied by Echeverria et al. [24]. Because of the lack of reference composting of SCG using soft rot fungi with temperature control, therefore in this study studied the composting of coffee grounds using fungi especially by Penicillium sp and Aspergillus sp.
Activator using fungi is a very promising method for increasing agricultural production, as well as reducing the release of chemical pesticides into the environment. This is because they are able to change and release many nutrients that play an important role in the nutrition cycle and maintain vegetation [25]. In addition, in this study also added cow dung and chicken manure to supply extra carbon for microbial activity, balancing C/N ratio, and providing most of the nutrients, including nitrogen (N), phosphorus (P), and potassium (K).
Composting method of this study is aerobic static batch composting with temperature control. Because can be generated in a short period of time [26] [27]. Temperature control is done to maximize the growth of microorganisms, especially Penicillium sp and Aspergillus sp during the composting process. This is because temperature is one of the most important environmental factors in the composting process. The objective of this study was to understand the effect of using combination fungi with temperature control in SCG composting.

Treatment and Composting Procedure
This study was conducted in Prefectural University of Hiroshima, Shobara campus, Japan. Each sample was produced in 1-liter plastic container with small holes for air circulation. 3 samples (control, C1, and C2) then put in incubator with temperature 30 degrees Celsius. Description of sample as explains below: 1) Control: SCG 150 g, chicken manure and cow dung each 100 g and adding water until Moisture content around 60%.
2) C1: SCG 150 g, chicken manure and cow dung each 100 g, adding commercial (dilution 100 times with water) to sample until Moisture Content around 60%.
3) C2: SCG 150 g, chicken manure and cow dung each 100 g, then adding fungi activator (dilution 100 times with water) to sample until Moisture Content around 60%.
The amount of activator used is 1 percent of the total weight of water. Spent coffee grounds were fermented Robusta coffee from Jember, East Java, Indonesia. Then dried in oven laboratory at 60 degrees Celsius to remove all the humidity and inhibit microbial processes. After, these materials were mixed and used in composting to achieve a mixture with C/N ratio below 10 (for horticulture plant), the idea being obtain compost rich in nitrogen for plant fertilization.
About 350 g dry material of each mixture was composted 28 days. The moisture was maintained around 60% of water content. The mixture was homogenized manually revolving each mixture almost every day. The temperature was controlled in 30 degree Celsius. Samples were taken on days 0, 7, 14, and 28 days. In all analyzes, 3 repetitions were performed for each sample. Sketch of the composting equipment can be seen in Figure 1 below.  mg of raw materials (Spent Coffee Grounds, cow dung, and chicken manure), added 1 M H 2 SO 4 1 ml mixed and put to water bath shaker 1 h. Then it was autoclaved 1 hours 121 degree Celsius. Then it was filtrated using filter paper. Then put in oven 105 degrees Celsius 24 hours. Weighed sample as lignin. Total of Polyphenol analysis uses spectrophotometry method [29]. Analysis of protein use Lowry method [29]. In addition, screening of macro and micronutrient content related to their potential to be composted was also carried out.

Physical and Chemical Analysis during Composting
The following analysis was carried out in fresh compost samples: electrical conductivity (EC), and pH were determined by "Soil analysis" standard procedures [30]; Total carbon and total nitrogen were determined also in dry sample by Macro corder (JM 1000CN). Then, the C/N ratio was calculated.

Macro Nutrient Analysis
The concentration of element macro nutrient Ca, Mg, P, and K were measured in dry samples by ICP-OES (inductively coupled plasma optical emission spectrometry) (Hitachi, PS7800) after nitric per chloric acid digestion [30].

Morphology of Starter
Morphology of fungi was observed under microscope using slide culture. While commercial activator was observed using Scanning Electron Microscopy (SEM) [31].

Germination Index
Percentage of seed germination, root growth and germination index (GI, a factor determined by both germination & root growth) were calculated based on the formula [33]: Seed germination (SG %) = SG % in each extracts/SG % in control × 100.
Root growth (RG %) = mean root length in each extracts/mean root length in control × 100.

Functional Group Identification
Functional group analysis is carried out by means of FTIR-ATR (Attenuated Total Reflection-Fourier Transform Infra-Red) [34].

