**Assessment of Agro-Environmental Impacts for Supplemented Methods to Biochar Manure Pellets during Rice (***Oryza sativa* **L.) Cultivation**

#### **JoungDu Shin 1,\*, SangWon Park <sup>2</sup> and Changyoon Jeong <sup>3</sup>**


Received: 19 March 2020; Accepted: 14 April 2020; Published: 21 April 2020

**Abstract:** The agro-environmental impact of supplemented biochar manure pellet fertilizer (SBMPF) application was evaluated by exploring changes of the chemical properties of paddy water and soil, carbon sequestration, and grain yield during rice cultivation. The treatments consisted of (1) the control (no biochar), (2) pig manure compost pellet (PMCP), (3) biochar manure pellets (BMP) with urea solution heated at 60 ◦C (BMP-U60), (4) BMP with N, P, and K solutions at room temperature (BMP-NPK), and (5) BMP with urea and K solutions at room temperature (BMP-UK). The NO3 −–N and PO4 −–P concentrations in the control and PMCP in the paddy water were relatively higher compared to SBMPF applied plots. For paddy soil, NH4 <sup>+</sup>–N concentration in the control was lower compared to the other SBMPFs treatments 41 days after rice transplant. Additionally, it is possible that the SBMPFs could decrease the phosphorus levels in agricultural ecosystems. Also, the highest carbon sequestration was 2.67 tonnes C ha−<sup>1</sup> in the BMP-UK treatment, while the lowest was 1.14 tonnes C ha−<sup>1</sup> in the BMP-U60 treatment. The grain yields from the SBMPFs treatments except for the BMP-UK were significantly higher than the control. Overall, it appeared that the supplemented BMP-NPK application was one of the best SBMPFs considered with respect to agro-environmental impacts during rice cultivation.

**Keywords:** Mitigation of CO2-equiv.; nutrient release; rice paddy water and soil system; slow-release fertilizer

#### **1. Introduction**

Developing methodologies to improve crop productivity and protect soil systems while mitigating environmental pollution is the current direction of research in sustainable agriculture [1–3]. Recently, biomass conversion from agricultural wastes to carbon-rich materials such as biochar has been recognized as a promising option to maintain or increase soil productivity [4], reduce nutrient losses [5], and mitigate greenhouse gas emissions [6] from the agroecosystem. It is estimated that 50 million tonnes of the 80 million tonnes of organic wastes produced in Korea originate from agriculture [7]. Carbon sequestration utilizing recycled organic wastes through biomass conservation technology can greatly mitigate greenhouse gas emissions and the environmental impact of organic waste in Korea. Biochar is made through the pyrolysis under high temperature in oxygen-limited conditions [8]. Converted biochar from agricultural biomass becomes recalcitrant carbonaceous structures. The structures and components of biochars are strongly related to the source of feedstock and the operating

conditions that are used in biochar production. Cantrell et al. [9] documented that the biochar made of poultry litter presented a relatively high nutrient content comparable to fertilizer. The reported analytical characterization of biochar is ranges between 5.2–10.3 in pH, 1.1–55.8% in ash content, 23.6–87.5% in carbon content, and 0–642 m<sup>2</sup> g−<sup>1</sup> in surface area [8,10,11]. Kim et al. [12] reported ranges of 10–69 cmolc kg−<sup>1</sup> in the cation exchange capacity (CEC) of biochar. Biochar application can significantly increase plant growth, crop yield, and root biomass by enhancing nutrient use efficiency [13,14]. However, few studies have reported a negative growth response in the early stages of plant growth [15,16]. Thus, research on the incorporation of biochar as a soil amendment in crop fields is still required to improve the production methods and application of biochar in soil. Drift of biochar occurs during field application due to the low density and irregular particle size of biochar. Husk and Major [17] reported that the biochar drift during field application was 25%, while the surface runoff losses due to intense rain events were estimated from 20% to 53% of incorporated biochar [18]. Pelletizing biochar can be a possible solution to minimize losses during field application, and it can also reduce handling and transportation costs [19].

Animal waste composts are recognized as valuable sources of major plant nutrients that reduce the need for synthetic fertilizers [20]. However, environmental problems such as nutrient loss due to surface runoff may arise if excess manure is applied to the agricultural land in sensitive catchment areas. One of the critical issues plaguing animal waste compost application is the lack of an environmentally safe application method to agricultural land in order to mitigate non-point source pollution [21,22]. Most of the nutrients losses from agricultural lands are caused by soil erosion from irrigated agriculture or runoff and leaching after rainfall events [23]. Hence, the top priority was to develop methods that would minimize rapid nutrient loss from animal waste manure application and mitigate nutrient runoff after irrigation or rainfall events. Major pathways of N losses are NH4 <sup>+</sup>–N and NO3 −–N leaching, NH3 volatilization, and runoff losses. New strategies such as biochar-manure pelletizing methods are available to minimize N loss from the application of animal-waste compost. New approaches that would improve the efficiency of compost are significant to agricultural production in Korea, because the amount of animal waste must be disposed in an effective manner with a minimal impact on agricultural eco-systems.

In general, the production of biochar pellets with poultry litter mixed with switch grass (BMP) is relatively simple. Pellet is blended poultry litter with powder of switchgrass, and then BMP is produced with slow pyrolysis [24]. Several scientists reported that the synergistic effects of biochar blended with inorganic fertilizer or biochar mixed with nutrient-rich compost were observed to improve crop yields [25–27]. There is only limited information on the field application of supplemented biochar manure pellets with inorganic fertilizers (SBMPFs). SBMPF provides supplemental nutrients and can also regulate nutrient loss or release rate by functioning as a slow release fertilizer. Slow-release fertilizers gradually discharge nutrients to the soil during the growing season and provide sufficient nutrients to crops while minimizing leaching losses [28], which can increase farmers' profits and minimize environmental impacts [29]. Ultimately, this application ameliorates the loss of income in agro-business and mitigates the potential contamination of agricultural watersheds. SBMPFs thus represent an efficient way to decrease field application costs and biochar loss during soil application [19].

However, only limited information on blended biochar pellets functioning as slow-release fertilizers is available. Kim et al. [30] indicated that the application of a combination of biochar and slow release fertilizers yielded the lowest methane emissions among the treatments due to the inhibition of methanogenic bacteria via increased soil aeration and improved rice yield compared to the control.

