Method for Rapid Labeling of Waste Sludge from a Food Factory with 15N-Glycine and Evaluation of N Use Using Komatsuna (Brassica rapa Var. perviridis)

The waste sludge from food factories has rich nutrients and useful material for fertilizer or animal feed, but quick treatments and recycling of the waste sludge are difficult due to its higher water content. We have developed a rapid composting system to make sludge fertilizer using mix of waste sludge and shredded newspaper (Sludge Fertilizer Made by Paper Mixing Method, SF-PMM). The mixture was incubated in a box reactor, continuously aerated with warm air around 35°C, and changed to mature SF-PMM, in only two weeks. To search movement of N from the SF-PMM to plants, we developed a new method to label small amounts of SF-PMM with 15N-glycine. 50 L of wastewater from a food factory was incubated with 1 L of active sludge and 3 g of 15N-glycine (98 atom% 15N), and 175 g of labeled sludge was obtained in a day. This sludge was mixed with 25 g of newspaper chips, packed between two steel meshes, and placed at 20 cm depth in the reactor composting 200 kg of unlabeled sludge-paper mixture. Composting was restarted, and after about 7 days of reaction, 15N-labeled SF-PMM 7.03 atom% 15N was obtained. The surrounding unlabeled compost contained 4.0, 4.0, and 0.8% of N, P2O5, and K2O, respectively. C/N and pH were 10 and 7.4, respectively. Komatsuna (Brassica rapa var. perviridis) was cultivated in a pot with 50 and 100 mg N of SF-PMM, and healthy plants were obtained as in the Control experiments containing 50 mg N ammonium sulfate. No growth inhibition was observed in these experiments. Even in 100 mg SF-PMM, excellent growth of the roots was observed. About 56% of the N in the plant was shown to come from 15N-SF-PMM, and about 6% of the total15N in the 15N-SF-PMM was also shown to be incorporated into the plant.


Introduction
In 2018, in Japan, 376 million tons (Mt) of industrial waste was disposed of, and in almost recent 20 years, 1996 to 2018, about 380∼430 Mt of industrial waste has been disposed of every year [1,2]. ese industrial wastes contain around 45% of sludge containing nonorganic materials and various heavy metals, so that the reduction of the disposition of the sludge is important issue for the sustainable industries.
On the other hand, in 2018, 8.8 Mt of the wastes was disposed of by the food industrial activity in Japan, along with 6.5 Mt of the waste sludge corresponding to 75% (w/w) of the total weight of the waste materials from the food industries [1]. e sludge from the food industry is generally not contaminated with dangerous materials, such as heavy metals and chemicals. erefore, the recycle of the sludge is very important issue for the development of the food industry. Waste sludge from food factories is rich in nutrition and contains organic materials and less amounts of dangerous contaminations, but the waste sludge in the food industries generally contains 80∼90% water [3]. Due to the higher water content, to use the waste sludge for animal feed or organic fertilizer is not easy. Waste sludge has been used as a combustion improver or an additive to fertilizer because principally landfill and incineration of sludge were prohibited in 1992 by the law, and the law for the advancement of recycles society was enacted in 2000.
Active sludge is used to clarify contaminated wastewater in food factories, as an eco-friendly and efficient method to treat a large volume of wastewater. Active sludge contains various microorganisms which uptake various compounds from wastewater for their growth and clarify the wastewater [4]. ese microorganisms eventually die and form sediments with the addition of coagulants (e.g., poly-aluminum).
e precipitate containing cadavers of microorganisms, along with some living organisms, is removed from the active sludge system as waste sludge. As mentioned above, the sludge is relatively free from harmful contamination. erefore, the dried active sludge is accepted as normal ordinary fertilizer by convention of the Japanese Government if the dried materials contained more than 4% N, 1% P, and 1% K. Besides, the N, P, and K contents of the sludge from various food factories in Japan are relatively consistent at about 7, 4, and 0.8% (based on dry weight), respectively, irrespective of the kinds of food factories [5].
