extfieldsofstudy: Chemistry Medicine

text:
Moderately low nitrogen application mitigate the negative effects of salt stress on annual ryegrass seedlings

Appropriate application of nitrogen (N) can alleviate the salt stress-induced damage on plants. This study explores the changes of nitrogen requirement in feeding annual ryegrass seedlings under mild salt concentrations (50 mM, 100 mM) plus its underlying mitigation mechanism. Results showed that low salt concentration decreased N requirement as observed from the increment in plant height and biomass at a relative low N level (2.0 mM not 5.0 mM). Under salt treatment, especially at 50 mM NaCl, the OJIP (Chl a fluorescence induction transient) curve and a series of performance indexes (PIABS, RC/CS0, ET0/CS0, ϕE0, ϕ0) peaked whereas DI0/RC, Vj and M0 were the lowest under moderately low N level (2.0 mM). In addition, under salt stress, moderately low N application could maintain the expression of NR (nitrate reductase) and GS (glutamine synthetase) encoding genes at a relatively stable level but had no effect on the expression of detected NRT (nitrate transporter) gene. The seedlings cultured at 2.0 mM N also exhibited the highest activity of CAT and POD antioxidant enzymes and the lowest MDA content and EL under relative low level of salt treatment. These results indicated that mild salt treatment of annual ryegrass seedlings might reduce N requirement while moderately low N application could promote their growth via regulating photosynthesis, alleviating ROS-induced (reactive oxygen species) damage and maintenance of N metabolism. These results also can provide useful reference for nitrogen application in moderation rather than in excess on annual ryegrass in mild or medium salinity areas through understanding the underlying response mechanisms.

requirement as observed from the increment in plant height and biomass at a relative low N level (2.0 mM not 5.0 mM). Under salt treatment, especially at 50 mM NaCl, the OJIP (Chl a fluorescence induction transient) curve and a series of performance indexes (PI ABS , RC/CS 0 , ET 0 /CS 0 , φE 0 , φ 0 ) peaked whereas DI 0 /RC, V j and M 0 were the lowest under moderately low N level (2.0 mM). In addition, under salt stress, moderately low N application could maintain the expression of NR (nitrate reductase) and GS (glutamine synthetase) encoding genes at a relatively stable level but had no effect on the expression of detected NRT (nitrate transporter) gene. The seedlings cultured at 2.0 mM N also exhibited the highest activity of CAT and POD antioxidant enzymes and the lowest MDA content and EL under relative low level of salt treatment. These results indicated that mild salt treatment of annual ryegrass seedlings might reduce N requirement while moderately low N application could promote their growth via regulating photosynthesis, alleviating ROS-induced (reactive oxygen species) damage and maintenance of N metabolism. These results also can provide useful reference for nitrogen application in moderation rather than in excess on annual ryegrass in mild or medium salinity areas through understanding the underlying response mechanisms.

INTRODUCTION
Feeding annual ryegrass (Lolium multiflorum Lam.) is an important forage grass with high yield, good palatability and high nutritive value (Castanheira et al., 2014). Salt stress is one of the major abiotic factors that limit annual ryegrass growth and productivity. The adverse effects of salt stress on plants include ionic toxicity, osmotic stress and secondary stresses, such as a decline in photosynthesis, oxidative stress and nutritional disorders (Allakhverdiev & Murata, 2004;Kalaji et al., 2011;Zhu, 2001). The forage quality such as crude protein, organic matter can be adversely affected by elevated salinity (Ibrahim et al., 2018;Robinson et al., 2004). In response, through long term evolution, plants have adopted a series of response mechanisms to resist and minimize salt stress-induced damage (Deinlein et al., 2014;Zhao et al., 2020). For example, in response to salt-induced oxidative stress, a series of antioxidant enzymes were induced to scavenge excessively produced reactive oxygen species (ROS) (Kohler et al., 2009;Baby & Jini, 2010), such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) (Apel & Hirt, 2004;Dong, Kim & Lee, 2001).
In saline environment, nitrogen (N) is usually the limiting growth macro-nutrient, thus N application is the most effective method of improving plant growth under salt stress. The mainly available inorganic N forms are ammonium N (NH 4 + ) and nitrate N (NO 3 − ), which are transported by AMT (Ammonium transporter) and NRT (Nitrate transporter) respectively (Giagnoni et al., 2015). The inorganic N is then assimilated and converted into amino acid via several enzymes such as nitrate reductase (NR), glutamine synthetase (GS) and glutamine 2-oxoglutarate aminotransferase (GOGAT) (Xu, Fan & Miller, 2012). Salt stress has also proved to inhibit the activity of many enzymes such as NR and GS/GOGAT, involved in N assimilation in maize (Zea mays), cowpea (Vigna unguiculata) mung bean (Vigna radiata), tomato (Solanum lycopersicum) and rice (Oryza sativa) (Chakrabarti & Mukherji, 2007;Debouba et al., 2007;Khan & Srivastava, 1998;Parul, Kumar & Kumar, 2015;Silveira et al., 2001;Wang et al., 2012), and then decreased the absorption and utilization of N in plants (Singh, Singh & Prasad, 2016). Increased salinity was also reported to significantly reduce nitrogen use efficiency (Murtaza et al., 2013). On the contrary, some studies showed that processes related to N uptake and assimilation in some plant species were stimulated under certain levels of salt stress. For instance, salt could induce the expression level of nitrate transporter genes such as McNRT1 (Popova, Dietz & Golldack, 2003). Under salt exposure, the nitrate uptake rate and activity of NR were promoted in glasswort (Salicornia europaea) (Nie et al., 2015). Thus, it has been speculated that the alteration of plant N nutrition level may hold great promise for regulating salinity response in different species under certain salt level (Chen et al., 2010).
Generally, N application can ameliorate the negative effects of salinity by compensating and correcting nutritional imbalances in higher plants (Esmaili et al., 2008;Gómez et al., 1996;Mansour, 2000;Villa et al., 2003). Several N-containing compounds were accumulated in plants that were subjected to salt stress (Dluzniewska et al., 2007;Ehlting et al., 2007;Sudmalis et al., 2018). The accumulates were then reported to participate in osmotic adjustment, promotion of photosynthetic capacity and mitigation of oxidative stress by scavenging excessive ROS (Homaee, Feddes & Dirksen, 2002;Mansour, 2000;Rontein, Basset & Hanson, 2002;Song et al., 2006;Todorova et al., 2013). Although many studies have shown that N plays an important role in the amelioration of salt stress, it has been confirmed that the alleviation of salt inhibition from N application shows concentration effects. For example, at the higher salinity levels, increasing the N content was found to be ineffective in resisting negative influences caused by the enhanced salt concentrations in tomato (Papadopoulos & Rendig, 1983). Another previous study reported that low levels of N could mitigate the negative effects whereas high N levels may exacerbate the detrimental effects of salt stress on photosynthetic rate of chickpea leaves (Soussi, Ocaña & Lluch, 1998). A recent study also found that, low to moderate N application could mitigate the adverse effects, while excessive N could elevate the negative effects of salt stress on cotton growth (Chen et al., 2010). In maize, reduction in N application could not cause additional damage on plant development under salt stress based on reductions in evapotranspiration (Lacerda et al., 2016). Other studies also pointed out that excessive nitrogen fertilization under high salinity levels might lead to more pronounced osmotic effect which in turn provokes the negative effect on crop yield (Beltrão et al., 2002). In addition, in highly saline soils, excessive application of N fertilizer will trigger secondary soil salinization, which in turn adversely affects crop growth (Chen et al., 2010). Moreover, over fertilization with N may contribute to N leaching in the saline soil, where plants are unable to utilize the supplied N fertilizer efficiently and leading to groundwater pollution (Pessarakli & Tucker, 1988;Shenker, Ben-Gal & Shani, 2003;Ward, 2013).
Therefore, N requirements for plants under saline environment might be inconsistent with those in normal environment. The difference might be attributed to distinct physical and chemical properties of growth medium and the alteration of plants nitrogen use efficiency plus other physiological response. Hence, in order to minimize the negative effects of salinity as well pollution of underground water, proper N fertilizer management in plants is necessary for different salt conditions. A previous study on annual ryegrass reported that increasing N concentration in the nutrient solution enhanced shoot biomass production under relatively high salinity levels (Sagi et al., 1997). However, at relatively low salt concentrations, the optimization of nitrogen demand and the possible mechanisms underlying this alleviation in annual ryegrass are still not fully explored. Based on the above studies, the objectives of this work were to assess the optimal N level under relatively low salt level and to investigate the possible salt stress-alleviatory mechanisms by analyzing physiological indexes and metabolism of N in annual ryegrass seedlings.