Additional benefit for cropland application of biochar is carbon sequestration [31,32]. Biochar has a much longer residency period (up to 1000 years) compared to raw materials because of its recalcitrance to biotic and abiotic degradation [33]. However, biochar is partly degraded and oxidized into CO2 when incorporated into soils [34] and up to 50% of feedstock carbon may be lost during pyrolysis [31,35]. Therefore, reduction of carbon during biochar production and increasing its stability in the soil would improve its potential for carbon sequestration. In terms of soil carbon sequestration

and the mitigation of CO2-equiv. (carbon dioxide equivalency) emission, biochar incorporated with cow manure compost can sequester 2.3 tonnes C ha<sup>−</sup>1, and ranges from 7.3 to 8.4 tonnes ha−<sup>1</sup> for mitigating CO2-equiv. emission in the cornfield [36]. Shin et al. [37] indicated that the application of biochar pellets blended with organic compost is a promising way to increase carbon sequestration during crop cultivation. For the application of BMP, carbon sequestration and mitigation of CO2-equiv. emission were 1.65 tonnes ha−<sup>1</sup> and 6.06 tonnes ha−<sup>1</sup> greater than those of the control, respectively, during rice cultivation [38]. Soil carbon sequestration from the application of biochar made of wood branch increased from 1.87 to 13.37 tonnes ha−1, while the plots with rice straw application demonstrated decreased soil carbon from 2.56 to 0.92 tonnes ha−<sup>1</sup> [39].

The objective of this study was to evaluate the agro-environmental impact of supplemented biochar manure pellet fertilizers (SBMPFs) application on the agro-ecosystems and soil carbon sequestration during the rice growing season. It is hypothesized that the SBMPFs can significantly mitigate non-point pollution sources and increase potential carbon sequestration in agro-ecosystems.

#### **2. Materials and Methods**

#### *2.1. Biochar Production*

Biochar derived from rice hull was purchased from a local farming cooperative society in Go-chang, JeonBuk, South Korea. The top to bottom pyrolysis method to produce biochar was employed, wherein rice hull is burned from the upper level to bottom, and reduces oxygen flux from the exterior of the pyrolysis system at 29.4 KPa of air suction rate. The maximum temperatures during pyrolysis were from 490 ◦C at the top and 550 ◦C at the bottom of the pyrolysis system. The loading volume in each batch was 1.5 m<sup>3</sup> of rice. The biochar was milled with a grinder to pass through a 2-mm sieve before chemical analysis. The same raw materials were used for both the biochar and pig manure compost, and their chemical properties are shown in Table 1 [37,38]. The moisture contents of the biochar and pig manure compost were 5.5% and 27.2%, respectively. The biochar was generally alkaline with a pH of 9.7 and low in total nitrogen (TN), 2.0 g kg<sup>−</sup>1.


**Table 1.** Chemical properties of biochar and pig manure compost used 1.

<sup>1</sup> EC; Electric conductivity, TC; Total carbon, TOC; Total organic carbon, TIC; Total inorganic carbon, and TN; Total nitrogen. The values were average of triplicates samples with standard deviation.

#### *2.2. Production of Supplemented Biochar Manure Pellet*

The processing of SBMPFs is described in Figure 1. Prior to pelleting, biochar was processed in a series of sieves (0.5–5 mm) to ensure even particle distribution. In producing biochar pellets, 40% biochar was mixed with 60% pig manure compost as a binder. The SBMPF was completely mixed by using an agitator while spraying different nutrient solutions in the mixtures, and then feeding it into a commercial pellet mill (7.5 KW, 10HP, KumKang Engineering Pellet Mill Co., Daegu, South Korea). Different biochar pellets (Patent number: 10-1889400) treated with (1) urea solution heated at 60 ◦C (BMP-U60), (2) N, P, and K nutrient solutions at room temperature (BMP-NPK), (3) urea and K solutions at room temperature (BMP-UK), and (4) pig manure compost only (PMCP) pelletized. The size of BMPFs was approximately Ø 0.51 cm × 0.78 cm. The total carbon, TN (total nitrogen), TP (total phosphorus), and TK (total potassium) contents of BMPF embedded with different treatments are described in Table 2. Their total carbon and nitrogen contents varied from 225 g kg−<sup>1</sup> to 289 g kg−<sup>1</sup> and from 29.1 g kg−<sup>1</sup> to 102.0 g kg<sup>−</sup>1, respectively. It was observed that the BMP-U60 had the highest nitrogen content of 102.0 g kg−<sup>1</sup> and BMP-UK had the lowest nitrogen content of 84.0 g kg<sup>−</sup>1.

**Figure 1.** Diagram of processing the supplemented biochar manure pellets with different types of fertilizer.

**Table 2.** Total carbon, total nitrogen, total phosphorus and total potassium contents of supplemented biochar manure pellet fertilizers 1.


<sup>1</sup> TC; Total carbon, TN; Total nitrogen, TP; Total phosphorous, TK; Total potassium; \* BMP-U60; BMP blended with urea solution heated at 60 ◦C, BMP-NPK; BMP blended with N, P and K nutrient solutions at room temperature and BMP-UK, BMP blended with N and P nutrient solutions at room temperature. The values displayed are averages of triplicate samples with standard deviation.

#### *2.3. Field Experiment*

The experimental field was cultivated with rice monoculture, and it has clay loamy soil. It is located at 35◦49.510 N of latitude and 127◦2.536 E of longitude in the National Institute of Agricultural Sciences (NIA), Rural Development Administration (RDA), Jeonju, Republic of Korea. The precipitation amount and average temperature were 718 mm and 22.3 ◦C during the rice cultivation season, respectively. Additionally, the solar radiation quantity and duration of sunshine are measured at 2753.2 MJ and 949.9 h during the cultivation period, respectively. The rice variety used in this experiment was Shindongjin, with a planting distance of 30 × 60 cm. The experimental design was a block design with five treatments consisting of (1) the control, (2) PMCP, (3) BMP-U60, (4) BMP-NPK, and (5) BMP-UK with three replications and 16 m<sup>2</sup> of the plot size. The amount of fertilizer and manure compost applied in the control and PMCP treatment were 90-45-57 kg ha−<sup>1</sup> (N-P-K) and 2600 kg ha<sup>−</sup>1, respectively, which was based on National Institute of Agricultural Sciences (NIA) recommended rates for rice cultivation [40]. The SBMPFs were incorporated into the soil based on 90 N kg ha−<sup>1</sup> for whole basal application at 5 days prior to rice transplanting. Water logging time was 6 days prior to rice transplanting. The date of rice transplant was May 23, and drainage times were 14 days, 35 days, and 93 days after transplanting with one-week drainage. Rice was harvested 154 days after transplanting period. To evaluate the agricultural impact of different SBMPFs, major plant nutrients were analyzed from the

surface water and soil in the paddy during rice cultivation. For rice growth responses, the plant height and number of tillers were measured about 100 days after rice transplanting, while the grain yield and dry weight of rice straw were weighed after harvest. For the effect of SBMPF applications in the paddy, the physicochemical properties of the soil used are presented in Table 3.


