Development of non-soil media capable of degrading organic nitrogen to nitrate, as in natural soil

Articial soil materials are unable to catalyse nitrication because added organic substances suppress nitrifying bacteria. We used a multiple parallel mineralization method, which enables the use of organic fertilizers in hydroponics, to support nitrication in non-soil materials. In this method, microorganisms immobilized on porous carriers produce inorganic nitrate from organic substances, as in natural soil. The carriers also released phosphate and potassium ions. Microorganisms produced nitrate from organic substances when immobilized on polyurethane resin, rockwool, vermiculite, oyster shell lime, and rice husk charcoal. The optimal amount of organic material added daily to 100 mL of carrier held 6 mg of organic N. Vegetable plants grew on inoculated materials but not on uninoculated materials. These results show that non-soil materials can be used to create articial soils in which plants can be grown with the addition of organic fertilizer, as in natural soil. Wako, Osaka, Japan) or sh-based medium (1 g/L sh-based soluble fertilizer, Yaizu Suisankagaku Industry, Yaizu, Japan), 15 g/L agar) in the dilution plate technique; and incubated the plates at 25 °C for a week in the dark. We analysed the microbial phase in inoculated AQ-14, uninoculated AQ-14, and liquid culture solution from each carrier incubated with 1 g/L of sh fertilizer in a ask for 2 weeks at 25 °C and 120 rpm. For inoculation, three tubes lled with 100 mL of AQ-14 were washed with 100 mL of water; 1 g of bark compost (Sanyo Chip Kogyo, Shimonoseki, Japan) was added on the top as inoculum, 1 mL of sh based soluble fertilizer diluted 10 times with distilled water was added as the organic substance, and the tubes were incubated in the dark overnight at 25 °C. The pellets were washed with 100 mL of water the next day and the leachate was tested for nitrate ions. The addition of sh fertilizer, incubation, and washing were repeated until nitrate ions were detected in the leachate, and then the microbial phase in the AQ-14 was analysed 25 by the Bioengineering Lab. Co., Ltd. The microbial phase of uninoculated AQ-14 and the precipitate collected by centrifuging the liquid culture solution were also analysed. in the and absence of immobilized microbes. We packed 100 mL of rockwool into tubes and added 1 g of bark compost as inoculum. The rockwool was washed with 100 mL of distilled water, 0.1 g of sh fertilizer was added, and the tubes were incubated for 24 h in the dark at 25 °C. The next day, the rockwool was rinsed with 100 mL of distilled water, and inorganic N in the leachate was measured. The addition of sh fertilizer, incubation, and washing was repeated for 3 weeks. Then we added 0.1 g (6 mg N), 0.2 g (12 mg N), 0.5 g (30 mg N), or 1 g (60 mg N) of sh fertilizer per tube and incubated the tubes for 24 h in the dark at 25 °C. The next day, they were rinsed with 100 mL of distilled water and the above amounts of sh fertilizer were again added. The addition of sh fertilizer, incubation, and washing were repeated for over 30 days. We measured inorganic N, ammonium, nitrite, and nitrate in the leachates.


Introduction
Soil is used to grow 98.8% of food 1 . It is common to use organic fertilizers made from food residues and livestock manure. Organic fertilizer ploughed into the soil is degraded to inorganic nutrients such as nitrate, ammonium, and phosphate. However, if organic fertilizer is ploughed into sterile regolith, nitrate is not generated 2 . For this reason, Moon and Mars regolith would not be able to degrade organic fertilizer to produce nitrate, even if microorganisms populated the fertilizer. Many crop plants require nitrate N, and if nitrate is not produced, they will not grow well [3][4][5] . In soil-less hydroponic culture, too, it is not possible to grow plants with organic fertilizers [6][7][8][9][10][11][12] , because nitrate is not easily generated in water 9 . Thus, organic substances can be used as fertilizers only in natural soils.
