Utilization of Wood Biomass for Organic Soil Based on the Soil Fertility Index (SOFIX)

Possibility of wood biomass for preparing organic soil was examined to construct reproducible and stable organic standard soil. Seven organic soils were constructed from base soils and additive materials based on the recommended values of the soil fertility index (SOFIX) (total carbon ≥ 25,000 mg/kg, total nitrogen ≥ 1500 mg/kg, total phosphorus ≥ 1100, and total potassium of 2500 to 10,000 mg/kg). Base soils were prepared from two types of wood biomass (big- and small-sized wood chips) at 50%, 60%, and 70% (v/v) and other organic materials such as peat moss, black soil, and mountain soil. Additive materials (soybean meal, oil cake, cow manure, and bone meal) were amended into all organic soils at the same amount. Incubation experiment showed that bacterial biomass in all organic soil was greater than 6 × 10 8 cells/g-soil after addition of 30% of water content for 1 week. In addition, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis re-sulted in a stable bacterial diversity of the organic soil prepared from the small size wood chip at 70%. Chemical properties of all organic soils were within the recommended values of SOFIX. The plant cultivation experiment showed that fresh Brassica rapa var. peruviridis weights in the organic soils with 50%, 60%, and 70% of small-sized wood chip were 5%, 16%, and 27% higher than that of the chemical fertilizer-amended soil. The organic soil with 70% of small wood chip was the best in the seven organic soils in this study.

company, Tochigi, Japan) were used as the additive materials. The base soils and additive materials were air dried for 1 week, and then sieved through a 2-mm sieve. The chemical soil (Hanachanbaiyodo company, Nagoya, Japan), which is amended with chemical fertilizer, was considered as a control treatment.

Analytical Methods
Total carbon (TC) was analyzed using a total organic carbon analyzer (SSM-5000A, Shimadzu, Kyoto, Japan). Total nitrogen (TN), total phosphorus (TP), and total potassium (TK) contents were analyzed by extracting soil samples using the Kjeldahl digestion method followed by analysis using the indophenol blue method, molybdenum blue method, and atomic absorption spectrophotometry, respectively [15] [16]. The total bacterial biomass of the soil was analyzed by quantifying the environmental DNA (eDNA) extracted by the slow-stirring method [17]. Nitrogen (N) circulation and phosphorus (P) circulation activities were examined according to our previous studies [13] [18]. The maximum water holding capacity (WHC) and bulk density were measured by the standard methods [19]. Soil pH (1:2.5 soil-to-water suspension, w/v) was analyzed using a pH meter (LAQUA. F-71, Horiba, Kyoto, Japan).

Preparation and Analysis of the Soil
The base soils and additive materials were dried at 37˚C for 1 week, and then these materials were sieved through a 2-mm sieve. Seven organic soils were prepared by blending the base soils and additive materials. To activate the microbial activities, 200 g of each organic soil was preincubated in a 400 ml pot and maintained 30% of water content for 1 week. The soil sample of each organic soil treatment was subsequently collected for SOFIX analysis [13]. The bacterial biomass was measured on days 0, 3, 5, and 7, while the other parameters were measured on days 0 and 7. The bacterial diversity of the different lots of ideal standard organic soil and the different lots of chemical soils was analyzed on day 0 with polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis. The organic soils were incubated in the plant factory with 12 h of light: 12 h of dark at 23˚C throughout the experimental period.

Plant Cultivation
Seven organic soils and the chemical soil (control) were used for plant cultivation experiment. A 2 L soil sample was put into a Wagner pot (1/5000a, Fujimoto Kagaku Kogyo company, Tokyo, Japan), and then preincubated at 30% of water content. Brassica rapa var. peruviridis (Komatsuna) seeds were sown in a nursery tray for 1 week, and four seedlings were then transplanted to each Wagner

PCR-DGGE Analysis
A best organic standard soil (among seven organic soils) and the chemical soil

Selection of the Base Soils and Additive Materials
Base soils and additive materials were selected to construct suitable chemical, physical, and biological characteristics in the organic standard soil. The properties of candidates for the base soil (mountain soil, black soil, peat moss, vermiculite, and wood chips) were measured (Table 1 and Table 2). The TC contents of peat moss and wood chips were higher than those of the other candidate base soils, while the TN and the TP contents of all candidates were low. The maximum WHC of black soil, vermiculite, and wood chips were relatively high but the bulk density of vermiculite, peat moss, and wood chips were low. The components  difference sizes of wood chips (wood chips 1 and wood chips 2) were almost the same but the bacterial biomass of a wood chip 2 was higher than that of a wood chip 1.
The total nitrogen contents of oil cake, soybean meal, bone meal, chicken manure, and cow manure were above 20,000 mg/kg. The TP contents of oil cake, bone meal, chicken manure, and cow manure were high. The bacterial biomass of all manures was above 6 × 10 8 cells/g. Among the three types of manure, cow