Because of the higher water content in waste sludge from food factories, the recycling rate is low; in addition, sludge burning in open fields is prohibited as mentioned above. To improve the recycling rate of waste sludge, we developed a rapid and easy method to convert waste sludge from the bean curd, confectionery, and fish meat factories into wellmatured fertilizer during only two weeks [6,7].
About 200 kg of waste sludge from food factories was mixed with newspaper chips through a shredder to reduce the water content to a certain level suitable for composting reaction. e mixture was placed in a 400 L reactor with continuous aeration at 35°C from the bottom of the reactor. A few days after the beginning of aeration, the composting reaction began, and the temperature throughout the reactor increased to 70°C in a few more days. After two weeks of composting reaction, maturation of the sludge fertilizer was completed. e composting period is very short compared with an ordinary composting method piling it in the open air. Our method of mixing waste sludge with paper chips was termed the paper mixing method (PMM); therefore, the resulting well-matured sludge fertilizer (SF) is termed SF-PMM [8,9]. e SF-PMM is produced from inexpensive small-scale equipment with low energy consumption; therefore, the system can be installed to produce SF-PMM in various small food factories. e waste management in food factories has become an important issue to establish zero emission of food industries in the world including developing countries; therefore, this new composting system may contribute to waste management in various food factories.
We have reported the effects after the application of SF-PMM to various plants and plant parts, for instance, root elongation [6,8,10], expansion of flowering period (unpublished data), and increase of flower bud formation of viola and marigold (unpublished data). However, the contribution of SF-PMM to vegetable growth as an N source has not been reported. erefore, we estimated the effects of the SF-PMM on the growth of komatsuna.
Using fertilizer labeled with 15 N is a reliable method for the evaluation of N use from fertilizer in soil to plants. Previous studies used 15 N-labeled manure [11] and dried active sewage sludge [12]. In the former, 15 N-labeled compost was prepared using a slow composting process from cow manure, and the authors clarified that tomato plant and rice plant adsorbed about 5.9% of the whole N from the cow manure [11]. In the latter, by using sewage sludge and 15 N-labeled ammonium chloride ( 15 N-NH 4 Cl), the efficiency of the uptake of N from the sewage sludge and the chemical fertilizer was determined as 20 and 12%, respectively. e relative efficiency of the sludge fertilizer to the chemical fertilizer was about 60% [12]. e 15 N-labeled urea was also used to investigate the remaining soil N content after crop harvesting [13]. 15 Nlabeled ammonium sulfate, (NH 4 ) 2 SO 4 , was used to label waste sludge from a food factory [14].
In this study, we report a new method developed to prepare a small amount of 15 N-SF-PMM. A small volume of 15 N-waste sludge (WS)-PMM was placed in a 400 L reactor performing the composting reaction to make SF-PMM, and after two weeks, labeled 15 N-SF-PMM was obtained, which was enough to determine the utilization of N from 15 N-SF-PMM by plants, komatsuna in a pot experiment. We expect that the results in this study might support the adoption of the PMM for waste sludge management in various small food factories.
In this report, we described the method for the production of SF-PMM and the easy and speedy way to label the SF-PMM with 15 N-glycine. We also reported briefly the evidence of the uptake of 15 N from 15 N-SF-PMM into komatsuna.