In natural soils, organic N is converted into nitrate N through a two-step degradation process of ammoni cation and nitri cation 13,14 . Nitri cation is carried out by nitrifying bacteria, ammonia-oxidizing bacteria such as Nitrosomonas spp., and nitrite-oxidizing bacteria such as Nitrobacter spp. [15][16][17][18][19] However, nitrifying bacteria tend to become inactivated when exposed to organic substances [20][21][22] . In arti cial culture, nitrifying bacteria must be grown in an organic-free inorganic salt medium 23,24 . In the presence of organic substances, it is di cult to culture nitrifying bacteria in a medium other than natural soil. Shinohara et al. (2011) developed a hydroponic technology that can use organic fertilizers by degrading organic fertilizers into inorganic nutrients in water 15 . This "multiple parallel mineralization method" allows co-culturing of nitrifying bacteria with heterotrophic microorganisms even in the presence of organic substances. By this method, it may be possible to add the function of degrading organic substances to nitrate in non-soil media. This would mean being able to grow crops in arti cial soil using organic fertilizer. However, to now, this has not been successful in non-soil materials 2,15 .
Here, we created soil-like media from non-soil media by using the technique of multiple parallel mineralization to immobilize microorganisms on a non-soil carrier in order to degrade organic N to nitrate Page 3/9 N, and tested plant growth in the media.
Chemical and microbiological analysis. To measure the generation of inorganic nutrients in each carrier, we measured ammonium, nitrite, nitrate, potassium, and phosphate ions with RQ-ex test strips (Merck, Darmstadt, Germany); pH by pH meter (C-73, AZ-ONE, Osaka, Japan); and electrical conductivity (EC) by EC meter (Twin Cond, Horiba, Kyoto, Japan). To estimate microbial density, we added 1 g (fresh weight) of sample carrier to 9 mL of water and vortexed the mixture for 5 min; applied 100 µL of sample to 1/10 NA medium (0.8 g/L Difco nutrient broth (BD, Franklin Lakes, New Jersey, USA), 15 g/L agar (Fuji lm Wako, Osaka, Japan) or sh-based medium (1 g/L sh-based soluble fertilizer, Yaizu Suisankagaku Industry, Yaizu, Japan), 15 g/L agar) in the dilution plate technique; and incubated the plates at 25 °C for a week in the dark. We analysed the microbial phase in inoculated AQ-14, uninoculated AQ-14, and liquid culture solution from each carrier incubated with 1 g/L of sh fertilizer in a ask for 2 weeks at 25 °C and 120 rpm. For inoculation, three tubes lled with 100 mL of AQ-14 were washed with 100 mL of water; 1 g of bark compost (Sanyo Chip Kogyo, Shimonoseki, Japan) was added on the top as inoculum, 1 mL of sh based soluble fertilizer diluted 10 times with distilled water was added as the organic substance, and the tubes were incubated in the dark overnight at 25 °C. The pellets were washed with 100 mL of water the next day and the leachate was tested for nitrate ions. The addition of sh fertilizer, incubation, and washing were repeated until nitrate ions were detected in the leachate, and then the microbial phase in the AQ-14 was analysed 25 by the Bioengineering Lab. Co., Ltd. The microbial phase of uninoculated AQ-14 and the precipitate collected by centrifuging the liquid culture solution were also analysed.
Nitrogen mineralization in each carrier. To immobilize microorganisms on the carriers 26 , we packed a weight equivalent to 100 mL of carrier in plastic tubes and added 1 g of bark compost as inoculum. The carriers were washed with 100 mL of distilled water, 0.1 g of sh fertilizer was added, and the tubes were incubated for 24 h in the dark at 25 °C. The next day, the carriers were rinsed with 100 mL of distilled water and the leachate was tested for nitrate ions. The addition of sh fertilizer, incubation, and washing were repeated until nitrate ions were detected in the leachate. Concentrations of inorganic N, ammonium, nitrite, and nitrate were measured.