**Table 3.** Soil physicochemical properties of experimental field 1.

<sup>1</sup> EC; electric conductivity, TC; Total carbon, TOC; Total organic carbon and ND; Non detected with 1 mg kg−<sup>1</sup> of detection limit. The values displayed are averages of triplicate samples with standard deviation.

#### *2.4. Chemical Analysis of Paddy Soil and Water*

After rice transplantation in the paddy, surface soil and water samples were collected every 20 days. The collected water samples were filtered through Whatman 2. The surface water was analyzed for NH4 <sup>+</sup>–N, NO3 <sup>−</sup>–N, K<sup>+</sup>, and SiO2 content using a UV spectrophotometer (C-Mac, Dae-Jeon, Korea) throughout the cropping season. The wet soil samples were extracted by using a 2M KCl solution (1:5, soil: extractant ratio). Those samples were analyzed directly for NH4 <sup>+</sup>–N and NO3 −–N by using the Bran-Lubbe Segmented Flow Auto Analyzer (Seal Analytical Ltd., Wisconsin, USA), and then the NH4 <sup>+</sup>—N and NO3 —N concentrations were calculated by compensation for moisture contents of wet soil. The extractant using the Mehlich III method [41] from dried soil samples that passed through 2 mm sieves were stored in a refrigerator at 4 ◦C until PO4 <sup>−</sup>, K<sup>+</sup> and SiO2 were analyzed using a UV spectrophotometer (C-Mac, Dae-Jeon, Korea). Total carbon (TC) in soils was analyzed with total organic carbon (TOC) analyzer (Elementa vario TOC cube, Hanau, Germany). The combustion temperature was 950 ◦C and tungsten trioxide (WO3) was used as the catalyst. With 350mg of soil samples, total nitrogen (TN) contents were determined by dry combustion with 250mg of L-Glutamic acid, standard compound, by using vario Max CN (Elementar, Hanau, Germany).

#### *2.5. Data Processing and Carbon Balance Calculations*

The soil carbon sequestration via BMPFs application was calculated from the difference of the residual amount of soil carbon between the control and different treatments after rice harvest by using the following equation [38]:

$$\text{SS}\_{\text{TC}} = \left\{ \sum\_{i=0}^{n} \text{T}\_{\text{TC}} \left( \text{Li} - \text{li} \right) - \text{NT}\_{\text{TC}} \left( \text{Li} - \text{li} \right) \right\} \times \text{SW} \tag{1}$$

where SSTC (kg ha<sup>−</sup>1) is the potential sequestration amount of soil carbon, T (kg ha−1) is the treatment of SBMPFs, NT (kg ha<sup>−</sup>1) is the control, TC is total carbon content (g kg<sup>−</sup>1), i is the sampling date, Li and Ii are carbon contents of the last and initial samplings which analyzed the soil carbon content (g kg<sup>−</sup>1), and SW is the soil weight (bulk density, 1.3; 10cm of plowing soil depth, kg ha<sup>−</sup>1).

The mitigation of CO2 emission for SBMPFs application was also estimated using equation [38]:

$$\text{CO}\_2 = \text{SS}\_{\text{TC}} \times \text{CF}\_{\text{SC}} \tag{2}$$

where SSTC is the amount of soil carbon sequestration (tonnes ha<sup>−</sup>1) and CFSC is the conversion factor of CO2 emission from soil carbon (1 kg C = 3.664 kg CO2-equiv.).

Profit analysis for the mitigation of CO2 emission was also calculated by using the equation [38]:

$$P = \text{AM} \times \text{MP} \tag{3}$$

where P is the profit of carbon dioxide trading (\$ ha<sup>−</sup>1), AM is the amount of mitigation of CO2 emission (tonnes ha<sup>−</sup>1), and MP is the market prices of CO2 offsets (\$ per tonnes CO2). Also, the trading prices of CO2 offsets in the European Climate Exchange (ECX) varied between \$4.1 and \$7.9 per tonnes CO2 in 2016 [42] while the Korean Climate Exchange (KCX) ranged from \$7.9 to \$19.3 per 1 Korean Allowance Unit (KAU) [43].

#### *2.6. Statistical Analysis*

Statistical analysis was conducted using SAS version 9.2 Software (SAS, Inc., Cary, NC, USA), with an ANOVA with Duncan multiple range tests for the comparison of treatments with carbon contents at 1st day of rice transplanting and day after harvesting, carbon sequestration, and growth components during rice cultivation. Standard deviation was used for comparisons of paddy water and soil chemical properties.

#### **3. Results and Discussions**

#### *3.1. E*ff*ects of Essential Nutrients in the Paddy Water and Soil*

#### 3.1.1. Paddy Water Quality

The NH4 <sup>+</sup>–N and NO3 −–N concentrations in the surface paddy water are presented in Figure 2. At the first day of rice transplanting, the NH4 <sup>+</sup>–N concentration of surface paddy water in the MBP-NPK was significantly higher than the other treatments, but its control showed nearly the same values than the other treatments. However, the NO3 −–N concentrations in the control and PMCP were only significantly higher than those in the SBMPF treatments. It was observed that NH4 <sup>+</sup>–N concentrations in the treatments were higher on the first day of rice transplants, but similar to the rest of the days. The loss of nitrogen under the application of SBMPF was almost complete within 21 days after rice transplantation. This might be due to the adsorption of NH4 <sup>+</sup>–N by the applied biochar in the soil. Regardless of the treatments at 112 days of rice transplanting, the NO3 −–N concentrations were higher compared with other sampling days (93 days) due to the start of drainage of the surface water in the rice paddy. The study showed that the application of SBMPs can be a solution to mitigate the loss of nitrogen and phosphorus [44].

The PO4 <sup>−</sup>–P, K<sup>+</sup>, and SiO2 concentrations in the surface paddy water under application of BMPFs are described in Figure 3. The measured PO4 - –P concentration in the control and PMCP treatment was 2.8–5.3 times higher than the value in BMP-U60, BMP-UK, and BMP-NPK, respectively, until 21 days after rice transplantation. The PO4 - –P concentrations were not significantly different (*p* > 0.05) from 41 days to 93 days after rice transplanting among the treatments. The greatest differences in K+ concentrations can be seen at 41 days after transplant. The higher values in the control and PMCP were 28.5 mg L<sup>−</sup>1, and the lowest in the BMP-U60 was 9.6 mg L−1, but not significantly different (*p* > 0.05) with that of BMP-UK.