Materials and Methods
We and the owner of the bean curd factory, located next to Niigata University, collaborated to recycle and reuse the sludge wasted from the factory. We equipped reactors in the factory producing 100 kg of waste sludge in wintertime and 200 kg in summertime every day. 15 N-Glycine. e 50 L of wastewater of the bean curd factory and 1 L of active sludge from the same factory were mixed by aeration at a 25 L·min −1 from the bottom of the cylindrical plastic tank (62 cmϕ × 72 cm high) at 25°C for 2∼5 days (Figure 1). At the start of aeration, 3 g of 15 N-glycine (98 atom% 15 N; Mass Trace Inc., Woburn, MA, USA) was added.

Preparation of Active Sludge Labeled with
During the 5 days of aeration, 1 L of aliquots of wastewater was frequently collected and allowed to stand for 1 h for floc sedimentation. In the first 24 h of incubation, samples were taken in 3 h intervals; in the next 48 h, they were taken in 12 h intervals; and in the final period of 48∼120 h, the sampling was done every 24 h. Various parameters such as pH and concentrations of amino acid, protein, and NH 4 + in the supernatant were measured. When agitation was ceased, 40 mL of 0.2% poly-aluminum chloride, [Al 2 (OH) n Cl 6-n ] m , was added to the suspension in the reactor and allowed to stand for 1 h to precipitate solid materials. e supernatant was discarded with gentle suction using a water pump, and the precipitate was finally recovered by centrifugation at 1,500 ×g for 10 min. e precipitate was filtered through filter paper on a Buchner funnel with suction until the water content of the precipitate reached around 85%. e water content was measured with a water content meter (FD-600; Kett Electric Laboratory, Tokyo, Japan), and 175 g of precipitate was obtained. e addition of the coagulant polyaluminum chloride did not increase the aluminum concentration in the SF-PMM (data not shown). 15  e nurse reactor was prepared according to the method in [6,8], and the air was blown from the bottom of the reactor at 40∼50 L·min −1 with an inverter-controlled blower (FVR-C11, Fuji Electric, Tokyo). e waste sludge, 170 kg, from the bean curd factory was mixed with 30 kg of shredded newspaper (WS-PM) and placed in a box reactor (60 cm l × 60 cm b × 120 cm h) (Figure 2). e reactor was continuously aerated with warm air (around 35°C) through the bottom of the reactor using a blower. e temperature was monitored with sensors in the center of the reactor at 10, 40, and 70 cm below the surface of the WS-PM, and temperature data were collected hourly for 20 days with a data logger. e nurse reactor had been started 2∼4 days before the insertion of the 15 N-WS-PM. e 15 N-WS-PM was sandwiched between two stainless steel mesh sheets (30 cm × 30 cm, 5 mm mesh) and embedded horizontally 10 cm below the surface in the nurse reactor warmed up beforehand, and aeration was restarted (see Section 2.3) (Figure 2). e timing of the embedding of the 15 N-WS-PM was flexible by a few days because the water contents and temperature of the waste sludge in the nurse reactor differed in each occasion. e WS-PM was composted for around 12 days, dried at 60°C overnight, and subjected to nutrient analysis. e N, P (as P 2 O 5 ), and K (K 2 O) contents were 4.0, 4.0, and 0.8%, respectively, based on dry weight; the C/N ratio was about 10; and the pH was about 7.4. ese values were repeatedly obtained with this reactor [6,8]. , because such a small amount of waste sludge could not spontaneously begin the composting reaction, even after mixing with shredded newspaper. e stainless mesh sheet containing 15 N-WS-PM was placed in the nurse reactor on day 3 or 4 after the starting of aeration when the temperature was increased to 40∼50°C, and aeration was restarted after the setting (see Section 2.2). e timing to set the mesh sheet differed a little bit from one batch to another. Figure 3 presents typical temperature fluctuations of WS-PM in the 400 L reactor. e timing to reach the highest temperature and duration of the plateau varied by a few days. Such differences were expected, because of the variations in various parameters in the WS-PM. Regardless of these differences, Figure 3 showed temperature fluctuations of 35 to 70 to 40°C over the course of two weeks; the highest temperature lasted for 2-3 days.  e diluted wastewater was adjusted to 1 L with the same buffer, and the dilution degree was factored into all subsequent calculations. Five aliquots of 170 mL suspension from the 1 L of diluted wastewater were transferred to five 170 mL screw-cap test tubes, and they were capped tightly after the removal of air bubbles. One 170 mL tube was used immediately for the measurement of dissolved oxygen as time zero, and the rest were kept in an incubator for 5 days at 20°C. Biological oxygen demand (BOD) was calculated as follows:

Preparation of
where DO 1 is the concentration of dissolved oxygen at time 0 and DO 5 is that at after 5 days of storage.
(2) Amino Acid and Protein Concentrations in the Wastewater. e wastewater was centrifuged (1,500 ×g, 10 min), and 10% (v/v, final concentration) trichloroacetic acid (TCA) was added to the recovered supernatant. To precipitate denatured proteins, the sample was centrifuged for 20 min at 12,500 ×g. To remove TCA, two volumes of ethyl ether were mixed vigorously in the supernatant. e ethyl ether washing was repeated two additional times, and the amino acid concentration was determined with the ninhydrin method [16] using a calibration curve of L-leucine.
Floc was removed by sedimentation (see Section 2.1), and the protein concentration in the supernatant was determined with the Bradford method [17] using bovine c-globulin as the standard.

(4) Weight of Dry Solid Materials in the Wastewater.
During the incubation of the wastewater in the tank, 1 L aliquot was taken, and solid materials were collected by centrifugation (1,500 ×g, 10 min) and dried for 24 h at 105°C. e weight was measured with an electric balance.

(5) Water Content. e water contents in various fractions
were measured with a water content meter (FD-600; Kett Electric Laboratory, Tokyo, Japan). 15 N Abundance. After completing the composting reaction, the SF-PMM was dried, and 50 mg of e changes of temperature in the reactor during composting of WS-PM. e waste sludge from a bean curd factory was mixed with newspaper chips as described in Figure 2, and the temperature of the reactor was monitored as described in Sections 2.2 and 2.3. On day 7, indicated with a broken line arrow, the aeration was ceased, and the WS-PM in the reactor was collected and replaced again after the thorough mixing of the sludge to check the subsequent 2 nd composting reaction. After setting the remixed WS-PM, aeration was started with the same condition as those in the first aeration. e broken line ---, the solid line -, and dotted line ···· represent fluctuation of the temperatures at depth of 10 cm, 40 cm, and 70 cm, respectively, from the surface of the mixture in the reactor. pulverized sample was hydrolyzed with 1 mL of concentrated sulfuric acid according to the Kjeldahl method [18]. e C/N ratio was determined with a C/N recorder (MT700 Mark II; Yanaco Co. Ltd., Kyoto, Japan). Phosphate was determined with the ammonium vanadomolybdate absorptiometry [19] and expressed as P 2 O 5 . K was measured with flame photometry using an atomic absorption photometer (Z-6100; Hitachi, Tokyo, Japan) and expressed as K 2 O.