Chemical Composition of Raw Material
Spent coffee ground (SCG) contains many polysaccharides such as hemicellulose (39.75%), and lignin (23.1%). Cow dung and chicken manure also contain hemicellulose and lignin which are also high. Cow dung has 18% hemicellulose, and 8.9% lignin, while chicken manure has 11% hemicellulose and 13.75% lignin (Table 1). These values are comparable to the others reported in the literature for SCG [35]. A little difference with some of the results of other studies may differ the type of coffee, method of roasting and also brewing techniques. It is very suitable for composting, mixing with spent coffee grounds to prevent nitrogen loss. The composition of manure is highly variable, according to animal The high content of hemicellulose and lignin is the basis for choosing the type of activator fungi in composting, namely Aspergillus spp and Penicillium spp.
Lignin is a major structural component of plants and is the one that is degraded the slowest. It has been claimed that humus is mainly formed from lignin, polysaccharides and nitrogenous compounds [36] [37] [38]. Therefore, in this study we choose specific fungus that can degraded lignin very well. The degradation of lignin is primarily accomplished by fungi. For hemicellulose, the main degrading enzyme is xylanase, produced by many bacteria and fungi [39].
This study also analyzed the caffeine content and total polyphenols in SCG.
This is because both of these compounds are toxic to soil and plants [4]. The caffeine content in the SCG sample was 1.83%. This value is lower than previous research of Musatto et al. (2011), because the sample used is Robusta coffee that has been fermented [35]. This fermentation process causes caffeine to degrade and its value is lower than other references.
Hakil et al. (1998) mention that Penicillium and Aspergillus are the more frequent caffeine-degrading genuses [40]. It therefore seems logical that the majority of the studies done on caffeine degradation by filamentous fungi are related to Aspergillus and Penicillium genuses.
SCG also contains Carbon and Nitrogen ratios 19.5/1. Cow dung (12.36/1), and chicken manure (9/1) ( Table 2) which approaches the C/N ratio of the soil, 20/1. Thus, SCG, cow dung, and chicken manure have the potential to be used as compost products because it has the macro and micronutrients needed by soil and plants.

Selection of Microorganism for Composting
2 fungal isolates from fertile soil were screened for their ability to produce celluloses. We also observed morphology of each strains. Fungi were observed under a light microscope with a magnification of 40× ( Figure 2 and Figure 3). This is because fungi have large cell sizes. From these observations, it is known that fungi are Aspergillus sp following characteristics: colony grows quickly, green color of colony, single row of phialides covering entire vesicle, conidiophore point out in all direction, and in variable length Rough, pitted, spiny. Besides, in activator also consist of Penicillium sp, with characteristic: old green color. Conidiophore hyaline upright, branched, tapered phialide, conidia pale green-shape flooding in place of hexadecyl trimethyl ammonium bromide or Congo red, gave a more rapid and highly apparent result [42]. Gram iodine was also used for the screening of cellulose producing microorganisms, i.e., fungi [43] and bacteria [32].
After observing the clear zone produced, the Relative Enzyme Activity (REA) was measured by comparing the diameter of the clear zone and the diameter of the colony. The REA measurement results are presented in Table 3 below.

Chemical Properties Changes during Composting
In the composting process, to determine the maturity, stability and quality of compost is to observe chemical changes in the material. Chemical changes in the material during composting process are described below. Observation of each parameter is done for the initial sample, 7, 14, 21, and 28 days composting is carried out aerobically and the sample is left in a container at incubator 30 degree Celsius. gen decomposition which is also affected by temperature. Some of the ammonia is released or converted to nitrate, then nitrate is denitrified by bacteria so that the compost pH becomes neutral at the end of composting [46]. This indicates mature compost suitable for most cultivated crops [47]. The EC values of matured compost samples in the range of 2.19 -9.32 ms/cm.

pH and EC
The evolution of EC for the three samples of composts is presented in Figure 5.
The general tendency of EC for all three samples of composts was to increase during the composting process. Usually a higher value of EC could be an indication of high nutrient elements presence, or a slower decomposition of the organic matter therefore a lower release of mineral salts into the solution in the process of biodegradation of biomass waste [48] [49].

C/N Ratio
The total of C content of the compost SCG was decrease during composting time. Because of the reduction in available carbon sources and synthesis reactions of the new complex and polymerized organic compounds or humification during the maturation phase [50], some researchers also indicated that the organic carbon content of compost samples has decreased during composting [51] [52] [53]. As shown in Figure 6, it can be seen that after one month the carbon has decreased. Compost sample with C2 has lowest total carbon average in final composting (6.99%), followed by C1 (7.04%) and control (8.33%). This means, C2 with fungi activator has the ability to degrade carbon better than others. Decreasing carbon of Sample C1 as much as 61.81%, while C2 60.56%. By using these three types activator, carbon reduction is better when compared to controls 56.51%. Our activator can compete with commercial. It is because the difference in the percentage of carbon reduction is only about 1.25%.
Decomposition rate of each compost can be seen in Figure 6. In the first week (mesophilic phase), C2 activator has a higher decomposition rate. Optimum temperature for Aspergillus and Penicillium sp growth is 28 -37 degrees Celsius [54]. This indicates composting with a temperature of 30 degrees Celsius, very good for the material using a fungi activator. This species is also able to grow in a wide range of pH from 3.0 to 7.0 with maximal growth rates at 4.0, the optimum range being 4.6 to 6.8 [54]. At the end of composting, all three samples had almost the same decomposition rate of carbon.
Total Nitrogen (TN) includes both organic nitrogen and inorganic nitrogen  (mainly ammonia nitrogen and nitrate) which are normally assimilated by microbes. The variations of TN for different treatments are also shown in Figure 8.
In the first week when the pH is alkaline, so there is an ammonification process in which inorganic N turns into ammonium. In Figure 8 below, nitrogen has dropped dramatically. However, in the second and fourth weeks, total nitrogen showed a slight increase. This is because the pH begins to return to near normal, there is nitrification where the ammonium changes to nitrate. Total N (TN) increased slightly as a consequence of concentration effect due to the mass loss during composting [50]. As which could be explained by volatilization and immobilization processes. As we can see in Figure 8, it shows the total nitrogen which has decreased compared to the initial.