**Figure 2.** Effects of different treatments on NH4 <sup>+</sup>–N and NO3 −–N contents in rice surface paddy water during rice cultivation. The values displayed are averages of triplicate samples with standard deviation.

Silicon (Si) in soil exists in an unavailable form, but the Si in crop residues is a useful structure (H4SiO4) compared with Si fertilizer for crop uptake [45]. This recycled Si is leached into soil after the decomposition of crop residues. It is observed that SiO2 concentration ranged from 10 mg L−<sup>1</sup> to 35 mg L−<sup>1</sup> during the cultivation period, and the highest SiO2 concentration was 34.4 mg L−<sup>1</sup> in the BMP-UK at after 41 and 112 days of rice transplanting. However, SiO2 concentrations in the paddy water under the application of SBMPFs were higher than those of the control and PMCP at 112 days after transplant. The most commonly used silicon fertilizer is wollastonite for soil application because of its high solubility for plant uptake (2.3–3.6%) [46]. Recently, much attention has been paid to biochar as an alternative soil ameliorant because it could slowly release 43 mg kg−<sup>1</sup> for the available plant uptake of silica [47]. The 1% KOH solution treated biochar application to soil significantly increased available form of silicon in the plant [48]. In this study, the SiO2 concentration was significantly increased at the harvesting time under the application of SBMPFs. Thus, the incorporation of SBMPFs had the potential ability to recycle silica. Overall, the PO4 —P, K<sup>+</sup>, and SiO2 concentrations were significantly higher than the other sampling days (93 days) due to the start of drainage of the surface water in the paddy field.

**Figure 3.** Effects of different treatments on PO4 <sup>−</sup>–P, K<sup>+</sup> and SiO2 concentrations in surface paddy water during rice cultivation. The values displayed are averages of triplicate samples with standard deviation.

#### 3.1.2. Nutrients in Paddy Soil

Urea application is usually the main source of ammonium ions because urea can be hydrolyzed into NH4 <sup>+</sup> and OH<sup>−</sup> by the ammonification reaction within short periods after application in the paddy soil. The major nutrient concentrations in the soil are described in Figure 4. NH4 <sup>+</sup>–N concentration in the BMP-NPK was highest among the treatments at 41 days after rice transplanting. Total nitrogen losses were reduced with the incorporation of rice straw in the rice paddy soil due to increasing immobilization [49] and denitrification [50]. P2O5 concentrations except the PMCP were not significantly different during 21 days after rice transplanting among treatments. The K2O concentrations in the soil treated with BMPFs continuously decreased during rice cultivation due to the K<sup>+</sup> solubility, except for the BMP-U60 treatment. Biochar application increased the availability of K<sup>+</sup> and P because it was a net source of cations due to increased soil capacity to hold exchangeable cations [51,52]. The application of biochar produced from rice straw increased the available P and K<sup>+</sup> by 15.3% and 28.6% in the soil, respectively. However, biochar application did not significantly increase total nitrogen compared with the control in the rice paddy [53]. Overall, the release of major nutrients to soil under the application of SBMPFs was significantly lower compared with those from the control and PMCP.

**Figure 4.** NH4 <sup>+</sup>–N, P2O5 and K2O concentrations under different treatments in the paddy soil during rice cultivation. The values displayed are averages of triplicate samples with the standard deviation.

#### *3.2. Carbon Sequestration and Profit Analysis*

Soil carbon sequestration was only considered after soil analysis from rice paddy incorporated SBMPFs at day 1 of rice transplanting and the day after harvesting. Changes of total carbon contents in paddy soil under different treatments at the initial stage and after harvesting are described in Table 4. The carbon contents on first day of rice transplanting and the day after harvesting were significantly (*p* < 0.001) different in the treatments. There was minimal difference in total carbon content in the control between the first day of rice transplanting and after harvesting.

The application of biochar incorporated to the soil has been suggested as a promising method for carbon sequestration as well as another method for mitigating greenhouse gas, increasing crop yields and enhancing the sorption of pollutants [49,54]. Regarding carbon sequestration, it might be distinguished that short term released CO2 refers to the retention time of sequestrated carbon in soil from organic matter decomposition, while long term, it is stored as biochar from thermal conversion materials [38].


**Table 4.** Carbon contents in the soils treated with different supplemented biochar manure pellet fertilizers on first day of rice transplant and day after harvest \*.

\* Mean values followed by different letters, which indicate significant differences (*p* < 0.05) among treatments with One way ANOVA by the mean comparison for all pairs using Tukey-Kramer HSD analysis for total carbon contents on first day of rice transplant and the day after harvest.

For the application of different types of SBMPFs, carbon sequestration, mitigation of CO2, and profit analysis were calculated by using Equations (1)–(3), respectively (Table 5). The analysis of carbon sequestration showed 2.67 tonnes C ha−<sup>1</sup> in the BMP-UK as the best treatment for carbon sequestration, and 1.14 tonnes C ha−<sup>1</sup> in the BMP-U60 as the worst. It appeared that their recovery rates varied from 25.4% to 48.5% of SBMPFs applied to the rice paddy. It was observed that the mitigation of CO2 increased with the application of BMPFs, and the highest was 5.09 tonnes C ha−<sup>1</sup> in the BMP-UK. The profit under SBMPFs application was estimated to range from \$6.56 ha−<sup>1</sup> to \$68.80 ha−<sup>1</sup> during rice cultivation for KAU. The target of the Korean government is to reduce greenhouse gas emissions by 1.48 million tonnes CO2-equiv. (5.2%) of the 28.49 million tonnes CO2-equiv. total greenhouse emissions in the agricultural sector by 2020 [55]. Therefore, it is estimated that the 482,085 ha−<sup>1</sup> (29.3%) of 1,644,000 ha−<sup>1</sup> total area of rice cultivation with the BMP-NPK application in Korea [56] is required to accomplish this goal.

In order to establish carbon trading in the agriculture sector, policymakers should prepare a draft policy specifically for mitigating greenhouse gas emissions by providing support to farmers of about \$58 per hectare of cultivated rice paddy through the application of BMP-NPK. The application of BMPFs did not only increase carbon storage, but also enhanced rice yield and soil fertility [38].


**Table 5.** Evaluation of carbon sequestration and its profit analysis for application of supplemented biochar manure pellet fertilizers during rice cultivation.

kg C = 3.664 kg CO2-eqiv., 1 tonnes CO2 = KAU = 23,000 (8.12) = \$13.53.