Plant materials and growth conditions
Annual ryegrass seeds were thawed in plastic pots that were filled with plant growth medium and then cultured in the greenhouse under natural sunlight. After one month, the seedlings were then transferred into Erlenmeyer flasks containing 585 mL of nutrient solution. The seedlings were then mowed to a height of 12.5 cm before the treatments were initiated. The experiment was set as control (0 mM NaCl) and NaCl treatment (50 mM or 100 mM). Both the control and NaCl regimes included different nitrogen application level (using NH 4 NO 3 as nitrogen source). The hydroponic culture media was processed in a growth chamber under the following conditions: 22/18 • C (day/night), 65% relative humidity, 300 µmol m −2 s −1 photons and a 16-h day/8-h night cycle. The culture solution was refreshed every two days.

Chlorophyll a fluorescence transient and the JIP-Test
A pulse amplitude modulation fluorometer (PAM2500; Heinz Walz GmbH) was used to measure the chlorophyll a fluorescence transient. In brief, the annual ryegrass leaves were put in dark place for 30 min, and the leaves were then exposed to 3,000 µmol photons m −2 s −1 red light condition. Each treatment consisted of at least three replicates. Based on the theory of energy fluxes in biofilm, the JIP test can further translate the primary data into other biophysical parameters (Force, Critchley & Rensen, 2003). Hence, the basic parameters were used to calculate a series of parameters (Yusuf et al., 2010).

Chlorophyll content and electrolyte leakage
The SPAD 502 Plus Chlorophyll Meter (SPAD-502Plus; Spectrum Technologies, Inc., Dallas/Fort Worth, TX, USA) was used to quantify the leaf chlorophyll content. The electrolyte leakage (EL) was measured according to the previous method (Blum & Ebercon, 1981).

Enzymes activity and lipid peroxidation
Fully expanded of 0.3 g leaves were immediately grounded into powder with liquid N. four mL ice-cold phosphate buffer (50 mM, pH 7.8) was added into the powder and the samples were centrifuged at 12,000 rpm for 20 min at 4 • C. The supernatant was collected to measure the activity of POD and CAT and the content of MDA based on the method as described by previous study (Fu & Huang, 2001).

Quantitative RT-PCR Analysis
The total RNA was isolated and reverse transcribed using the RNeasy kit (Qiagen) and TaqMan reverse transcription kit (Applied Biosystems). Quantitative real-time RT-PCR analysis was conducted using SYBR Green real-time PCR master mix (Toyobo, Japan) and ABI real-time PCR system (Applied Biosystems, Foster City, CA, USA). The primers used are listed in Table S1. The ryegrass Actin gene was used as an inner control, and comparative Ct method was applied for gene expression level analysis.

Statistical analysis
One-way ANOVA was performed using SPSS17.0 for Windows (SPSS). All of above tests had at least three independent replicates. Results were expressed as mean ± SD, and letters show significant differences (P < 0.05) by Student's t -test.

Effect of different N level treatment on the growth of annual ryegrass seedlings under NaCl stress
Under control condition, the plant height and the relative increase of biomass of annual ryegrass seedlings peaked under 5.0 mM N, and then slightly declined under 10 mM N. As compared to 5.0 mM N, the height of seedlings grown under 2.0 mM N and 10 mM N declined by about 10% and 15% respectively (Figs. 1A-1D). However, under 50 mM or 100 mM NaCl treatment, the plant height reached maximum under 2.0 mM N and then declined slightly under 5.0 mM N. In addition, salt treatment dramatically reduced the height of plants grown under 5.0 mM or 10 mM N compared with their respective control plants. However, under 2.0 mM N condition, the plant height had no significant difference with or without salt treatment (Fig. 1D). When exposed to 50 mM NaCl, the relative increase of biomass had no significant difference among different N levels. After the Figure 1 Morphological parameters of annual ryegrass seedlings grown under different nitrogen and salt conditions. Morphological parameters of annual ryegrass seedlings grown under different nitrogen and salt conditions. The seeds of annual ryegrass were cultured in soil for one month, and the seedlings cut to the same height were then transferred into different nitrogen level (2.0, 5.0, 10 mM) under NaCl (0, 50, 100 mM) stress in a hydroponic culture. After being grown for 10 days, the plant height and biomass were measured. (A-C) Images of seedlings at 10 days after transferred. (D) Plant height at 10 days after transferred. (E) The relative change of biomass (% of biomass which was measured before treatment). Different letters above the columns indicate significant differences at P < 0.05 by Student's t -test.
Full-size DOI: 10.7717/peerj.10427/fig-1 plants were exposed to 100 mM NaCl for 10 days, the biomass only increased when 2.0mM N was applied whereas the biomass decreased when extra nitrogen (5.0 or 10 mM N) was applied (Fig. 1E). These similar alteration trends in biomass and plant height suggested that salt stress might change the N requirement of annual ryegrass and moderately reducing N application might alleviate the inhibitory effect of salt stress on annual ryegrass seedlings growth. In order to investigate whether ultra-low N treatment still had a moderating function on the growth of annual ryegrass seedlings under salt stress, we added a lower concentration (0.5 mM) of N treatment and conducted another experiment. The results showed that the plant height of annual ryegrass seedlings achieved maximum under 5.0 mM N without salt treatment. However, when exposed to 50 mM or 100 mM NaCl, the plant height reached maximum under 2.0 mM N (Figs. S1A, S1B), showing the similar tendency with the first experiment (Figs. 1A, 1B). In addition, without NaCl treatment, there was no significant difference of plant height when seedlings were supplemented with lower N concentration (0.5 mM or 2.0 mM). However, under NaCl treatment, the plant height was significantly increased under 2.0 mM N compared with 0.5 mM N (Fig. S1). The results of these two experiments at different times together confirmed that the alleviatory effect of N application on the growth inhibition of annual ryegrass under salt stress might have a certain range. Moderately low N could alleviate the inhibition of annual ryegrass growth by salt stress through a series of response mechanisms, whereas ultra-low N could not promote, but seriously inhibit the growth of annual ryegrass.

Impact of N on the OJIP transient curve in the leaves of annual ryegrass under NaCl stress
In order to decipher the effect of moderate N-mediated alleviation of salt stress on annual ryegrass, the impact of N levels on photochemistry of photosystem II (PS II) of NaCl treated annual ryegrass seedlings were determined through chlorophyll a fluorescence transient-JIP test. The step O to J represents the reduction process of Q A by PSII. Due to the brimming of the plastoquinone pool, the curve then rose to I phase. The step I to P was an account for the blockage of electron transfer to the acceptor side of PSI. Under control condition, the fluorescence of I and P phase of seedlings leaves grown with 2.0 mM N or 5.0 mM N was stronger than that grown with 0.5 mM N ( Fig. 2A). However, after NaCl exposure, the chlorophyll fluorescence curve of annual ryegrass leaves grown with 2.0 mM N from I to P step was higher than that under 0.5 mM or 5.0 mM N (Figs. 2B, 2C). In particular, the OJIP curve was much higher when plants exposed to relatively low NaCl treatment (50 mM) under 2.0mM N level compared to other two N levels (Fig. 2C). The results suggested that deficient or excessive nitrogen application under salt stress might lead to the photosynthetic electron transport traffic jam, especially beyond Q A − . In addition, under NaCl treatment, the leave chlorophyll content of the plants grown with 2.0 mM and 5.0 mM N was significantly higher than that grown with 0.5 mM N. However, there was no significant difference in the chlorophyll content between 2.0 mM and 5.0 mM N-supplied plants (Fig. 2D).