Determination of the C/N Ratio, Phosphate and K Concentrations, and
An aliquot of hydrolyzate in the Kjeldahl method [18] was mixed with an equal volume of 1 N NaOH and adjusted to 300 μL with water. 200 μL of phenyl nitroprusside solution (0.64 M C 6 H 5 OH, 0.76 mM Na 2 [Fe(CN) 5 NO] in 18 mM Na 3 PO 4 , 12 mM Na 3 C 6 H 5 O 7 , and 8.1 mM EDTA) and 300 μL of alkaline sodium hypochlorite (25 mL NaClO in 1 L 0.4 M NaOH) were added and reacted for 45 min. e N concentration was measured calorimetrically at 635 nm with a calibration curve based on ammonium sulfate (NH 4 ) 2 SO 4 . e 15 N concentrations in the compost and plant materials were determined by an emission spectrometry with a 15 N analyzer (NIA1; Jasco Inc.) [20].

Measurement of the Uptake of the SF-PMM and Evaluation of N Use from 15 N-Labeled Compost in
Komatsuna. SF-PMM or 15 N-SF-PMM was dried and finely powdered with a vibration mill (TI-100; Irie-Shokai, Tokyo, Japan) and mixed well with sand dune soil from Ikarashi, Niigata, Japan. e texture of the soil was analyzed as follows: loamy sand, pH (H 2 O): 6.75; cation exchange capacity: 4.60 cmolc·kg −1 ; total N: 0.03%; available N: 20.4 mg·kg −1 ; available P 2 O 5 : 72.8 mg·kg −1 . e available N is low and good to estimate the N movement from exogenous fertilizer to plant. We assumed that the application of fine powder fertilizer is more effective than coarse particle or flake formulations; future experiments are required to evaluate this assumption and optimize the formulation. Each pot in the treatment, 590 g of the sandy soil was used (see Section 2.4.4).
In the preexaminations, the culture system with the soil amended with (NH 4 ) 2 SO 4 -50 mg N supported the growth of komatsuna for more than 5 weeks without any abnormal inhibitory effects. According to these observations, we designed experiments to estimate the fertility of the SF-PMM for 5-week culture as follows: Control: (NH 4 ) 2 SO 4 50 mg N; experiments: SF-PMM-50 mg N and SF-PMM-100 mg N·kg −1 soil designed to estimate the plant growth.
In these treatments, calcium dihydrogen phosphate (calcium phosphate, monobasic) (CaH 4 O 8 P 2 ) and potassium chloride (KCl) were also added to make up 50 or 100 mg of P and K kg −1 soil if they were less than 50 or 100 mg, respectively. In the Control treatment, no exogenous nutrient was used.
e effects of these three treatments in supporting the growth of komatsuna were estimated with increase of the weight in the shoot (above ground part) of the plants.

Culture of the Plants.
In the culture of the plants for the three treatments, six seeds of komatsuna were sown in one pot (10,000 −1 a, Neubauer pot), and the pots were incubated for 5 weeks, in the longest case, in a climate chamber under a 12 h photoperiod with 130 μmol·m −2 ·s −1 photons. In the measurements of the weight of shoots, the 4 plants were used to make an average and statistical analyses.
At 5 weeks after the sowing, 4 plants in each treatment were harvested randomly from 4 pots. e total four plants were used to calculate the average in each treatment. ese averages of each weight coming from four plants weights were statistically analyzed using group comparison analyses. e water content in the soil was maintained at 50∼60% of the field capacity with deionized water throughout the experiment.
e percentage of N derived from 15 N-labeled compost ( 15 N%) in the plants was calculated using the following equation [21], where 15 N atom% excess is 15 N atom% minus natural abundance of 0.37 atom%. 15 N% � 15 N atom% excess in komatsuna 15 N atom% excess in compost × 100.
e N content derived from 15 N-compost in the plant was calculated as 15 N% in komatsuna × total N content.

Statistical Analyses.
e data of the weight of four plants in each treatment described in Sections 2.4.3 and 2.4.4 were analyzed with Tukey-Kramer multiple comparison procedure in P < 0.05 [22]. To determine P value, Student's t-test was also done, but data was not shown.