#### *3.3. Rice Growth Responses to Supplemented Biochar Manure Pellet*

Growth responses to the application of SBMPFs are shown in Table 6. The plant height in BMP-U60 was 15.2% higher than the control, and rice yield in the BMP-U60 was increased by 15.7% compared with the control, even when the application amount of pig manure compost applied was reduced to about 1000 kg ha<sup>−</sup>1. This result might be due to the enhanced nutrient use efficiency under application of BMPFs functioning as a slow release fertilizer. Min et al. [4] reported that supplemented BMPFs application enhanced rice yield. Shin et al. [38] also reported similar results in their study. With the whole basal application of SBMPFs in the rice field prior to rice transplanting, it could prevent additional fertilizer application. Puga et al. [57] conducted similar research to evaluate the effects of biochar-based N fertilizers on nitrogen use efficiency (NUE) and maize yield. Their results showed that an average maize yield was increased 26% in the application of biochar-based N fertilizers (51% biochar with 10% N) compared with urea only treatment, and the NUE was 12% improved. Pokharel and Chang [58] also reported that manure pellet with wood chip biochar significantly increased plant grain yield by 36.3 and 16.1%, compared to the control, while woodchip with biochar applications significantly decreased plant grain yield.


**Table 6.** Characteristics of rice growth to supplemented biochar manure pellet fertilizer application.

#### **4. Conclusions**

Different supplemented biochar manure pellet fertilizers were tested to assess their agroenvironmental impacts on paddy water and soil systems during rice cultivation. With regard to the water quality of paddy, the NO3 <sup>−</sup>–N and PO4 −–P in control and PMCP were relatively higher than those of the SBMPFs applied plots. Non-point pollutants in runoff water to small stream near the rice cultivation area were reduced with application of SBMPFs. Considering the soil chemical properties, NH4 <sup>+</sup>–N concentration in control was lower compared with the SBMPFs treatment at 41 days after rice transplant. However, the available P2O5 concentrations were almost stage-state among all the treatments from 21 days after rice plant until the harvest period, except for the first day of rice transplant in the PMCP. It is possible that the SBMPFs can be applied with whole basal application without additional application of chemical fertilizers. Also, the highest carbon sequestration was 2.67 tonnes C ha−<sup>1</sup> in BMP-UK treatment, and the lowest was 1.14 tonnes C ha−<sup>1</sup> in the BMP-U60 treatment. The grain yields from the SBMPF applied plots, except for BMP-UK, were significantly higher than the yield from the control even though amounts of pig manure compost applied were decreased from 1881.8 kg ha−<sup>1</sup> to 2070.8 kg. Therefore, the application of SBMPFs can contribute to reducing the agro-environmental impacts of runoff as well as enhance carbon sequestration and rice yield in agro-ecosystems.

**Author Contributions:** Project leader and original draft writing, J.S.; Statistics and visualization, S.P.; review and editing, C.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded beyond Research Program of Agricultural Science & Technology Development (Project No. PJ013814012020) in Korea.

**Acknowledgments:** We are thankful to the National Institute of Agricultural Sciences, Rural Development Administration in Korea.

**Conflicts of Interest:** The author certifies that there are no affiliation with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

#### **References**


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### *Article* **Natural Grasslands as Lignocellulosic Biofuel Resources: Factors Affecting Fermentable Sugar Production**

**Linda Mezule 1,\*, Baiba Strazdina 2, Brigita Dalecka 1, Eriks Skripsts <sup>3</sup> and Talis Juhna <sup>1</sup>**


**Abstract:** Semi-natural grassland habitats are most often limited to animal grazing and low intensity farming. Their potential in bioenergy production is complicated due to the heterogeneity, variation, accessibility, and need for complex pre-treatment/hydrolysis techniques to convert into valuable products. In this research, fermentable sugar production efficiency from various habitats at various vegetation periods was evaluated. The highest fermentable sugar yields (above 0.2 g/g volatile solids) over a period of 3 years were observed from habitats "xeric and calcareous grasslands" (Natura 2000 code: 6120) and "semi-natural dry grasslands and scrubland facies on calcareous substrates" (Natura 2000 code: 6210). Both had a higher proportion of dicotyledonous plants. At the same time, the highest productivity (above 0.7 t sugar/ha) was observed from lowland hay meadows in the initial stage of the vegetation. Thus, despite variable yield-affecting factors, grasslands can be a potential resource for energy production.

**Keywords:** fermentable sugar; enzymatic hydrolysis; lignocellulosic biomass

#### **1. Introduction**

Worldwide attention towards application of waste materials for energy and highvalue chemical production has become a standard. Extensive use of agricultural and wood processing waste in lignocellulosic biofuel production increases the overall turnover of this industry annually. Furthermore, the use of lignocellulosic biomass for biofuel production is now facilitated by the European Union (EU) Renewable Energy Directive 2018/2001 [1]—the resource is included as alternative raw material under Annex IX. Regrettably, biomass recalcitrance towards saccharification is often the major limitation in the conversion of the resource to valuable end-products. Effective and economically feasible extraction of fermentable sugars is closely linked to the selection of an appropriate pretreatment/hydrolysis technique and to the type of biomass used. A tremendous amount of studies have been performed to evaluate the potential of certain biomass resources, e.g., wheat or barley straw, corn stover, with various technologies and their combinations [2,3], resulting in an extensive amount of data and laboratory scale research. Furthermore, it has been demonstrated that the combination of climate, soil fertility, and grassland biomass type can influence the overall bioenergy potential, i.e., hydrolysis efficiency and fermentable sugar yields [2,4].