Impact of N on Chlorophyll fluorescence parameters in the leaves of ryegrass under NaCl stress
The chlorophyll fluorescence parameters were used to quantify the photosynthetic behavior of the samples. Under the control condition, the PI ABS value, which represents the overall activity of PSII, increased with the N level, and peaked under 5.0 mM N (Fig. 3A). However, under 50 mM or 100 mM NaCl treatment, the PI ABS value under 2.0 mM N were higher than that under other N levels (Figs. 3B, 3C). The variable fluorescence at J phase (V j ) and the relative speed of Q A deoxidation (M 0 ) of NaCl-treated leaves grown with 2.0 mM N were smaller than those grown with 0.5 mM or 5.0 mM N, and the difference was most significant under 50 mM NaCl treatment (Fig. 3B). Under normal condition, 0 and E 0 had no significant difference among three N levels (Fig. 3A). When exposed to 50 mM NaCl, the proportion of energy used for photochemical reaction and energy electron transport in leaves (φ 0 , φE 0 ) grown with 2.0 mM N were larger than those grown with other N levels, together with greater reaction center density RC/CS 0 and electron-transfer energy ET 0 /CS 0 and lower DI 0 /CS 0 (the energy consumed in unit cross-sectional area) (Fig. 3B). However, φP 0 , which represents the maximum quantum yield for primary photochemistry, displayed no changes. When exposed to 100 mM NaCl, the PI ABS value under 2.0 mM N was higher than that under other N levels, whereas the other parameters showed no significant change (Fig. 3C). These results suggested that the optimum amount of N might promote primary photochemical reactions of PSII, especially under relatively low NaCl level.

The lipid peroxidation levels and activities of antioxidant enzymes in the leaves of the annual ryegrass seedlings under NaCl stress
Malondialdehyde (MDA) is one of the products of membrane lipid peroxidation which can be used to assess the degree of the salt-induced damage to plants. The results showed that, in the absence of salt stress, there was no significant difference of MDA content among the three N levels. When the plants grown under 2.0 mM N were exposed to a relatively lower NaCl treatment (50 mM), the MDA content declined significantly compared to the control. In addition, the MDA content of plants grown under higher N concentration were significantly lower compared to those grown under 0.5 mM N (Fig. 4A). The electrolyte leakage (EL) in the leaves of ryegrass increased with the increase of NaCl concentration under all three N levels. When exposed to 100 mM NaCl, The EL in the leaves of ryegrass grown under higher N concentration was significantly lower compared to that grown under 0.5 mM N. Moreover, under 100 mM NaCl stress, the EL of ryegrass grown under 2.0 mM N was significantly lower than that grown under 5.0 mM N (Fig. 4B). The lipid peroxidation levels and activities of antioxidant enzymes of the leaves were also determined. With the increase of NaCl concentration, the CAT enzyme activity rose under all N levels. Under NaCl treatments, the CAT activity of ryegrass seedlings cultured at 2.0 mM N was the highest compared with that of plants cultured at 0.5 mM N or 5.0 mM N (Fig. 4C). The POD enzyme activity had no obvious regularity with the N levels. However, when exposed to 50 mM NaCl, the POD activity of seedlings grown under 2.0 mM N was higher than seedlings grown under 5.0 mM N (Fig. 4D). These results suggested that ryegrass cultured in 2.0 mM N solution might improve the activities of certain antioxidant enzymes and enhance the salt-tolerance ability of ryegrass, especially at relatively low NaCl level.

Effect of different N treatment on the N content and N assimilationrelated genes under NaCl stress
To investigate the influence of different N concentrations on N assimilation under NaCl stress, we assessed the expression level of NR gene in the leaves of ryegrass. Without NaCl treatment, the level of the NR expression in the leaves increased with the increase of N concentration. A reduction in N (2.0 mM) caused a significant decline in mRNA expression of NR (Fig. 5A), as compared with 5.0 mM N-applied plants. When plants were cultured with 5.0 mM N, the level of NR gene expression showed a significantly decrease with the increase of salt concentration. However, under 2.0 mM N, the suppression degree of NR expression by salt stress was relatively lower. Compared with 0 mM NaCl, the expression level of NR of 2.0 mM N-supplied plants showed no significant decrease when exposed to 100 mM NaCl (Fig. 5A). Under the combined treatment of nitrogen and salt, the homolog gene of GS showed a similar expression response pattern with NR (Fig. S2A). The expression of the NRT gene was induced when plants were exposed to a relatively low NaCl level (50 mM). However, there was no significant difference in the homolog of one NRT gene expression between plants grown with 5.0 mM N and 2.0 mM N under NaCl treatment (Fig. S2B). When exposed to NaCl, the nitrogen content of leaves grown under 2.0 mM N or 5.0 mM N showed a significant decline compared to the control condition, respectively. Without NaCl treatment, the N content of ryegrass leaves grown under 5.0 mM N was higher. However, under salt treatment, the N content of leaves showed no significant difference between 5.0 mM N and 2.0 mM N application (Fig. 5B).