Ratio
Ammonium nitrate ratio is also one of parameter to check maturity of compost.
The decrease of

Degradation of Lignocellulose
On the world lignocelluloses are the main part of biomass, because it is a renewable resource and the prominent structural component of plant cell wall as well. Cellulose is the dominant part of lignocellulose and consists of a linear chain of D-glucose linked by β (1)(2)(3)(4)-glycosidic bonds to each other. The cellulose strains are connected to each other deliver cellulose fibril. A number of intraand intermolecular hydrogen bonds are linked cellulose fibers together. Hemicellulose is the second plentiful constituent of lignocellulose, is comprised of diverse pentoses (arabinose, xylose) and hexoses (mannose, galactose, glucose) [59].
Previous study has explained about transformation of macromolecule such as cellulose, hemicellulose, and lignin as a consequence of biological activity during composting [60]- [69]. It has been claimed that humus is mainly formed from lignin, polysaccharides and nitrogenous compounds [36] [37] [38]. Another's studies also confirm the extensive loss of cellulose and hemicellulose, while confirming the increasing proportion of humus. Therefore, in this study we observed changes of cellulose, hemicellulose and lignin during composting. Figure 8 summarized the rates of lignin, and cellulose decomposition during composting, and showed a relatively high initial proportion of lignin (around 41% -46%) and a low cellulose content (around 4%). The greater lignin degradation in C2 could be explained by added to fungi when inoculating at t 0 . This led to better global lignin degradation in C2 (40.28%) compared to similar rates in C1 (35.56%) and control (31.1%). For cellulose degradation, Sample C1 decreased 83.47%, Sample C2 74.39%. According to previous study, Aspergillus Figure 7. Ammonium-Nitrate ratio after composting. and Penicillium is a soft rot fungus which show preference for cellulose and hemicellulose. Soft rot fungi can tolerate a wide range of temperature, humidity and pH conditions, and attack a variety of wood substrates [70]. They are usually thought to degrade mainly carbohydrates in soil, forest litter and compost, but they may also degrade lignin in these environments [71] [72] [73] [74]. Thus, some of them were found to be able to mineralize grass lignins [75] and e.g. Penicillium chrysogenum, Fusarium oxysporum and F. solani [71] mineralized in 28 days up to 27% of a 14C-labelled lignin prepared from milled wheat straw.
Protein is the first degraded to peptides by the enzyme's proteases and then into amino acids by the enzyme's peptidases. These enzymes are produced by many species of Bacillus. Further decomposition yields NH 3 , NO 3 , CO 2 , and water. This process (ammonification) occurs as a result of hydrolytic and oxidative enzymatic reaction under aerobic conditions by heterotrophic microbes such as fungi and bacteria. As shown in Figure 9, the percentage of decompose of protein samples C2 85.44% and C1 83.02% is higher than the control 81.82%.

Mineral
Phosphorus plays a role in cell division, fruit, flower and seed formation, plant maturity, stimulating the development of root hair, quality of crop yields and disease resistance [78] [79]. Enzyme activators, cell turgor regulation, nutrient transport and water and increase plant endurance [78]. Calcium is essential for the growth of meristems, and particularly for the proper growth and functioning of root tips. While magnesium is a specific constituent of chlorophyll, in which one atom of magnesium is bound to four pyrrole rings. Magnesium also plays a major role in numerous enzyme reactions [80].
Compost spent coffee ground has a high mineral element (Table 4) and is very useful for plant growth. This value is higher than before composting. This increase in concentration is due to a decrease in carbon during composting, as in previous studies in the literature [81] [82]. This is an advantage of spent coffee ground compost with high mineral elements.

Phytotoxicity
The seed germination index (GI) has been defined as a factor of relative seed     Compost control and C1 also have almost same functional group with C2 like The strong band at 1650 cm −1 can be assigned to amide I, carboxylates and C = C from aromatic and alkenes [88]. Of the four samples, C2 had slower peaks and lower wavenumber. This indicates that the aromatic structure is both lower than control and C1.

FTIR Analysis
Components rich in proteins of compost can be identified by a strong band between 1570 and 1540 cm −1 [88]. From Figure 9 above, all three samples, con-

Conclusion
Combination of some fungi and bacteria activators with temperature control in composting SCG can improve quality compost produced, with the physical characteristics of compost black and crumb, and normal pH. While the chemical characteristics of compost produced is a C/N ratio below 10 with and far difference from the control. Compost is also rich in minerals, such as phosphorus, potassium, calcium, and magnesium, as well as rich in humic acid as shown from the results of the FTIR analysis. Addition of a combination of activator fungus such as Aspergillus sp, and Penicillium sp can compete with commercial activators, likewise with the use of activator lactobacillus sp. This is also evidenced from the results of the phytotoxicity analysis, where the Germination Index of the compost sample with the addition of fungi activator (C2) is 191.86% greater than the commercial activator (C1) 183.88%.