Currently in the EU, more than 61 million hectares are occupied by permanent grasslands [5] where temperate semi-natural grasslands with a long extensive management history represent the richest species ecosystems on earth. At a small spatial scale, their vascular plant diversity exceeds tropical rainforests, which are normally considered as global maxima [6]. Ref. [7] described the trend of grassland management abandonment due to economic reasons in Europe, leaving huge amounts of this resource unused. The abandoned areas are predominantly semi-natural and nature conservation grasslands, bearing

**Citation:** Mezule, L.; Strazdina, B.; Dalecka, B.; Skripsts, E.; Juhna, T. Natural Grasslands as Lignocellulosic Biofuel Resources: Factors Affecting Fermentable Sugar Production. *Energies* **2021**, *14*, 1312. https:// doi.org/10.3390/en14051312

Academic Editor: Alberto Coz

Received: 4 January 2021 Accepted: 23 February 2021 Published: 28 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

a large variety of plant and animal species. Most of these grasslands are characterized by low productivity, but the optimal management regime includes low-intensity agricultural practices. In many cases, this means controlled grazing or late seasonal harvest that leads to the creation of patchiness, selection of particular species, or high amounts of lignin and cellulose in the biomass, respectively. Thus, forage quality is reduced [8,9]. Therefore, it is necessary to find alternative management regimes to maintain the biodiversity in European manmade landscapes [10] and at the same time to facilitate sustainable use of this resource. Unfortunately, semi-natural grasslands cannot be evaluated on a species level due to the high diversity and variability of the vegetation. Species composition and, especially, the coverage and distribution of particular species can vary even within one vegetation class or small grassland plot. Furthermore, it is influenced by environmental conditions [11–13], management [14,15], surrounding areas [16], land use history [17], and other factors. Thus, it is crucial to investigate and perform proper evaluation of the local grassland variations, their productivity and variability to estimate the costs and possible yields of biomass that can be further converted into high value chemicals, including biofuels [18].

Grass co-digestion with other waste streams to produce biogas has been shown to be efficient [19]. It is estimated that 8–17% of the current grassland biomass could provide up to 1% of EU transport fuel [20]. However, the high effect of area-specific biomass diversity, cutting time, accessibility, and need for pre-treatment have limited the potential use of grass in biogas production at an industrial level [21,22]. As an alternative to methane production via complex anaerobic digestion process, the use of lignocellulosic grassland biomass has been demonstrated for fermentable sugar production [23], which is an intermediate stage to produce various liquid biofuels, e.g., bioethanol or biobutanol, high value chemicals, used as an additional feedstock in biogas stations or regarded as a first step towards biorefinery [20]. The aim of this study was to evaluate fermentable sugar yields and overall productivity potential from various grassland habitats that are common in a temperate climate and classified under EU habitat codes. To aid towards biorefinery, non-commercial enzymes extracted from white rot fungi were used in the hydrolysis. The assessment involved not only the evaluation of habitat type but also seasonality, cutting time, species diversity, and solid content in the biomass. In-house made enzymes were preferred to commercial products due to their potential onsite production capacity and, thus, minimization of manufacturing costs. To the best of the authors' knowledge, this is the first study where the Natura 2000 grassland habitat classification [24] is linked with fermentable sugar productivity in the Baltic region, thus offering new grassland management practices by facilitating the of use of these resources for high value chemical production.

#### **2. Materials and Methods**

#### *2.1. Biomass Sampling*

In total, 162 grass biomass samples were collected from 67 randomly selected seminatural grassland plots in Sigulda and Ludza municipalities (Latvia) over a 3 year period (Supplementary Materials Annex 1), corresponding to 6 habitat types of Community importance (the most common habitat types within these municipalities), and classified under the EU (Table 1).


**Table 1.** Description of analyzed semi-natural grassland habitats.

\* Includes several vegetation types which vary according to the moisture (flooding) gradient: *C. acuta* or *C. aquatilis*-alluvial meadows, Calamagrostis-alluvial meadows, *Phalaris*-alluvial meadows, *Deschampsia caespitosa*-alluvial meadows.

> Most of the samples (89) were collected in June–August of 2014. Thirty-nine and 34 samples were collected in 2015 and 2016, respectively (Table S1). Sampling in June (almost half of the samples) corresponded to a vegetation period when grassland biomass has the highest fodder value. August samples: period of late mowing.

> The selection of semi-natural grassland sampling plot locations was based on visual assessment of the area. One most representative 1 × 1 m vegetation plot was selected and biomass was clipped at 2 cm above the ground level within the 1 x 1 m square using hand shears (Figure 1). First samplings were performed before the first cut or at the beginning of the grazing period (late June or early July). The second sample was collected in late July or August in sites managed by late mowing. In unmanaged sites, the third sample was also collected in September 2015 (9 samples in total). To evaluate the fermentable carbohydrate potential of early biomass, one sample from each habitat was collected in early June (season of 2015).

> Prior to clipping, a description of the vegetation (vascular plant species richness) in each square was prepared. Then, the collected material was stored in pre-weighed plastic bags and brought to the laboratory for further analyses. If the biomass was not processed within one day, the samples were cut to fractions <20 cm, manually homogenized, and kept frozen (–18 ◦C) in sealable bags.

**Figure 1.** 1 × 1 m square frame sampling plots before (**A**) and after (**B**) collection of grass samples.

#### *2.2. Dry Matter and Ash Content Analyses*

A representative set of grass biomass was cut to pieces below 10 mm. Total dry weight (DW) was determined as weight after drying of sample at + 105 ◦C (laboratory oven 60/300 LSN, SNOL, Utena, Lithuania) for 24 h. Total ash content was measured according to a modified EN ISO 18122 [27]. In brief, the samples were heated at + 550 ºC for 2.5 h (Laboratory furnace 8, 2/1100, SNOL). Volatile solid (VS) percentage was calculated as the difference between total dry mater and ash.

#### *2.3. Enzymatic Hydrolysis*

For enzymatic hydrolysis, a previously described method was used [23]. In brief, all biomass samples (fresh or frozen) were ground (Retsch, Grindomix GM200) to fractions below 0.5 cm. Then, 0.05 M sodium citrate buffer (mono–sodium citrate pure, AppliChem, Germany) was added to the biomass samples (final concentration, 9% w/v wet biomass) and mixed by vortexing. Then, the samples were boiled for 5 min (1 atm) to eliminate any indigenous microorganisms. After cooling to room temperature, a laboratory prepared enzyme (0.2 FPU/mL, obtained from white rot fungi *Irpex lacteus* (Fr.) Fr.) was added to the samples and incubated on an orbital shaker (New Brunswick, Innova 43) for 24 h at 30 ◦C and 150 rpm. Enzyme efficiency was compared with a commercial enzyme product (Viscozyme, Novozymes) and substrate control—hay (obtained in Latvia, 2015, DW 92.8 ± 1.3%).

Samples for reducing sugar measurements were collected after the addition of sodium citrate buffer, prior enzyme addition (both as zero time controls), and after 24 h of hydrolysis. All biomass samples were analyzed in six repetitions.