Changes of Various Parameters in the Wastewater during Incubation with Active Sludge.
e BOD was 900 mg·L −1 at the start of the incubation and drastically decreased to 200 mg·L −1 in the first 24 h. e weight of floc increased slightly to 0.5 g·L −1 after a 12 h lag in the first 24 h of incubation and decreased to 0.4 g·L −1 during further 5 days of incubation (Figure 4(a)). We assumed that an increase and subsequent decrease in protein concentration would follow a decrease and subsequent increase of amino acid concentration and that these trends would mirror one another. is assumption was verified, as shown by the changes in protein and amino acid concentration over time in Figures 4(a) and 4(b). Because it is easier to measure amino acid concentration than to measure protein concentrations, we used the fluctuations in amino acid concentration to determine the optimal time to stop the incubation. e NH 4 + concentration was low, around 1 mg·L −1 , and steady in the first 24 h but increased dramatically to 9 mg·L −1 in the subsequent 24 h (Figure 4(c)). Importantly, increase of the NH 4 + was similar in pattern to that of amino acid as shown in Figure 4(b).

Effects of the Addition of Glycine to the Mixture of
Wastewater and Active Sludge. We assessed whether the addition of 3 g of glycine to 50 L of wastewater altered the fluctuation trends in the parameters shown in Figure 4. e International Journal of Agronomy trends obtained in the experiment with glycine ( Figure 5) were similar to those without glycine (Figure 4). Of particular importance, the inverse trends between the amino acid and protein concentrations and the sharp increase in NH 4 ( Figure 5) were essentially the same as those without glycine and were reproducible in three experiments (data not shown). Interestingly, the minimum amino acid concentration was observed 12 h earlier in the experiment with glycine than that without glycine (Figures 4(b) and 5(a)). However, the time required to reach the maximum or minimum values of each parameter differed among the replicates (data not shown). e amino acid concentration at the start of aeration ( Figure 5(a)) was 90 mg·L −1 in the experiments with exogenous glycine and decreased to about 40 mg·L −1 during the initial 12 h of incubation. erefore, a large amount of added glycine, 2.5 g, was estimated to be incorporated into the microorganisms. is amount of incorporated amino acid was about 4 times higher than the value of about 13 mg·L −1 estimated from the results presented in Figure 4(b), in which the lowest concentration reached at 24 h after the start of amino acid incorporation.
is suggested that the addition of glycine significantly enhanced the proliferation of microorganisms during the initial 12 h of incubation, and that autolysis of microorganisms and protein degradation occurred 12 h earlier in the presence of glycine than in its absence. ese results suggested that fluctuations in amino acid concentration can be used to monitor the appropriate timing to obtain a higher yield of 15 N-labeled sludge.

Incorporation of 15 N-Labeled Glycine into
Microorganisms. In the sludge labeling with 15 N-glycine, along with the expected fluctuations in amino acid and protein concentrations, the NH 4 + concentration increased slightly during 18 and 21 h of incubation (data not shown). As described in Section 3.2, after adding 15 N-glycine in the mixture of the wastewater and the active sludge, the amino acid content rapidly decreased in the initial 12 h. At 18 h after the addition of 15 N-labeled glycine, the rapid decrease in amino acid ceased (data not shown). e incubation was terminated at 24 h after the addition of 15 N-glycine, and 175 g of labeled sludge containing 85% water was recovered. Using emission spectrometry, the resulting precipitate was evaluated to have a 15 N content of 10.2 ± 0.03 atom% excess (n = 3), and approximately 0.9 g of 15 N-glycine was suggested to be incorporated into the sludge. Consequently, about 30% of the added 15 N-glycine appeared to be assimilated into microorganisms, suggestive of high incorporation efficiency. e total N content of the waste sludge was about 6% and the same as the value of the sludge routinely produced in this factory (data not shown).

Changes in the Reactor Temperature during the Composting of Waste Sludge-Paper Mixture, WS-PM.
e changes of temperature in the WS-PM during composting in the reactor were similar to those reported previously [6] and reached the highest temperature, 60∼70°C, on day 5 after the starting of aeration (Figure 3). e maximum temperature, around 70°C, was recorded for 2 days (on days 4∼5) at 10, 40, and 70 cm below the surface of the mixture, and then the temperature acutely decreased to around 30°C during the subsequent 2 days at all sensing points. Although a Although not all parameters were always tested, the experiment was repeated several times as described in Figure 1. For the details about the method to determine the various parameters, see Section 2.4.2.
difference of a few days was often observed, the fluctuation of the temperature was observed with very high fidelity in many experiments.
At time indicated with arrow with a dotted line in Figure 3, the sludge in the reactor was remixed and a second composting reaction was attempted, but almost no second composting reaction was observed. ese observations indicated that the reactor could be used as a nurse reactor for the labeled fertilizer in terms of high temperature and short period reactions. erefore, 15 N-WS-PM was placed at the center of the nurse reactor on day 3 or 4 after starting aeration (see Section 2.3).