Construction and Characterization of the Organic Standard Soils
The candidates of a standard soil based on SOFIX recommended values (Table  3) were prepared to construct a stable and reproducible organic standard soil. Seven candidates of the organic standard soil were prepared using the base soils and additive materials at different ratios (Table 4 and Table 5). Cow manure, oil cake, soybean meal, and bone meal were added in base soil at 5%, 0.25%, 0.25%, and 0.05% w/w, respectively. Chemical and physical properties of the seven prepared organic standard soils are shown in Table 6. The TC, TN, TP, and TK contents, and the C/N and C/P ratios of the seven candidate standard soils were 24,000 -34,740 mg/kg, 1580 -1840 mg/kg, 1040 -1160 mg/kg, and 6450 -9660 mg/kg, and 14 -20 and 22 -31, respectively. The bulk density and the WHC of the seven organic standard soils were above 0.5 g/cm 3 and 1200 ml/kg, respectively. The chemical and physical properties of the seven organic soils were around SOFIX recommended values. Among seven organic soils, T7 was showed the lowest bulk density but the highest WHC.
The biological properties of the seven candidate organic soils after controlling the water (30% of water content) for 1 week are shown in Figure 1 and Table 7. The bacterial biomass of all candidate organic soils exceeded 6 × 10 8 cells/g-soil on day 3, and the bacterial biomass of T2, T3, T4, T5, T6, and T7 was greater than 11 × 10 8 cells/g-soil on day 7. This result indicates that the wood chips increase the bacterial biomass. Among the seven organic soils, T7 showed the highest value of the bacterial biomass. The nitrogen and phosphorus circulation activities of the seven candidates of the organic soil were close to the SOFIX recommended values.

Plant Growth in the Organic Standard Soils
To compare the plant growth, B. rapa cultivation experiment was conducted ( Table 8). The performance of Brassica rapa in the seven organic soils was similar      Means followed by the same letter do not significantly differ (p < 0.05). Value followed by ± is standard deviation. Figure 1. The bacterial biomass in the seven organic soils (T1-T7) during 7 days.
or better than that in the chemical soil. An increase of wood chip 2 led to a higher fresh weight and shoot length of B. rapa than that in the chemical soil and in the organic soils with wood chip 1. Especially, B. rapa growth in the organic soil T7 containing 70% (v/v) of wood chip 2 was the highest. These findings suggest that wood chip 2 is the most suitable for B. rapa cultivation. Chlorophyll of plants in the chemical soil used was 19% -29% higher than those in the organic soils, suggesting that the inorganic nitrogen in the chemical soil was richer than that in the organic soil. As a result, the organic soil T7 was identified as the best organic standard soil.
In the next experiment, comparison of the bacterial diversity between the organic standard soil (T7) and the chemical soil was conducted.

Analysis of the Bacterial Diversity in the Organic Standard Soil
The comparison of the bacterial diversity between the organic standard soil (T7) and the chemical soil were conducted in this study. The bacterial diversities of different lots of the organic standard soil and different lots of the chemical soil were compared (Figure 2). The bacterial diversities of the organic standard soil and the chemical soil were different, even though the same base soil was used in the organic standard soil and the chemical soil. The bacterial diversities of the organic standard soil were similar, but those of different lots of the chemical soil were unstable. The number of bacterial species in the organic standard soil was higher than that in the chemical soil. The organic standard soil was controlled not only by the bacterial biomass but also by the bacterial diversity, suggesting that the bacteria biomass and bacterial diversity seem to be a positive relationship.

Discussion
Based on SOFIX database [13], the values of TC (≥25,000 mg/kg), TN (≥1500 mg/kg), TP (≥1100 mg/kg), TK (2500 to 10,000 mg/kg), and C/N ratio (8 to 25) were controlled by mountain soil, black soil, wood chips, peat moss, vermiculite, and additive materials. The bacterial biomass of the organic soils with wood chips was higher than 6 × 10 8 cells/g-soil after controlling the water content to 30%. Wood chips, especially the small particle size (wood chips 2), were found to Journal of Agricultural Chemistry and Environment be most suitable for the bacteria growth and diversity. The surface area and pore size of wood chips may be suitable for soil microorganisms [21] [22]. In fact, the bacterial biomass in the organic soils with wood chips 2 were obviously higher (≥14 × 10 8 cells/g) than that in vermiculite after 7 days. Moreover, the bacterial biomass in the organic soils was also higher than those in organic farming soils [23].
The growth of B. rapa in the organic standard soil was higher than that in the chemical soil. Soil microorganisms play an important role in soil nutrientcycling [24] [25]. In addition, the bacterial diversities in the organic standard soil became almost the same within the PCR-DGGE experiment [34], indicating that the preparation of the organic standard soil was reproducible. The bacterial diversity was also controlled reproducibly by the addition of the water.
In this study, the main elements (nitrogen, phosphorus, and potassium) in the organic standard soil were successfully controlled by biomass resources based on the SOFIX database. Other factors, such as micronutrients, will be considered in the next stage of the organic soil construction, which is currently in progress.

Conclusion
The reproducible and stable organic standard soil was constructed in this study.
All organic soils showed the suitable values of chemical and biological properties according to SOFIX recommended values. Out of those, T7 (with 70% of smallsized wood chip) had the highest bacterial biomass and stable bacterial diversity.
In addition, T7 led to the increase of the fresh weight of B. rapa.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.