#### *2.4. Reducing Sugar Analyses*

The Dinitrosalicylic Acid (DNS) method was used to estimate the reducing sugar quantities in the collected samples [28]. First, the samples were centrifuged (6600× *g*, 10 min). Then, 0.1 mL of the supernatant was mixed with 0.1 mL of 0.05 M sodium citrate buffer and 0.6 mL of DNS (SigmaAldrich, Taufkirchen, Germany). Distilled water was used as blank control. To obtain the characteristic color change, the samples were boiled for 5 min and transferred to cold water and supplied with 4 mL of distilled water. Absorption measurements were performed with a spectrophotometer (Camspec M501, Leeds, UK) at 540 nm. For absolute concentrations, a calibration curve against glucose was plotted.

#### *2.5. Statistical Analyses*

For data analysis, MS Excel 2013 *t*–test (two tailed distribution) and ANOVA single parameter tool (significance level ≤0.05) were used for analysis of variance on data from various sample setups.

#### **3. Results and Discussion**

#### *3.1. Assessment of Biomass Resources*

Biochemical parameters such as total solids (TS), volatile solids (VS), and ash content were analyzed for grass biomass samples collected from 6 habitats to evaluate the overall composition of the biomass and its changes over time. These parameters characterize the biomass as a potential energy source and indicate its absolute energetic value. Fast growing biomass can have ash content above 20%; woody biomass has typically 1% ash content. Each 1% increase in ash translates roughly into a decrease of 0.2 MJ/kg of heating value, making it an unpopular resource for combustion [29]. At the same time, the presence of inorganic chemicals can be a good source of microelements along with sugars in the fermentation processes.

The average dry matter from grassland samples in respective Community Importance habitats ranged roughly from 1.0 to 6.0 t/ha (Figure 2) and 93 ± 2% from the dry matter were volatile solids. The highest average yields were obtained from Lowland hay meadows (6510), but the lowest were from Xeric sand calcareous grasslands (6120). That corresponds to yields from semi-natural grasslands in Estonia [30], central Germany [31], and Denmark [32].

The harvesting time had a significant impact on the total amount of the biomass. On average, 5% to 32% less biomass was harvested in June than in July and 17.5 to 42.6 % less in June than in August.

Moreover, variations were observed among the harvesting years. The amount of the biomass (t/ha) in 2016 was 33% to 19% less than in 2015 and up to 27% less than in 2014 (Table 2). Assessment of average daily temperature did not present any significant fluctuations among the years (Figure S1). At the same time, total precipitation in both sampling locations during the summer months was lower in 2015 when compared to 2014 and 2016 (Figure S2). This, to some extent, could explain the differences between these years. A similar influence of annual weather conditions on yield in multi-species grassland has been reported from Estonia and Denmark [30,33].

**Figure 2.** The average biomass dry matter (t/ha) collected from various grassland habitats at different sampling months over a three year period.

The ash content ranged from 3.84 to 9.62% from DW. The lowest ash content was observed in samples from Xeric sand calcareous grasslands (6120) (5.72 ± 1.03%) and the highest for semi-natural dry grasslands and scrubland facies on calcareous substrates (6210) (7.41 ± 1.10%, *p* < 0.05 among other biotopes). This corresponds to the results of other studies—the highest ash concentrations are typically identified in samples from the habitats with larger proportion of dicotyledonous plant species. Typically, ash content is associated with the concentration of minerals in plant organs [34] and dicotyledonous plants tend to accumulate greater quantities of minerals compared with monocotyledonous plants [33].


**Table 2.** The average quantity of biomass (t/ha as dry matter) collected from grassland habitats at various sampling years.

#### *3.2. Enzyme Potential to Release Carbohydrates*

Prior to application of a non-commercial enzyme from *I. lacteus*, its efficiency to release fermentable sugars from hay was compared with a commercial enzyme product. The results demonstrated that a commercial preparation was able to release 0.39 ± 0.05 g/g hay DW after 24 h of incubation. Due to the variable species composition, the amount of cellulose and hemicellulose in hay can vary from 35–45% and 30–50%, respectively [35]. However, prolonged incubation (48 h) did not yield any significant increase (*p* > 0.05) and reached only 0.409 ± 0.048 g/g DW. At the same time, a crude non-commercial product (un-concentrated, un-purified) yielded 0.183 ± 0.03 g/g DW after 24 h and 0.199 ± 0.045 g/g DW after 48 h. In both cases, the amount of sugar released after mechanical and thermal pre-treatment was not significant. Despite lower yields (*p* < 0.05), the observed extractable sugar concentration was still higher than reported for various grass materials [36]. Due to lower costs and potential wide scale application, a non-commercial preparation was used for all future tests and 24 h incubation was set as the optimal.

#### *3.3. Fermentable Sugar Yields*

To evaluate the amount of fermentable sugar released from various grassland biomass resources, enzymatic hydrolysis with the non-commercial enzyme product at optimal conditions was performed. The results of 2014 showed significantly higher (*p* < 0.05) sugar yields (w/w) in June than in July or August (Table 3, Figure 3).

The length of the vegetation season had an overall tendency to decrease the amount of produced sugar. This was observed for all habitats in both 2014 and 2015 sampling seasons where June produced the highest sugar yields (*p* < 0.05) when compared to August or September. The samples from August and September demonstrated no significant sugar yield difference (*p* > 0.05).

Semi-natural dry grassland and scrubland facies on calcareous substrates (6210) and Lowland hay meadow (6510) samples produced the highest fermentable carbohydrate yields in 2014, e.g., 0.235 and 0.165 g per g VS, respectively. In 2015, the highest sugar yields were attributed to Xeric sand calcareous grasslands (6120) and 6210, but the lowest ones were in the samples of 6510 and Northern boreal alluvial meadows (6450) collected in September (Table 3). This slightly contradicted the results obtained in 2014, when from 6210, the highest yield (w/w) was obtained. One of the reasons for this could be the higher proportion of dicotyledonous plants in samples from 6210 collected during 2014. Similarly, as observed before, biomass with dominant monocotyledonous plant proportion showed lower carbohydrate yields due to higher crystallinity, lower hydrolysability, and potential presence of enzyme activity interfering substances [37].

The assessment of the overall producible sugar quantity from one ha exhibited a high potential of 6510 which from all tested habitats had the highest productivity in all vegetation periods, and in June, more than 0.7 t of fermentable sugar per ha could be produced. Other habitats that have demonstrated high sugar yields had lower productivity, e.g., 6210 having only 0.45 t/ha in June (Figure 3, Table 3) and 6120 even having below 0.2 t/ha.