Composting of 15 N-SF-PM.
We obtained about 175 g of 15 N-labeled waste sludge from the tank and composted it in the nurse reactor as described in Section 2.2. e temperature of the nurse reactor was measured as shown in Section 2.3, and a similar temperature fluctuation to that in Figure 3 was observed at the three sensing ports (data not shown). Finally, 300 g of 15 N-SF-PMM was obtained. Total N content was 6.06%, and 7.03 atom% of 15 N abundance was shown with the Kjeldahl method and the 15 N analyzer, respectively. Of note, although glycine seemed to enhance the proliferation of the microorganisms (see Section 3.2), almost the same amount of sludge (floc) was produced with or without glycine, i.e., at 4 h and 48 h of incubation, about 180 and 250 g flocs, respectively (data not shown).

Effects of the SF-PMM on the Growth of Komatsuna.
e effects of the SF-PMM on the growth of komatsuna were estimated in comparison with the treatment of Control of (NH 4 ) 2 SO 4 -50 mg N. All data were statistically analyzed.
Komatsuna was cultivated in the SF-PMM-50 mg N and -100 mg N treatments for 5 weeks (Table 1). e weights of the shoots in SF-PMM-50 and -100 treatments at 5-week were 3.136 ± 0.326 and 4.299 ± 0.445 g, respectively. Compared to the weight in Control, the shoot weights in SF-PMM-50 and -100 treatments were 2.2 and 3.1 times heavier. ese values of the weights in the three treatments were statistically significant with Tukey-Kramer analysis [22]. e photographs of the plants cultured in 5-week experiments are shown in Figure 6. e three plants were harvested at 5 weeks in the treatments of Control (Figure 6(a)), SF-PMM-50 ( Figure 6(b)), and SF-PMM-100 ( Figure 6(c)), respectively. e plants in SF-PMM-50 ( Figure 6(b)) and SF-PMM-100 ( Figure 6(c)) treatments looked very healthy and showed luxuriant growth in roots. Compared to these two photos, the plant in Control was slightly small, but it was average feature.

3.7.
e Movement of the 15 Figure 7). ese data suggested that our labeling method and nurse composting system offered useful tools for analyzing N movement in plants and soil and could be adopted in future fields experiments. However, it will be necessary to increase the amount of the N released from the SF-PMM.

e Composting Sludge from Food Factory Wastewater.
e composting of the waste sludge from food factories to well-matured fertilizer usually takes long time, due to its  Figure 5: Effects of addition of glycine on the change of various parameters in the wastewater during incubation with the active sludge in the plastic tank. 50 L of the wastewater was treated as described in Section 2.1, and 3 g of glycine was added at the start of the incubation. (a) Concentrations of protein ( ) and amino acid ( ) were measured according to the description in Section 2.4.2. (b)NH 4 + concentration ( ) and pH ( ) were measured according to the methods described in Section 2.4.2. e experiment using cold glycine was done three times as mentioned in Figure 1, and the experiment of the addition of 15 N-glycine was done one time. International Journal of Agronomy higher water contents [3]. Besides, the higher water content in the waste sludge interferes with the start of composting reaction. However, it is not difficult to start the composting reaction using sawdust or rice husk mixing method [3,6,23]. In these treatments, the temperature often reached around 70°C, but it was very difficult to convey these high temperatures all over the reactor [23], because these subsidiary materials, sawdust and rice husk, are too rigid to make "flexible thin air streams" all over the reactor. ese subsidiary materials are also not fluffy to keep sufficient air in the mixture of sludge having large volume water which is necessary for the growth of microorganisms. erefore, these materials cannot make a long random air stream in the mixture [23].
On the other hand, thin paper chips, such as shredded newspaper, were a good subsidiary material to get stable starting of a composting reaction even in the mixture with 75% water. In this paper, with the chips mixing method, it was possible to convey the high temperature all over the reactor [7]. Generally, in the waste sludge-paper mixing method, WS-PMM, after slow increase of the temperature next to the lag time, the temperature was going up steeply regardless of the length of lag time (Figure 3). e highest temperature in the reactor of WS-PMM was found throughout the reactor, but as mentioned above, in the ordinary composting reactions, such high temperature could be detected only in the surface area of the reactor (unpublished data).
It is important to attribute the reason for the high fidelity to the rapid and stable increase of such high temperature in the WS-PMM for the stable sludge recycle. Here, we point out a few theories. Fujita indicated the importance of a porous structure in the substrate in the reactor [23]. He insisted that the porous structure in the sludge could make longer random air streaming, and through this stream, aerobic atmosphere could be spread all over the reactor. e PMM reduced the water contents to around 75% from around 90%; under this water content, the air pumping urged the composting reaction voluntarily in the whole area of the reactor; and shortly 70°C temperature was recorded ( Figure 3) [6,8]. Several paper chips in a large amount of the chips added make a tiny compound composed of a small volume of the sludge and paper chips in the first mixing step. In other words, the SW-PMM in the reactor is the gigantic Chem-50 mass of aggregates of those particles made by several paper chips and a tiny sludge particle. Inside the aggregate, an infinite flexible random stream exists like tiny capillary vessel. en, pumped air can move along the stream randomly all over the reactor, and homogeneous aerobic conditions in the reactor could be maintained during the whole process of the composting reaction [23]. "We think this is a porous structure," Fujita said.
Porous structure guarantees even distribution of heat, water, and air which are necessary for the proliferation of the microorganisms in a reactor. Bacteria can catabolize the nutrition in the all particles, and this guarantees the homogeneous characteristics in the mass. To obtain highquality sludge fertilizer, the composting reaction must reach and keep around 70°C for at least 2∼3 days in the whole area of the reactor [3]. During these periods, various thermophilic bacteria digest the sludge and secrete various metabolites including unknown molecules. Future work is required to analyze precisely the mechanisms of SF-PMM and determine the utility of sludge fertilizer in agricultural applications. Undoubtedly, the PMM will be applied to other organic materials such as livestock feces.

