*2.1.1.2 Measuring C-organic content using the Walkley and Black method*

The procedure carried out in this test was taken from several references, namely, Black (1965); Graham (1948); Page et al. (1982); Rayment et al. (1992) in Sulaeman et al. (2005) "Technical Guidelines for Soil, Plant, Water, and Chemical Chemical Analysis of Soil Research Institute Indonesian Ministry of Agriculture." With the following method of work, 0.500 g soil of size <0.5 mm was weighed and put in a 100 ml volumetric flask. 5 ml of K2Cr2O7 1 N was added and then mixed. 7.5 ml of concentrated H2SO4 was added, mixed, and let to sit for 30 min. Diluted with ion free water, the mixture was allowed to cool and squeeze. In the next day, absorbance of the clear solution was measured with a spectrophotometer at a wavelength of 561 nm. As a comparison standard, 0 and 250 ppm were made, by piping 0 and 5 ml of the 5000 ppm standard solution into a 100 ml volumetric flask with the same treatment as the working sample [21].

### *2.1.1.3 Measuring N-total levels using a spectrophotometer*

In this test, the procedure was taken from several references, namely, Black, (1965); Page et al. (1982); Burt (2004); and Lisle et al. (1990) in Sulaeman et al. (2005) "Technical Guidelines for Soil, Plant Chemical Analysis, Water and Fertilizers, Indonesian Ministry of Agriculture Soil Research Institute." This test was divided into two stages: the destruction stage and the measurement stage [21].

### *2.1.2 Biogas measurement*

Samples that had been researched in preliminary methods could be inserted into the reactor and mixed with other components that are related; then the reactor must be sealed in order to obtain anaerobic digestion. During the treatment process, the volume of biogas production was observed in an interval of 2 days throughout 60 days.

To find out the amount of biogas, place the reversed cylinder glass in the container that is filled with water (reversed cylinder glass must be filled with water). Place the plastic tube into the reversed cylinder glass. Record the initial volume from it. Open the clip that clipped the plastic tube (the clip's function was to avoid the oxygen entered into the digester). The biogas will go out through the plastic tube and will make the water volume to decrease. Record the final volume. Lastly, record the biogas volume by counting the water level difference. The digester is shown in **Figure 2**.

### **2.2 Liquid anaerobic digestion (L-AD) method**

### *2.2.1 The effect of total solid (TS)*

TS content of liquid anaerobic digestion (L-AD) systems ranges from 0.5 to 15% [4]. The research about "Biogas Production from Water Hyacinth (*Eichhornia crassipes*): The Effect of F/M Ratio" [22] was conducted to know about the effect of F/M ratio to biogas production from water hyacinth leaves using the liquid anaerobic digestion (L-AD) method. In biogas production anaerobically, the value of F/M shows a comparison between the amount of substrate that is contained in waste (medium)

### **Figure 2.**

*Schematic diagram of series laboratory batch assessment of L-AD and SS-AD [23].*


### **Table 1.**

*Initial and final total solid of water hyacinth leaves.*

and the amount of microorganism used [18]. The variation of F/M ratio depends on the existence of rumen volume variation and total solid from each materials.

The main substrate used in the research [22] was water hyacinth leaves as much as 200 g. The initial total solid of water hyacinth leaves that has been calculated using (Eq. (1)) was 13.52. When it is combined with a different cow rumen fluid volume in each reactor, the total solid of water hyacinth leaves will be changed. To find out the F/M ratio, a comparison of the total solid of water hyacinth leaves with cow rumen fluid was multiplied by the weight and volume of each ingredient. The data is shown in **Table 1**.

After the research had been done, results show that the biogas production with F/M ratio of 10.01 and TS of 6.76% produced more biogas in the amount of 127.071 ml/g TS. **Figure 3** shows the graphic of cumulative biogas yield.

A research has also been conducted [20] using water hyacinth leaves as much as 200 g as the main substrate combined with water and rumen fluid. The combination is shown in **Table 2**.

In this study to get a low total solid content, rumen was added to the first variable, and water and rumen were added to the second variable. This is consistent with the research conducted by Astuti (2013) which states that the stuffing material must contain about 6–9% dry matter. This situation can be achieved by dilution [20]. From the graphic below, the final result of biogas production with a TS variable of 6.76% was 177.33 ml/g TS and for a TS variable of 3.38% was 369 ml/g TS. The graphic of cumulative biogas yield/TS is shown in **Figure 4**.

### *2.2.2 The effect of C/N ratio*

In addition to total solid and F/M ratio, biogas production is also affected by carbon to nitrogen (C/N) ratio. Various C/N ratio researches have been done [23] by adding organic compound that contained high nitrogen such as urea.