Addition of organic substances and generation of inorganic N. We examined rates of N mineralization and e ciencies of conversion of organic N to inorganic N in the presence and absence of immobilized microbes. We packed 100 mL of rockwool into tubes and added 1 g of bark compost as inoculum. The rockwool was washed with 100 mL of distilled water, 0.1 g of sh fertilizer was added, and the tubes were incubated for 24 h in the dark at 25 °C. The next day, the rockwool was rinsed with 100 mL of distilled water, and inorganic N in the leachate was measured. The addition of sh fertilizer, incubation, and washing was repeated for 3 weeks. Then we added 0.1 g (6 mg N), 0.2 g (12 mg N), 0.5 g (30 mg N), or 1 g (60 mg N) of sh fertilizer per tube and incubated the tubes for 24 h in the dark at 25 °C. The next day, they were rinsed with 100 mL of distilled water and the above amounts of sh fertilizer were again added. The addition of sh fertilizer, incubation, and washing were repeated for over 30 days. We measured inorganic N, ammonium, nitrite, and nitrate in the leachates.
Optimal conditions for N mineralization. To compare conditions, new tubes packed with 100 mL of rockwool with nitri cation ability were prepared by repeating the addition of 0.1 g of sh fertilizer, incubation at 25 °C overnight, and washing with 100 mL water for 2 to 3 weeks.
To compare the effects of different organic substances, we added sh fertilizer (6% N), corn steep liquor (CSL, OAT Agrio, Tokyo, Japan; 3.3% N), or rapeseed oil cake (Sun and Hope, Kitakyushu, Japan; 6% N) as sources of organic N in amounts equivalent to 6 mg N per tube and incubated the tubes for 24 h in the dark at 25 °C. The next day, the tubes were rinsed with 100 mL of distilled water and the above amounts of organic materials were again added. The addition of organic substances, incubation, and washing were repeated every day for 3 weeks. We measured inorganic N, ammonium, nitrite, and nitrate in the leachates in triplicate.
To determine the optimal incubation temperature, tubes with nitri cation ability were incubated at 15, 20, 25, 30, 37, 42, or 45 °C and washed with 100 mL of water, and the addition of 0.1 g of sh fertilizer, overnight incubation, and washing was repeated for 2 weeks. We measured inorganic N, ammonium, nitrite, and nitrate in the leachates in triplicate.
Test plants. On inoculated rockwool we sowed 5 seeds of komatsuna (Brassica rapa var. perviridis, ATU121), added 0.1 g of sh fertilizer to each carrier, incubated the carriers in chamber MLR-352 (Panasonic) at 25 °C in 12-h light/dark, and then added 100 mL of water the next day. The addition of sh fertilizer, incubation, and washing was repeated for 11 days. We also grew lettuce (Lactuca sativa var. capitata), saradana (L. sativa, Melbourne MT), radish (Raphanus sativus var. sativus, Akamaru Hatsuka Daikon), and turnip (Brassica rapa var. glabra, Shogoin Kabu) on rockwool and nursery soil (Naeichiban; Sumirin Agro-Products, Aichi, Japan) in a greenhouse at Tsu, Mie Prefecture, from September to December, 2013. During these experiments, light and temperature were not controlled.

Results
Nitrogen mineralization on inoculated carriers. The chemical properties of leachates from rockwool differed signi cantly between inoculated and uninoculated materials (Fig. 1). The generation of nitrate was stable for 2 weeks after the start of generation (Figs. S2, S3 online). By 30 days, 1.7 mg of K and 1.4 mg of phosphate were generated (Fig. S4).
Chemical, physical, and microbiological analyses. Oyster shell lime, rice husk charcoal, and rockwool were effective at producing inorganic nutrients and large amounts of nitrate (Fig. 2). Oyster shell lime achieved an e ciency of 69% in net N mineralization. However, phosphate couldn't be detected. Physical properties of carriers are shown in Table S1.
Optimum amount of organic substance to add and generation of inorganic N. The generation of inorganic N increased as the amount of organic N applied increased, but the quantity of nitrate and the e ciency of conversion of organic N to inorganic N decreased (Fig. S5 online).