Changes of Various Parameters in the Wastewater during
Incubation with Active Sludge. We obtained 175 g of labeled sludge with 10.2 ± 0.03 15 N atom% excess and with 85% water content from the mixture of 50 L wastewater, active sludge, and 15 N-glycine. Around 0.9 g of 15 N-glycine was incorporated into the sludge, so about 30% of the added 15 N-glycine was calculated to be assimilated into microorganisms, suggestive of high incorporation efficiency. In the labeling experiments, we noticed that a rapid increase of NH 4 + occurred almost concurrently with the decrease of proteins (Figure 4). e monitoring of NH 4 + is very easy and accurate, so improvement of the incubation method to get higher yield of the labeled materials will be possible by monitoring NH 4 + . Microorganisms in the sludge are essentially steady even under frequently changing conditions in the water clarifying system. is hypothesized theory is supported by the fact that the relatively stable N, P, and K contents were observed among the food factory waste sludge, FF-WS [5]. at said, it is slightly difficult to expect that the wastewater from such different factories would have stable pH, BOD, COD, ingredient concentrations, organic molecules, etc. In either case, it is very important future work to determine quantitatively and qualitatively what compounds are labeled with 15 N in our labeling system. To determine those molecules will bring epoch in the research about the N movement among soils, fertilizers, and plants.

Development of a Nursing Reactor to Compost Small Amounts of 15 N-WS-PM.
A small volume of reactor cannot make a matured small volume of compost, because the heat rapidly dissipates and it is difficult to maintain an appropriate high temperature without an addition of heat [23]. In our reactor, the temperature around 60°C was maintained for around 4 days (Figure 1), and this was thought to fit a condition of nurse reactor for the small sized materials. Using our tank system shown in Figure 1, about 30% (w/w) of the 15 N-glycine was incorporated into microorganisms. In the subsequent nursing system, 15 N-labeled SF-PMM resulted from 15 N-labeled WS-PMM, and the contents of the label were estimated as 7.03 atom% 15 N. us, during the composting in the nursing reactor, about 3% of the 15 N was lost (see Sections 3.3 and 3.5). It is important to reduce these losses during the nursing to get highly labeled SF-PMM.
Takahashi et al. [11] estimated the 15 N content in the manure compost as 1.81 atom%. Meanwhile, Sǿrensen [24] evaluated the N mineralization in the sheep manure compost using 15 N-labeled compost with 15 N of 3.70 atom% and found that 12% of N was mineralized in 7 days. Sǿrensen and Amato [25] searched 15 N movement using 15 N-labeled pig manure with 15 N of 3.07 atom%. Compared to these reports, content of the label in the 15 N-SF-PMM was more than two times higher.
To get more highly labeled fertilizer, longer labeling period with 15 N seemed to be better. In our labeling system, 12∼24 h was required for the incorporation of 15 N into the waste sludge, and in the subsequent nursing reaction, 15 N-SF-PMM 10 atom% 15 N was obtained. To increase the contents of 15 N in the SF-PMM, higher amount of 15 Ncompound and longer incubation will be better. Moreover, in the subsequent nursing reactor, higher temperature and short incubation are necessary. In future, to elucidate precise 15 N movement between plant, soil, and fertilizer, it is necessary to produce the fertilizer highly labeled with 15 N. PMM with nursing reactor must be one of the useful technologies to get such highly labeled compound.
As mentioned above, we suggested the possibility of applying the PMM to composting of livestock manures. In this case, the point to get such highly labeled fertilizer is to develop the method to mix homogeneously the 15 N-compounds and shredded paper chips with a small amount of feces having appropriate fluidity.