DISCUSSION
Plant response to salt stress is a complex phenomenon that involves both morphological, physiological, and biochemical processes. It has been reported that the application of N may alleviate salt stress-induced phytotoxicity (Correia et al., 2005;Siddiqui et al., 2012;Singh, Singh & Prasad, 2016). Salt stress effect on plants consists of Na + and Chl − ion toxicity, osmotic effects, and nutrient imbalance (Kohler et al., 2009;Shannon, 1997). Nitrogen fertilization plays a critical role on the growth and development of the many plants, and appropriate N could help to mitigate the damage caused by nutritional imbalances due to saline irrigation (Al-Rawahy, Stroehlein & Pessarakli, 1992;Borzouei et al., 2014;Fan et al., 2013;Duan & Chang, 2017). However, related researches showed that rather than N alone, plant growth was significantly affected by the interaction between soil salinity and N (Papadopoulos & Rendig, 1983;Chen et al., 2010). In this experiment, exogenous N application to annual ryegrass seedlings significantly increased the in planta N content, plant height and the biomass, but there was a concentration effect. In the absence of exogenous salt, the increment of plant height and biomass was directly proportional to the N level, and peaked at 5.0 mM N. However, after exposure to NaCl, the plant height and the relative increase of biomass peaked at the N level of 2.0 mM (Fig. 1). Moreover, ultra-low N seriously inhibited the growth of ryegrass under both control and salt conditions (Fig. S1). These results were consistent with results detected in cotton (Chen et al., 2010). Also a previous study on annual ryegrass reported that increasing N application could promote shoot growth under under salt concentrations of 2.0 dS/m (below 20 mM) and 11.2 dS/m (over 100 mM) (Sagi et al., 1997). However, we noticed that moderate reduction of N application had a maximum promotion effect on plant growth. The salt concentration used for our treatments are 50 mM and 100 mM, which are different from the salt concentration used in Sagi's experiments. This difference may be attributed to different culture conditions as well as levels of salinity. In this study, we focus mainly on the optimization of nitrogen application at lower salt concentration. Moreover, N content was also positively correlated with the amount of N applied which peaked at the N level of 5.0 mM without salt treatment. However, under salt treatments, there was no significant difference in N content of ryegrass leaves between 5.0 mM and 2.0 mM N application (Fig. 5B). External cues such as salt can stimulate the production of ROS which can further damage lipids in plant cells (Kohler et al., 2009). Accumulation of N-containing compounds has been reported to participate in salt response such as osmotic adjustment and ROS scavenging (Dluzniewska et al., 2007;Ehlting et al., 2007;Homaee, Feddes & Dirksen, 2002;Mansour, 2000;Song et al., 2006;Sudmalis et al., 2018). In this study, by reducing MDA content and elevating certain antioxidant enzyme activities, moderately low N application could reduce the damage to the membrane of ryegrass seedlings caused by salt stress, especially at relatively low NaCl treatment. Together, these results indicated that saline habitats might change the N requirement of ryegrass seedlings. Excessive or ultra-low N applications both have counter effects on the growth or salt resistance of annual ryegrass under low level of salt stress.
Under salt stress, chlorophyll a fluorescence transient is a useful parameter for reflecting the primary reaction alternations of PSII, which is more sensitive than photosystem I (PS I). To investigate PSII behaviors in O-J-I-P transient, JIP test is always used to quantify the derived photochemical parameters (Apostolova et al., 2006;Sayed, 2003;Stirbet et al., 2014). In this study, after exposure to NaCl, the nitrogen application level had a significant effect on fluorescent transients, especially the J and P steps (Fig. 2). N deficiency and N over application under salt stress might lead to the photosynthetic electron transport traffic jam, especially beyond Q A − (Figs. 2B, 2C). With the increase of N level, the PI ABS , which could accurately reflect the state of plant photosynthetic apparatus, increased without NaCl treatment ( Fig. 2A), indicating that N could promote the primary photochemical reactions of PSII in the waterside. However, under salt stress, excessive or deficient N application slowed down the promotion of the primary photochemical reaction of the PSII (Fig. 2B, Fig. 2C). In addition, the accumulated amount of Q A − (V j ) and the relative speed of Q A deoxidation (M 0 ) (Strasser, 1997;Strasserf & Srivastava, 1995;Force, Critchley & Rensen, 2003) of plants grown under moderately low N were lower than those grown under other N conditions, indicating that leaves grown under moderate N level have a higher electron transport rate between Q A and Q B , thus reducing the accumulation amount of Q A − andincreasing the photochemical reaction efficiency (Allakhverdiev & Murata, 2004. The increase of 0 and E 0 of plants grown under 2.0 mM N indicated that leaves use more energy for photochemical reaction and electron-transfer process, thus producing more NADPH for carbon assimilation and confirming that leaves have the optimal energy distribution under certain salt level (Strasser, Tsimilli-Michael & Srivastava, 2004). The leaves of annual ryegrass grown under 2.0 mM N also had a greater reaction center density RC/CS 0 and higher ET 0 /CS 0 but lower DI 0 /CS 0 than those grown under other N conditions. This pattern indicated that the specific activity of a unit cross-sectional area of leaves grown under moderately low N was stronger than that grown under other N conditions, reducing the energy burden of a unit reaction center. We also noticed that, under the lowest NaCl treatment (50 mM), the application of 2.0 mM N had the best effect on alleviating salt stress. Under 50 mM NaCl treatment, the physiological indexes of annual ryegrass seedlings seemed to be less affected, and therefore the seedlings might be more sensitive to the promotion of nitrogen application. Thus, we proposed that the optimum amount of N might promote primary photochemical reactions of PSII under certain level of NaCl treatment.
After absorption as ammonia, N can be directly assimilated by plants. While after absorption as nitrate, N must first be reduced by nitrate reductase and sub-acid reductase enzymes. NR can reduce nitrate N to ammonium, which has also has important effects on photosynthesis and other processes related to N metabolism (Xu, Fan & Miller, 2012). Reports have shown that NO 3 − has a significant effect on the induction of NR expression. From this experiment, under control condition, the level of NR gene expression in leaves increased with the increase of N concentration (Figs. 5A, 5B), which is consistent with the previous reports (Oaks, 1993). However, when the seedlings were treated with NaCl, the NR expression level was significantly declined at a higher N level (5.0 mM). On the contrary, at a moderately low N level (2.0 mM), the NR expression level is relatively low without NaCl treatment, but the degree of reduction is moderate when exposed to NaCl. The GS gene expression displayed a similar trend with the NR gene under the interaction between salt and nitrogen conditions, indicating an interactive response mechanism between N assimilation-related genes (Fig. S2A). Therefore, it can be observed that moderate N application might help annual ryegrass maintain the expression level of N assimilation-related gene (Fig. 5A) and further maintain the nitrogen content under salt stress (Fig. 5B). However, when excessive N was applied under salt stress, the NR expression was significantly increased, indicating that N assimilation was strengthened; it might then compete with photosynthetic carbon for ATP and NADPH and increase the burden of photosynthetic electron transfer. The competition result might lead to a decline in the overall activity of PSII of annual ryegrass (PI ABS ) (Fig. 2). Under nitrogen deficiency, the reduced absorption of nitrogen might reduce the consumption of nitrogen assimilation reducing power, most of which are derived from photosynthesis, thus resulting in the accumulation of chloroplast NADPH. The over-accumulation of NADPH could inhibit the photosynthetic efficiency and cause excessive production of ROS (Fig. 3A), leading to increased cell membrane damage, which may in turn lead to reduced photosynthetic efficiency (Figs. 2B, 2C, 2D). Nitrogen is also a constituent of chlorophyll which is not only the most important pigment molecules of photosynthesis involved in energy absorption and transmission but also the essential electron mediator during electron transport. Studies showed that the nitrogen content of leaves is constant with the photosynthetic capacity (Grassi et al., 2005;Kattge et al., 2009). Through this experiment, we can deduce that under different salt stress condition, the appropriate addition of N can increase relative chlorophyll content of plants. However, the relative chlorophyll content is only positively correlated with N levels within a certain range (0.5-2.0 mM) and should be reduced beyond a certain range (Fig. 2D). The moderately supply of N under salt stress increased the content of chlorophyll and might increase the light-harvesting ability, partly contributing to the up-regulated photosynthetic performance index. Based on the above studies, it can be seen that moderately low N application under low level of salt stress might help annual ryegrass maintain the expression level of N assimilation-related gene and then maintain the leaf N content of the plant, which might in turn cause changes in chlorophyll content, further avoiding the negative effect on photosynthetic capacity.

CONCLUSION
To investigate the possible mechanism of moderately low nitrogen-mediated alleviation of NaCl stress, the degree of lipid peroxidation, antioxidant enzyme activity alternation, changes of photosynthesis performance and nitrogen assimilation were analyzed in this study. In summary, under low salt stress, the demand for N may have declined, while moderately reducing N application could help to alleviate the salt-induced damage in annual ryegrass mainly by alleviating the damage caused by ROS and promoting the performance of photosynthesis and nitrogen metabolism. Further, in order to enhance plant growth and increase nitrogen use efficiency, the optimum application of nitrogen fertilizer needs to be controlled to match the plant needs at each growth stage and to adapt to different saline environment.


s2fieldsofstudy:
Agricultural And Food Sciences
Chemistry

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Fractionation of Cellulose-Rich Products from an Empty Fruit Bunch (EFB) by Means of Steam Explosion Followed by Organosolv Treatment

In this study an empty fruit bunch (EFB) was subjected to a two-step pretreatment to defragment cellulose-rich fractions as well as lignin polymers from its cell walls. First pretreatment: acid-catalyzed steam explosion (ACSE) pretreatment of EFB was conducted under the temperature range of 180–220 °C and residence time of 5–20 min. The ACSE-treated EFB was further placed into the reactor containing 50% aq. ethanol and NaOH as a catalyst and heated at a temperature of 160 °C for 120 min for the second pretreatment: alkali-catalyzed organosolv treatment (ACO). The mass balance and properties of treated EFB were affected by the residence time. The lowest yield of a solid fraction was obtained when the residence time was kept at 15 min. Xylose drastically decreased, especially under the ACSE pretreatment. However, the crystallinity of cellulose increased by increasing the severity factor of the pretreatment and was 47.8% and 57% udner the most severe conditions. The organosolv lignin fractions also showed the presence of 14 major peaks via their pyrolysis-GC analysis. From here, it can be suggested that this kind of pretreatment can indeed be one potential option for lignocellulosic pretreatment.