Optimal conditions for N mineralization. Fish fertilizer was better mineralized than CSL and rapeseed oil cake (Fig. 3). The generation of nitrate in rockwool was in uenced by temperature (Fig. S6 online). The production of inorganic N was low at 15 and 20 °C, and very little nitrite and nitrate were produced at 15 °C. The production of nitrate was high at 25-42 °C but poor at 45 °C. No nitrate was generated at 20% RH, and the production of inorganic N was much lower at 20% than at 51% and 92% RH (Fig. S7 online).
Plant growth. Komatsuna seedlings grew well in inoculated rockwool but not in uninoculated rockwool (Fig. 4). Lettuce, saradana, radish, and turnip grew as well in inoculated carrier as in nursery soil (Fig. S9  online). These results suggest that the inoculated carriers acquired the microbial function of degrading organic N into nitrate suitable for plants.

Discussion
Non-soil carriers usually do not allow organic substances to be used as fertilizer (Fig. 4). Rather, the presence of organic substances suppresses nitrifying bacteria 15,27,29 (Fig. S4 online), perhaps because heterotrophic microorganisms cannot completely degrade the substances that inactivate nitrifying bacteria. If we want to grow crops with organic fertilizer in non-soil media, materials capable of degrading organic N to nitrate are needed. Here we achieved nitri cation in non-soil media by immobilization of microbes.
Nitrate production stabilized by 3 weeks of incubation (Fig. S2 online). The nitri cation ability of inoculated carriers was high at 25-42 °C (Fig. S6 online). In natural soils, the conversion of organic N to inorganic N increases from 5 °C to 35 °C [29][30][31] and is highest at 35 °C 32 . Our results indicate that the N mineralization ability of the inoculated carriers is similar to that of soils. The amount of inorganic N is in uenced more by soil temperature than by soil humidity 33 , but our results show that the N mineralization ability decreased in drier air. It will be necessary to investigate how the temperature and moisture content of carriers affect N mineralization. The NA medium that we used to determine bacterial density is frequently used to culture common soil microorganisms (Fig. S10 online) 34 . However, our assay probably excluded nitrifying bacteria, the growth of which is signi cantly inhibited on media containing organic substances 28 . We tested arti cial resins (polyurethane), mineral-derived materials (vermiculite and rockwool), biological materials (oyster shell lime and charcoal), and natural materials (coconut husk). Even on these non-soil carriers, it proved possible to induce the same inorganic nutrientproducing ability as in natural soil. This shows the possibility of creating arti cial soil from materials with intentionally designed pore size, particle size, water retention, and ion exchange capacity. The oyster shell lime produced a high amount of inorganic N (Fig. 2), but the e uent did not contain phosphate (data not shown). Since the main component of the oyster shell is calcium carbonate, it is possible that phosphate was immobilized.
We used bark compost as the inoculum, but many bark composts are likely to be free of nitrifying bacteria on account of being composted on concrete. We con rmed that the bark compost we used was aged on soil. We recommend that future studies use soil from organic farms, where nitrifying bacteria are sure to live.
An inoculated carrier could be used to produced inorganic fertilizers from organic substances. Adding an organic substance such as sh fertilizer to an inoculated carrier and ushing it with water the next day could allow the inorganic nutrient solution to be collected daily. Inorganic fertilizers, in general, are chemically synthesized from minerals or air through the use of large amounts of energy. Our technique offers a new method for producing inorganic fertilizers with little energy input.
Bacillus was the most abundant genus in the inoculated carrier (42.1%), uninoculated material (39.6%), and culture solution (77.5%). Among plant growth-promoting rhizobacteria, Bacillus is one of the most extensively studied genera and promotes plant growth and development 34 . Out test plants did not grow on uninoculated material (Fig. 4), suggesting that they cannot grow without nitrifying bacteria, even in the presence of Bacillus.
In summary, this study showed the ability of inoculated carriers to mineralize organic N to nitrate and their potential to replace natural soils. Technology to create arti cial soils will help clarify the ideal physical and chemical properties of soils and contribute to increased food production.