Effects of SF-PMM Treatments on the Growth of Komatsuna and Photo Observation.
To determine the efficiency of the SF-PMM, we used the sandy dune soil which had no history of exogenous fertilization and had a relatively low fertility. e soil elements are shown in Section 2.4.3.
e weights of komatsuna in 50 mg N and 100 mg N experiments are higher than that of the Control treatment, Chem-50, 2.36 and 3.06 times, respectively (Table 1). ese three weights were statistically significant and clearly suggested that 50 and 100 mg N of SF-PMM supported the growth of komatsuna shoots for 5 weeks without any inhibitory effects compared to that in 50 mg N Control treatment. Any significant abnormal growth was not shown in the photo observations ( Figure 6). Interestingly, very magnificent roots were observed in the plants grown in SF-PMM-50 and -100 treatments (Figures 6(b) and 6(c)), suggesting that, even in the rather higher concentration of SF-PMM-100, komatsuna might not receive inhibitory effects from SF-PMM in the growth of the roots.
Here, we introduce research showing the SF-PMM fertilizer's advantage in root growth. Honma et al. [10] used SF-PMM to proliferate and grow the cuttings of the Japanese cider Sugi, Cryptomeria japonica D. Don, which was a mutant making no pollen. erefore, the ordinary proliferation of the Sugi was the cutting, and the fertilization was started one year later after the cuttings. In the simultaneous application of any fertilizers, including commercially available organic fertilizers, almost all cuttings were killed. However, even in simultaneous application of the SF-PMM with the cutting, more than 90% of the cuttings survived, and they gained statistically significant increase in the height of twigs and the root weight [10].
Rooting in the cutting must be one of the most delicate phenomena in the agricultural proliferation. e report of Honma et al. [10] suggested that the SF-PMM was something mild, and even in SF-PMM-200 it allowed the rooting even in simultaneous application with cuttings. is must be the most difference point from the Control treatment with (NH 4 ) 2 SO 4 . 15 N-SF-PMM into Komatsuna. In the culture of plants with 15 N-SF-PMM-100 mg N treatment, 6.2 mg of 15 N was accumulated into a plant, and this amount was about half of the total amount of N in a plant grown in the pot. is suggests that SF-PMM was easily decomposed in the soil and provided N for the plant growth. However, the total amount of 15 N incorporated into the plant was only 6.2% of whole amount of 15 N-SF-PMM applied in the treatments, suggesting that only several percent of the compounds labeled with 15 N in the 15 N-NF-PMM were adsorbed into a plant. We believe that, under more appropriate conditions, higher amount of 15 N can be incorporated into the plant.

N Use from SF-PMM and Uptake of 15 N from
In a previous study, Matsuda et al. [14] attempted to describe 15 N-labeled compounds in a plant. As future work, it is important to determine the 15 N-labeled molecules in both the plants and the soil. ese experiments to determine the compounds will explain the movement of N from SF-PMM to plants. To estimate the appropriate amount of nutrients that should be applied in the field, it is necessary to measure the amount of the residual fertilizer in farmland on a yearly basis [13,26]. Such research must be undertaken using highly labeled 15 N-SF-PMM in future pot experiment as well.
About 50% of N was incorporated from SF-PMM applied; this relatively higher value could have resulted from the use of low fertility soil in this study. In this pot tests, we used the sandy soil having a value of available N as 20.4 mg·kg −1 . On the other hand, the question of why the availability rate was as low as 6% must be answered. e answer must be very important to obtain well designed SF-PMM, but we can just speculate now. First, the volume of the SF-PMM applied was too high, and almost 90% of the N in SF-PMM remained. Second, very high nitrification and/or denitrification occurred during the pot treatment. We must determine the amount of remaining 15 N in the pots. is must be very important to design useful SF-PMM.

Conclusion
In this study, we developed a rapid and efficient method to label the sludge in the wastewater of the food factory with 15 N-glycine. A small volume of wastewater was mixed with active sludge and 15 N-glycine for half a day, and the resulting active sludge was embedded into 400 L reactor tank carrying high temperature composting reaction at ∼70°C. After around 10 days of more composting, sludge fertilizer labeled with 7.03 atom% 15 N was obtained. From the labeled 15 N-SF-PMM, about 6% of the label applied was incorporated into komatsuna. Nonlabeled SF-PMM at 50 or 100 mg N supported the growth of komatsuna, and healthy shoots with flourishing roots were obtained in 5 weeks. e method proposed herein and related results were expected to establish the recycle of waste sludge from food factories with efficient sludge fertilizer.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.