Introduction
Over the recent decades, the utilization of empty fruit bunch (EFB) to produce renewable energy source has been studied extensively. This, EFB typically, is a leftover of the fresh fruit bunches after their fruit harvesting. Moreover, it is the major downstream residue from the Southeast Asian palm oil industry. This industry, recently, has produced 70 million metric tons of crude palm oil (CPO) just within one production year 2017-2018 amounting their production even up to 35% of the total production of edible vegetable oils [1]. Theoretically, palm oil plantations can produce more than 100 million metric tons of EFB since 1.42-1.88 tons of EFB are generated for every one ton of CPO produced [2]. However, unfortunately, most of these EFB is dumped and pose serious environmental problems. Therefore, it is highly desirable to utilize these biomasses for sustainable energy production.
Biochemical conversion of lignocelluloses involving chemical pretreatment followed by enzymatic hydrolysis and finally anaerobic fermentation is a well-established production line for lignocellulosic bioethanol production. In this classical approach, due to an inherited complex cell wall structure, effective fractionation of major lignocellulosic biomass components (cellulose, hemicellulose, and lignin) is crucial for effective subsequent processing. Undoubtedly, there have been several pretreatment studies involving EFB to deconstruct its native web into its constituents. In this regard, steam explosion, which uses high-pressure steam to breakdown biomass structure, has been widely adapted strategy [3]. The effects of several factors like biomass particle size, moisture, pressure, residence time, reaction temperature and catalysts on the steam explosion were consistently investigated for several decades [4]. Previously, Choi et al. used alkaline reagent (3% NaOH at 160 • C) to catalyze steam explosion pretreatment and dissolved out C5 sugar-based components as well as lignin components from biomass [5]. Their yields, after enzymatic hydrolysis, for glucose and xylose heightened up to 93% and 78% respectively. In another study, the steam explosion of rose gum tree (Eucalyptus grandis) was carried out under the temperature range of 200-210 • C and residence time of 2-5 min after impregnation with 0.087% and 0.175% (w/w) H 2 SO 4 [6]. Their results showed that 0.175% (w/w) H 2 SO 4 -impregnated chips at 210 • C for 2 min was the optimal condition for hemicellulose extraction. Today steam explosion is seen as one of the most widely employed and cost-effective process for lignocellulose pretreatment, yet the process still needs to be improved to realize industrialization [7]. In addition to this steam explosion strategy, another effective fractionation scheme for lignocelluloses is organosolv treatment, so-called organosolvation. This process, in principle, extracts lignin from lignocellulose with organic solvents, such as alcohols, acetone and organic peracids [8]. Previous studies have shown considerable effects of organosolv treatment (diethylene glycol) of rice straw and EFB as compared to soda pulping by Gonzalez et al. [9] keeping in view the heterogeneous yet complex cell wall structure of EFB, it is highly likely to investigate an appropriate pretreatment strategy for commercial production of biofuels [10]. Therefore, it has to be investigated the proper pretreatment process of EFB. In this study we adapted a two-step pretreatment strategy to fractionate EFB biomass into its sugar and non-sugar components. For this two-step pretreatments, EFB was first subjected to acid-catalyzed steam explosion (ACSE) followed by alkali-catalyzed organosolv treatment (ACO). Pretreatment efficacy was evaluated in terms of yields, crystallinity, and chemical composition of each solid fraction.

Feedstock Analysis
As raw biomass for this study, EFB (≤6% moisture) was provided by the Korea Institute of Science and Technology, in a setting of 0.5-2 mm fiber. Holocellulose, lignin, and ash content in the EFB sample were analyzed according to the National Renewable Energy Laboratory (NREL) standard procedures [11][12][13]. Carbon, hydrogen, and nitrogen contents were measured by using a CHNS-932 analyzer (LECO Corp., St. Joseph, MI, USA) and inorganic elements were analyzed by inductively coupled plasma emission spectroscopy (iCAP7400 Duo, Thermo Fisher Scientific, Waltham, MA, USA) [14]. Thermogravimetric analysis was performed under an inert atmosphere (50 mL/min N 2 flow) at a heating rate of 10 • C/min up to 800 • C with a TGA/DSC 3+ (METTLER TOLEDO, Columbus, OH, USA) [14]. All the results with the corresponding values are listed in Table 1.

ACSE-First Pretreatment
Initially, we screened out experimental conditions based on our initial trials and proceeded accordingly as described below for the pretreatment. The complete fractionation processes are delineated in Figure 1. Briefly, for ACSE treatment, EFB was first washed to remove inorganic elements using a method suggested by Moon et al. [15] Later on it was impregnated in distilled water for 120 min with slow stirring, and finally the feedstock was dried in an oven at 75 • C. It was then impregnated in dilute sulfuric acid (0.5, 1, 2 wt%) for 4 h to make it ready for steam explosion pretreatment. The effect of dilute acid concentration on impregnation was examined by the component analysis (holocellulose, lignin, and sugar) [12].
For ACSE experiments, autoclave reactors with 2 L high pressure steam were loaded first with 150 g of raw EFB biomass samples on dry basis as a control. The final temperature in the reactor was applied to reach in the interval of 180-220 • C and the residence time was 5-15 min. After the intended reaction time, a valve was opened to release the pressure immediately. Solid fractions (E-S) from the steam explosion was washed with distilled water, and the resulting aqueous fraction (E-L) was also collected. Then, acid impregnated EFB, with the same solids loading, was steam-treated under the temperature of 220 • C and 20 min of residence time, the most severe conditions that could be stably maintained with the reactor. After the reaction and cooling, solid fraction (EA-S), and aqueous fraction (EA-L) were also collected.

ACO Second Pretreatment
Organosolv treatment experiments, as a 2nd treatment for both the E-S & EA-S solid fractions obtained from 1st treatment, were performed as follows: both the solid fractions were placed, one by one, in a solvent-circulating reactor with a solvent of a 50:50% (w/w) ethanol:water mixture maintaining a (10:1 (w/w) solvent:solid ratio) along with a 2 wt% of NaOH as a catalyst. The reaction was carried out at a temperature of 160 • C and 120 min residence time. After the desired reaction time, cellulose-rich solid fractions were collected and labelled as E-S-CS and EA-S-CS and liquid fractions were labelled as E-S-OL and EA-S-OL corresponding to E-S and EA-S biomass samples, respectively. The organosolv lignin was precipitated from E-S-OL and EA-S-OL by acidification with a 2 M of hydrochloric acid maintaining pH = 2. The precipitated organosolv lignin was freeze-dried and collected as a powder and saved for further experiments.

Analysis
Yields of each solid fraction were calculated using the following equation: Yield (%) = weight of a solid fraction (g)/weight of dry feedstock (g) × 100 Holocellulose, lignin, and sugar content in each solid fraction were analyzed by the same process mentioned in Section 2.1. X-ray powder diffraction patterns (XRD) were analyzed by a Bruker D8 Advance with DAVINCI using Cu Kα radiation (λ = 1.5418 Å), operated at 40 kV and 40 mA with a scan speed of 0.5 s/min in a range of 2-50 degrees (2 thetas). Chemical compounds in liquid fraction were quantified and qualified using gas chromatography-mass spectrometry systems (5975C Series GC/MSD System, Agilent Technologies, Santa Clara, CA, USA) [14]. Organosolv lignin was analyzed by a coil-type CDS Pyroprobe 5000 (CDS Analytical Inc., Oxford, PA, USA) [16].

Effect of Residence Time and Temperature on Fractionation
The effects of residence time and temperature on the fractionation process were studied at various intervals (residence time 5, 10, 15 min and temperature 180, 200, 220 • C). Figure 2 shows the mass distribution of solid fractions obtained from EFB at various temperatures and residence time conditions.  [17]. The severity factor obtained ranged from 3.1 to 4.7. There was a scant negative correlation between the severity factor and the solid fraction yield, showing that increasing temperature and residence time results in the removal of compounds from solid fractions.
In general, both mechanical and chemical effects take place when an acetyl group derived from hemicellulose acts as an acid at high temperature during the steam explosion [18]. These effects play important roles in the hydrothermal degradation of holocellulose. Hemicellulose and lignin are isolated by the high temperature in the steam condition. Organosolv treatment, however, on the other hand, solubilize lignin and provide holocellulose by using organic solvent mixture and inorganic catalysts [7]. Therefore, holocellulose and lignin compositions of solid fractions were also analyzed to examine the fractionation efficiency of the process.
Holocellulose and lignin contents of E-S and E-S-CS were calculated for each experiment ( Table 2). Percentage of holocellulose composition decreased with increasing severity of steam explosion. A maximum of 24.1 wt% of holocellulose (220-15) was dissolved in E-L. On the contrary, the percentage of lignin composition increased with increasing severity factor. It can be anticipated that lignin melts under a high temperature and pressure, then condensed with other compounds like extractives or ash while cooling. Solid fractions which have undergone organosolvation showed an unremarkable change of holocellulose contents. In addition, a considerable amount of lignin content still remained in the solids. Maximum 7.5% of lignin (220-15) was solubilized in 50% ethanol mixture. Lignin fractionation efficiency of organosolv treatment from EFB was falling short of our expectation because of its low lignin removal. These results showed EFB presents a high level of recalcitrance to the fractionation process. Thus, dilute acid pretreatment to level down the EFB recalcitrance had considerable effects and the results are discussed in Section 3.1.2.

Effect of Dilute Acid Impregnation on Fractionation
Acid impregnation has been considered to play a vital role for steam explosion treatment [4,19]. Keeping this in view, the effects of dilute acid on the fractionation process was studied with the feedstock impregnation in dilute sulfuric acid (0.5, 1, 2 wt%) for 4 h. Steam explosion process was further operated at residence time of 20 min and temperature of 220 • C, which showed the highest severity score so that the effect of dilute acid concentration can be compared clearly. Figure 3 shows the mass distribution of solid fractions obtained from EFB at various acid concentrations.
Mass composition of EA-S did not affect by the acid concentration clearly until the concentration reaches 2.0%. Moreover, at 0.5 and 1.0% dilute acid concentrations, EA-S from ACSE also showed no remarkable changes in the mass distribution. In the range of 0%-1.0% dilute acid concentration, yields of EA-S decreased from 73.0 to 70.8 wt%. However, EA-S-CS from ACO decreased with an increase in the acid concentration. In the same range of 0%-1.0% dilute acid concentration, EA-S-CS decreased from 59.0 to 45.9 wt%. At 2.0% dilute acid concentration, EA-S and EA-S-CS decreased with an increase in the acidity.  Table 3 shows the holocellulose and lignin contents of EA-S and EA-S-CS calculated for each experimental run. The total amount of holocellulose harshly decreased with increasing dilute acid concentration. At 0.5% dilute acid concentration, 41.3 and 26.8 wt% of holocellulose still remained in EA-S and EA-S-CS, respectively. When the concentration increased to 2.0%, however, only 28.1 and 16.0 wt% of holocellulose remained in EA-S and EA-S-CS, respectively. Lignin exhibited somewhat different behavior in mass decrease. The amount of lignin did change slightly by the steam treatment. Hwoever, after ACO, 4.3%-6.7% of lignin was removed from the solid fractions. The removal efficiency of lignin between 0.5% and 2.0% acid concentration respectively. Similar studies were reported for the dilute acid treatment of rice straw [20] and corn stover [21]. However, the proper acid concentration or removal efficiencies of hemicellulose and lignin were different by feedstock properties. Since the purpose of this experiments is to lower only hemicellulose and lignin contents in EFB, so a high acid concentration was not adapted to avoid the loss of holocellulose contents.  Tables 4 and 5 show the total sugar contents of each product fractions (EA-S, EA-S-CS, EA-L, and EA-S-OL). Glucose content in the solid fractions showed no significant change while lignin content increased compared to the raw material. It means that the glucose in the EFB took limited damage by the steam explosion process and the organosolv treatment. It is possible that long reaction times increased the accumulation of degradation by-products and mass losses by volatilization [22]. As the acid concentration increased, however, the amount of glucose in the solid steadily decreased. When the acid concentration increased to 2.0%, the glucose amount decreased from 415 to 380 mg/g fraction. In contrast, concentration of glucose in EA-L dropped with 0.5 wt% acid pretreatment but increased by increasing acid concentration. Xylosyl groups were the most affected hemicellulose components during acid impregnation and the pretreatment process. The amount of xylose in the solid drastically decreased with increasing dilute acid concentration. As a result, concentration of xylose in the liquid fraction increased to twice of the control (10.1-21.9 mg/mL liquid). At 2.0% dilute acid concentration, more than 70% of the xylan in the EFB was hydrolyzed during two-step pretreatments. Arabinosyl, galactosyl, and mannosyl residues were removed from the solids by steam explosion with more than 0.5% acid concentration (Table 5).

Cellulose Crystallinity
The change of cellulose crystallinity as a result of the steam explosion and organosolv treatment was determined by analyzing the x-ray diffraction patterns ( Figure 4 and Table 6). By the XRD peak height method, crystallinity index was calculated from the ratio of the height of the 002 peaks (22.7 • ) and the height of the minimum (18.3 • ) between the 002 and the 101 peaks [23]. The crystallinity of EFB fiber decreased due to the steam explosion and organosolv treatment. The lowest crystallinity was shown in E-S-CS under the condition of 180-10 (35.9%). The crystallinity of EFB increased by increasing the severity factor. This might be caused by the removal of the amorphous structure of cellulose or components like hemicellulose.
Cellulose crystallinity after the dilute acid impregnation followed by two-step pretreatment was determined by analyzing the X-ray diffraction patterns. The crystallinity of EFB fiber also decreased by both ACSE process and ACO. There was little change of crystallinity, however, between the raw and organosolv treatment.

Chemical Properties of Organosolv Lignin
Qualitative and quantitative analysis of obtained organosolv lignins from E-S-OL were performed via pyrolysis-GC-MS analysis, and the results are listed in Table 7. Organosolv lignin obtained under steam explosion conditions of 200 • C and 5-10 min exhibited more than 14 peaks. Phenolic compounds, such as toluene (1), phenol (2), guaiacol (5), syringol (8), and isoeugenol (10), were the major compounds. As shown in Table 7, the number of chemical compounds and the overall peak size in the organosolv lignin decreased by increasing residence time. This might be explained by the fact that the lignin compounds can be condensed in harsher conditions and less decomposed by heat.

Conclusions
Two-step pretreatment, ACSE followed by ACO of EFB, was performed under a temperature range of 180-220 • C and residence time of 5-20 min after impregnation with 0.5%-2.0% dilute sulfuric acid. The mass balance and the chemical composition of pretreated EFB fractions were noticeably affected by the reaction temperature, residence time, and acid concentration during the steam explosion process. Moreover, glucose and xylose contents decreased under the acid-catalyzed pretreatment conditions. All the analyses together with the XRD peaks showed that the crystallinity of cellulose increased by increasing severity factor. In this experiment, the effect of two-step pretreatment was in no way remarkable but showed potential to advance it. Further study is needed to sort out the optimal conditions for this two-step EFB pretreatment to conclusively suggest its potential for EFB fractionation.


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Manipulation of Rumen Fermentation and Microbial Diversity for Live-Weight Gains of Sheep as Influenced by Ginger Powder and Lime Peel

The study was conducted to determine the influence of ginger powder and lime peel on manipulation of rumen function for live-weight gains of sheep. Twenty four West African dwarf sheep of about 8 – 9 months old with average weight of 7.00 ± 0.95kg were randomly assigned to four treatment diets in a completely randomized design. The prepared diets contained; ED1 (0% ginger powder and 0% lime peel powder that served as control group), ED2 (0% ginger powder and 3% lime peel), ED3 (1.5% ginger powder and 1.5% lime peel) and ED4 (3% ginger powder and 0% lime peel). In all the diets examined, ED1 showed higher significant (p<0.05) values in total fungi, Acetobact xylinum, Methanogens, total protozoa, Entodiniomorphs, Holotrichs, ammonia-nitrogen, total volatile fatty acids, acetate, butyrate and feed conversion ratio. However, total bacteria, Ruminococcus albus, Ruminococcus fibrisolvens, Bacillus spp, propionate, feed intake, nutrient digestibility, final and daily weight gains indicated significant (p<0.05) higher values in diet ED3 than diets ED1, ED2 and ED4. Rumen pH was significantly (p<0.05) higher in ED4 as compared with other diets. There was no significant (p>0.05) difference in Fibrobacter succinogens, iso-butyrate, valerate, iso-valerate and initial weight among diets. It can be concluded that using 1.5% ginger powder and 1.5% lime peel in the diets enhanced rumen fermentation and ecology for growth performance of sheep.

I. INTRODUCTION
Small ruminant production suffers insufficient feeds supply in the tropics, due to mainly low rain fall which can barely support natural pasture production in most years. This problem is further compounded by the competition between livestock and human feed industries for the available conventional feed ingredients. However, crop residues and agro by-products are considered as the most highly abundant and important available feed resources that are widely distributed at low cost with no competitive in their usage [1]. These feeds can be used to alleviate feed deficits and increase economic efficiency of feed production by reducing the pressure on cereal crops, particularly in regions where forages are scarce. Their high fiber content that is associated with low nutrient intake and digestibility limits their optimal utilization and productivity in ruminants [2], [3]. Thus, there is a strong incentive to search for natural feed additives with good potentials that can improve feed efficiency for enhancing ruminant performance.
Recently, there has been much interest in the utilization of plant herbs or extracts as source of additives to aid digestion of poor quality feeds due to their secondary metabolites. Natural herbs are classified as phytogenic additives that have anti-microbial, anti-oxidant with anti-inflammatory substances that can improve immune system and stimulate digestive enzymes of animals for better productivity. [4] reported that phytogenic additives possess great potentials in rumen modulation for regulating micro-flora due to their several bioactive components such as flavonoids and phenolic compounds.
Ginger powder and lime peel are such natural additives that can act as antimicrobial, antioxidant, and bioflavonoids agents for improving poor quality feeds digestion, fight infection, reduce inflammation and eradicates toxic elements in grazing herbivores. They are locally available, and their products can be stored for longer periods without deterioration in nutritive and secondary components. Several authors [5], [6] and [7] have reported that ginger powder as additive in ruminant diets can modify overall rumen fermentation to inhibit methane production and increase rumen escapes of nutrients for more efficient enzymes digestion of protein and starch in the intestine. Notwithstanding, there has been limited research to date on the use of lime peel as a dependent potential substitute for antibiotics in sheep diets. Thus, the objective of the study was to assess the manipulation of rumen fermentation and microbial population for live weight gains of sheep as influenced by ginger powder and lime peel.

A. Study Location
The study was conducted at the sheep and goat unit of the livestock Teaching and Research Farm, Ambrose Alli University, Ekpoma, Nigeria. The location lies on longitude 6.09 0 E and latitude 6.42 0 N within the humid climatic zone of South-South Nigeria. It has an average annual rainfall and temperature of about 1556mm and 31 0 C respectively with relative humidity of about 78%. The vegetation represents an interface of between the tropical rainforest and derived savannah.

B. Experimental Diets
Ginger root and lime peel were obtained within Ekpoma, cut into smaller surface area, air dried for five days before ginger was milled into powder form and lime peel was crushed into meal. Thereafter, they were incorporated into the supplementary diets at different and equal proportions (Table, 1). Guinea grass was cut at a re-growth age of between 7 and 8 weeks, allowed to wilt overnight and chopped into smaller sizes. However, guinea grass and supplementary diets were used in a ration of 60:40 in form of total mixed ration. The four prepared diets contained; ED1 (ginger powder 0% and lime peel powder 0% as control group), ED2 (ginger powder 0% and lime peel 3%), ED3 (ginger powder 1.5% and lime peel 1.5%) and ED4 (ginger powder 3% and lime peel 0%).

C. Animals, Design and Management
Twenty four West African dwarf male sheep of about 8-9 months old with initial average live weight of 7.00±0.95 kg were used for the study. They were quarantined and placed on prophylactic treatments for two weeks during which they were also subjected to control of external and internal parasites.
At the end of the adaption period, the sheep were randomly allocated to the four dietary treatments that were replicated three times with two sheep per replicate in a completely randomized design. The sheep were housed adjacent to each other in individual semi-open pens with feeders and water troughs available. They were offered feeds twice daily at about 8:00am and 4:00pm prompt. The quantity of the diet offered to each sheep was calculated on the basis of 5% dry matter of their body weight. The amount of feed given was adjusted every week according to live weight change. The sheep had free access to drinking water and the experiment lasted for 90 days exclusive of the two weeks adaptation period.

D. Intake and Live-Weight Change
Diets offered and leftover were measured for individual animals daily in the morning prior to feeding to compute the feed intake. Animals were weighed at the commencement of the study before giving the experimental diets, subsequently on weekly basis to estimate live-weight changes for the individual animals.

E. Digestibility
At the end of growth study four sheep per treatment were selected and transferred into individual metabolic cages made for separate collection of faeces and urine. They were acclimatized for the first 7 days, before another 7 days collection of faeces voided per sheep daily. The collected faecal samples for each sheep was bulked, properly mixed and about 10% sub-sample was pooled and kept in the freezer (-20%) for laboratory analysis. Thus, nutrient digestibility was calculated by standard procedures reported by [8].

F. Rumen Fermentation and Ecology
Fore-stomach sampling was preceded once weekly during the feeding trial of the sheep. About 100 ml of rumen liquor samples were collected from each sheep using oral stomach tube and inserted through the oesophagus approximately 3 hours after the incoming morning feeding. The fluid was monitored visually to ensure that they were not polluted with saliva. Rumen pH of the fluid was measured immediately after sampling using a portable digital pH meter (Mettler-Toledo Ltd, England). However, the liquor was squeezed through four layers of cheese-cloth to remove the feed coarse particles. The filtered liquor was divided into fractions for the determination of rumen fermentation and microbes. Sub-samples of 10ml were acidified with a 1m sulphuric acid solution to measure ammonia nitrogen (NH3 -N) concentration and total volatile fatty acid (VFA) as previously reported by [9]. Another 10ml sub-samples were treated with 2ml of 25% meta-phosphoric acid (prepared 5N sulphuric acid) kept overnight at 4 °C and centrifuged at 584 rpm for 15min to measure individual volatile fatty acids using gas chromatography according to the procedures reported by [2].
The other fraction of the rumen liquor was used for fungi and protozoa counts with preparation of bacteria culture before counting. Six strains of cellulolytic bacteria were isolated from rumen fluid and were grown in a purely cultural. The indentified and separated strains were Ruminococcus albus, Ruminococcus fibrisolvens, Bacillus spp, Fibrobacter succinogens, Acetobact xylinum and Methanogens. Pour-plate technique for pure preparation of cultures, according to [10] was used for the isolation of species. The samples were immediately gassed with carbondioxide viable counts of rumen cellulolytic bacteria and their classification were done as reported by [11] and [12] respectively. About 20 ml of the rumen fluid were also stored in 40ml of 10% formal saline for direct counts of One ml of the filtered rumen fluid was added to four ml of methyl green-formalin-saline solution for the counting. The number of protozoa was microscopically (100x) counted in a drop of rumen liquor with the defined volume with the division on the Entodiniomorphs and Holotricha groups as noted by El-Essawy (2019). Measurements were done in duplicate from each sample and the average of these measurements was used to estimated the number of protozoa present in the initial sample.

G. Chemical and Statistical Analyses
Feed samples were dried for 48 hours at 60 °C in a forced air oven to determine the dry matter (DM) content before ground in a wiley mill to pass through a 1mm sieve for proximate and phyto-chemicals composition according to the method described by AOAC (1990).
Data obtained from the study were subjected to analysis of variance (ANOVA) using the general linear model (GLM) procedures. Where significant difference between treatment effects occurred, means were separated by Duncan multiple range test [14]. The mathematical model used for the analysis is as follows: where Yijk = independent variable (live-weight gain, rumen fermentation........). G = overall mean of the observed variable. Ai = effect due to i th treatment diets. eijk = effect of random residual error.

III. RESULTS
In the three feed ingredients examined (Table 2), guinea grass (GG) recorded the highest content of crude protein and crude fibre but relatively lower in dry matter and nitrogen free extract values as compared with ginger powder (GP) and lime peel (LP). However, LP marked higher values in ether extract and ash than the values obtained in GP and LP. Notwithstanding, supplementary diets were varied according to the type and amount of feed ingredients used in the diets. The test diets had relatively higher dry matter, crude fiber and nitrogen free extract than the control diet. Generally, similar values were showed for crude protein, ether extract and ash with no remarkable difference among treatment diets. Phyto-chemical components had highest contents in GP, LP and test diets than GG and control diet as shown in Table 2. There were differences in the distribution of rumen microbial community (Table, 3) in sheep as influenced by inclusion of feed additives in diets. The levels of total fungi, cellulytic bacteria diversity of Acetobact xylinum and Methanogens in the rumen fluid were significantly decreased (P< 0.05) in counts as ginger powder and lime peel were added in the diets. On the other hand, their effects as additives showed extremely significant (P<0.05) higher values for total bacteria, Ruminococcus albus, Ruminococcus fibrisolvens and Bacillus spp as compared with control diet. However, Fibrobacter succinogens diversity indices observed in the study had no significant (P>0.05) difference irrespective of the additives included in the diets. Interestingly, higher significant (P<0.05) abundance spices of total protozoa, holotrich and entodiniomorphs were found in control diet than the test diets.
The use of natural feed additives in the study showed no significant (P>0.05) effects in values recorded for isobutyrate, valerate and iso-valerate among diets (Table,4). However, remarkable differences were observed in the rate of ammonia nitrogen, total volatile fatty acids, and acetate with butyrate production in the rumen fluid of sheep. It was avidly noticed that diet with no additives had significant (P<0.05) greater values than those with additives in their diets. The rumen pH and molar proportion of propionate were significantly (P<0.05) higher in diets with mixture of ginger and lime or solely ginger or lime inclusion than the control diet.
Feed intake, apparent digestibility and live weight change of sheep were significantly (P<0.05) influenced by feed additives in the diets except initial live weight that was not significantly (P>0.05) affected (Table 5). Total daily feed intake, digestibility of dry matter and crude protein were found to register higher values in test diets than the control group. Similarly, digestibility coefficient of crude fibre appeared to be higher in diet ED3 followed by ED4 and ED2 but considerably lower in control diet ED1. Higher values of final live weight and weight gains of sheep were recorded for diets ED2, ED3 and ED4 as compared with diet ED1 that had extremely lower values. However, feed conversion ratio was more pronounced in control diets than the test diets in terms of mean values.

IV. DISCUSSION
Herb plants are phytogenic in nature as they contain secondary metabolites and anti-microbial properties that improve health benefits and modify rumen fermentation to stimulate digestion in ruminants [15], [16]. When use as additives in livestock feeds, they can increase appetite and feed utilization efficiency to optimize performance [5]. The phyto-chemical components of ginger powder (GP) observed in this study was higher than the recorded values in lime peel (LP). However, their tannin levels were considered low and safe with regards to tannin poisoning critical values of 7.3 -9.0 mg/g reported by [6].
Nutrient contents obtained in guinea grass, GP and LP were comparable with the range values reported in literature by [16] and [7] respectively. Variations registered in nutrient composition of experimental diets were reflection of different feed ingredients used in the diets. It is important to note that the formulated diets crude protein values observed in the treatments were above 8% crude protein minimum value required for small ruminants' performance in the tropics [17].
The influences of ginger powder or lime peel and their mixture in diets were effective in reducing total fungi, Acetobact xylinum and Methanogens population in sheep. This could probably because of their phyto-chemical and poly-phenol components that were not appropriate for their thrived, hence affected their production. Feeding additives that are rich in secondary metabolites had been reported to reduce methane emission directly by inhibition of Methanogens and indirectly by reduction of fiber digestion fungi in the rumen [18]. Thus, the higher diversity of cellulolytic bacteria of Ruminococcus albus, Ruminococcus fibrisolvens and Bacillus spp in the test diets indicates that the additives used were the main contributors for their increased in production. This evidences demonstrated the effects of bioactive compounds in the diets which likely be the mechanism encouraging the increase of their outflow as bacteria spices that improved the utilities fermentation as part of digestive process and supply of energy to the host animal. Some researchers have proved that flavonoid in herb plants can improve the levels of Ruminococcus albus and the diversity of Ruminococcus fibrisolvens with Bacillus spp in the rumen of cows without any negative effect on their fermentation [4], [19]. Fibrobacter succinogens appeared to be dominant species in the study irrespective of the diets. The reason could be traced to their ability to tolerate moderate concentration of the secondary compounds in the diets. However, ciliate protozoa are important in ruminants but not essential in the rumen ecosystem and to the well-being of the host animals. They account for about half of the total microbial biomass in the rumen with 50% of the total fermentation products which make them a significant hydrogen producer that encourage methane emissions [20]. Their presences in rumen degrade dietary protein for production of ammonia nitrogen concentration. They also engulf bacteria for their amino acid requirement in the rumen ecosystem. Notwithstanding, the reduction of protozoa (defaunation) in the rumen of sheep on diets with additives in this current study might translated to the decrease in endo-symbiotic methanogens with low hydrogen availability that could led to reduction in energy wastage from digestion of grass cell walls. Perhaps positive effect of eliminating methanogens that existing as endo and ecto-symbiotic with ciliate protozoa in the rumen of sheep have been reported by [21] to increase ruminant average weight gain by 9% and reduced entric methane emission by 11%.
Fibre and starch digesting microbes thrive well at rumen pH of 6.0 to 6.8 and 5.5 to 6.0 respectively, with their balance level occurring at rumen pH of 6.0. Several authors [22], [23] have considered a rumen pH greater than 5.9 to be normal for fibre digestion. However, the rumen pH (6.12 to 6.83) of sheep obtained in this study were within the optimal normal range considered for microbial digestion activity but higher than the pH of 5.6 and 5.8 levels suggested to be marginal for developing problem of acidosis [23]. The decreased of ammonia nitrogen concentration in diets with ginger and lime peel or their mixture inclusion were consequence of defaunation and decreased degradability of protein feed that increased bacteria biomass in the rumen. It is noteworthy that, this could be an advantage to the sheep as it associated with greater rumen escape of microbial protein out flows into the duodenum which translated to higher supply of protein for efficient digestibility observed in sheep. This is in line with the report of [24] who observed that tannin component in herb plants can form complexes with protein and protect it from degradation in the rumen which eventually flows and dissociated under the acid condition of the abomasums and in turns leading to better digestion of protein in host animals, especially when feed is deficient in protein rather than energy content.
The strong correlation between defaunation and methanogens counts observed in the test diets had no negative impact on rumen fermentation and degradability but resulted in modulating rumen digestion of grass cell and starch feeds which were considered to be the two sources of energy supply for the host sheep. However, the beneficial effect of this valuable reason was seen in greater proportion of propionate and decreased fractions of acetate with butyrate in the rumen, leading to low rate of total volatile fatty acids production. This scenario could responsible for the decreased in the digestibility of fibre components in control diet. This is excepted as the connection between higher molar propionate with low fraction of acetate improved feed intake, digestibility and weight gains in sheep on test diets (Table, 5). It could also be relatively linked to the increase in rumen pH of sheep on test diets which was earlier stated by [25] as mechanism that may likely decreased the incidence of metabolic disorder such as acidosis in ruminants. This is implicated in recent review of [21] who found that decrease in acetate and butyrate will reduce methane emission to release hydrogen for incorporation into propionate acids that is the main precursor of glucose synthesis for efficient energy production require for metabolism and absorption in ruminants.

V. CONCLUSIONS AND RECOMMENDATION
The study discovered that, influence of ginger powder and lime peel in the diets of sheep did not show any adverse effect on rumen function but leads to variation in rumen ecology which affected the fermentation pattern and performance of sheep positively. These changes mostly reduced methanogens, protozoa numbers, acetate and butyrate with increased in propionate proportion. These positive results promoted feed intake and digestibility of nutrients which translated to better weight gains of sheep.
However, this enhancement that occurred in test diets was more efficient in diet that contained mixture of ginger powder 1.5% and lime peel 1.5% (ED3) than diet with either ginger powder or lime peel. Furthermore, long-term experiment is required in this study to determine the production benefits of using mixture of ginger powder and lime peel in the diets of sheep.