In 2016, samplings were performed only in June with the aim to determine if there was any trend in-between habitats over the years. Again, the highest sugar yields (w/w) were produced from the habitat 6120, followed by Molinia meadows on calcareous, peaty, or clayey-silt-laden soils (6410) and 6510. Assessment of the total sugar quantity per 1 ha revealed that 6510 was able to generate more than 0.78 t of sugar per ha; however, 6120, only 0.186 t/ha. Similarly, as in previous seasons, this difference was due to the low total biomass quantity in 6120; thus, low correlation between fermentable sugar yield (per g biomass) and total amount of sugar per ha of habitat was observed.

The evaluation of the vegetation period showed a strong decrease in sugar yields with increasing vegetation time (Figure 3). No significant decrease (*p* > 0.05) was observed only between the samples collected in August and September. Similar observations have been made for methane yields in biogas production, where the increase in crude fiber at the end of the vegetation period has been set out as one of the main factors influencing the methane production [38]. Others have pointed out that to grasses harvested after October, an extra carbohydrate source must be added if applied for energy production purposes [36]. No influence of specific habitat type has been observed or recorded previously.

**Figure 3.** The amount of fermentable sugar produced per g volatile solid (VS) from biomass collected at various community importance habitats during 2014–2016 vegetation periods. Each bar represents the average value from at least two samplings with six individual measurements of reducing sugar.


**Table 3.** The Reducing sugar yield (mg/g volatile solid (VS) or t/ha) that can be produced from natural grassland habitats at various sampling period.

n/d—not determined; VS—volatile solids.

In some cases, discrepancies from general observations have been detected. Molinia meadows on calcareous, peaty, or clayey-silt-laden soils (6410) did not produce the observed decrease in sugar yields with the progression of the vegetation season. This could be linked to the fact that 6410 includes Molinion grasslands, grasslands with low height sedge species like *Carex flacca, Carex hartmanii, Carex hostiana, Carex panicea, Carex buxbaumii*, as well as grasslands lacking any predominant species. Usually these habitats are represented with high species diversity and located in periodically drying soils [25]. One of the possible explanations can be related to the fact that in July 2014 and June 2015, the samples were collected mainly in sedge grasslands, while in August 2014 and July 2015, in Molinia grasslands. Furthermore, both sugar yield and productivity in 6270 was higher in August 2014 than in July—0.081 and 0.101 g/g VS or 0.22 and 0.31 t/ha, respectively. Apart from the general view (the increase in biomass and carbohydrate yields progresses with the vegetation time) that is challenged within this study, we hypothesize that the observed trend in 6270 is more linked to the environmental conditions, species composition in each individual sampling plot, and vegetation structure in general. Even in one habitat, multiple subtypes with diverse plant communities can be found. Nevertheless, to give the precise explanations of these variations, a more sophisticated classification and evaluation of species compositions would be required.

The average amount of the fermentable sugars highly varied not only seasonally, but also among the years. Sugar yields from the biomass harvested in June 2016 (a month with the most comprehensive data set) were 3% to 58% higher than in those collected in June 2014 and June 2015 for all habitats except 6210 (Table 3). Furthermore, it was estimated that the sugar yields tend to fluctuate (*p* < 0.05) even on a monthly basis, e.g., samples collected within the first ten days of June and at the end of June. The rationale for these differences within one habitat can be explained by the habitat's heterogeneity. The habitats listed in the annexes of the EU Habitats Directive are not classified in a single hierarchical system. Habitats can be separated by the phytosociological classification of plant communities or by habitat groups that include several similar habitats. These can be further divided by specific environmental conditions. Moreover, weather conditions could affect the productivity in single habitat on a yearly basis.

The management of natural grasslands in Natura 2000 classified territories is generally restricted to low-intensity agricultural practices and strict regulations related to grazing, mowing, and cutting [9]. Despite grazing being seen as one of the simplest strategies, follow up on over- or under-grazing, formation of patchiness, preference of certain species by animals, or maintenance of cattle are limiting factors. Mowing at the same time requires the selection of correct timing and frequency; e.g., late moving is preferred to protect animal species and late-flowering plants. At the same time, early cutting and removal of cut grass help to maintain low nutrient levels, keep plant diversity, and avoid alien species [9,39]. On average, the amount of sugar produced from the various grassland habitats at various vegetation periods was comparable to the data obtained with hay (~0.2 g/g DW) and the strategy was shown to be applicable in both high productivity grasslands and at early cutting periods. Upgraded enzymes, adjustment of the technology, e.g., introduction of more intense pre-treatment, could further facilitate the release of the energy stored into grassland biomass. Nevertheless, as demonstrated by this study, multispecies presence, quantities, and applicability under variable conditions set grassland resources as highly sustainable when fermentable carbohydrate production is foreseen.

#### **4. Conclusions**

A simple pre-treatment/hydrolysis technique with non-commercial enzymes made from *I. lacteus* was demonstrated to be efficient for the production of fermentable sugars from the biomass of community important grassland habitats classified under Natura 2000 that have to follow restricted farming practices.

The results showed that fermentable sugar yields from semi-natural grassland habitats are closely linked to vegetation period and plant species variation (monocotyledonous/dicotyledonous species proportion). Dicotyledonous plant rich habitats (6120, 6210) at the beginning of vegetation generated the highest amount of fermentable sugar per mass of biomass—above 0.2 g per g VS. At the same time, habitats rich in total biomass (6510) yielded higher sugar quantities per ha. The lowest yield and productivity in all habitats were observed in August–September, indicating potential bottlenecks of bioenergy production when biomass is collected at a late vegetation period. Overall, the study demonstrated that fermentable carbohydrate production from multispecies biomass of natural and semi-natural grasslands can be used as an alternative management strategy to currently practiced grazing. Thus, fuel production technologies can be merged with sustainable environment management.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1996-1 073/14/5/1312/s1, Annex 1: Location of biomass sampling plots, Table S1: Number of collected biomass samples per sampling year and habitat type; Figure S1: Average daily temperature in sampling months of 2014, 2015 and 2016 at 2 locations; Figure S2: Total precipitation (mm) in sampling months of 2014, 2015 and 2016 at 2 locations and the whole period (Total).

**Author Contributions:** Conceptualization, writing, and data analysis, L.M.; validation, T.J.; formal analysis and data collection, B.D., E.S.; sampling, B.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was supported by the IPP3: INNO INDIGO Programme Project B-LIQ 'Development of an Integrated Process for Conversion of Biomass to Affordable Liquid Biofuel', No. ES/RTD/2017/18, and the National Research Programme "Energetics" Project "Innovative solutions and recommendations for increasing the acquisition of local and renewable energy resources in Latvia" No. VPP-EMAER-2018/3-0004.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We acknowledge the EU LIFE+ Nature & Biodiversity program Project "GRASSSERVICE"—Alternative use of biomass for maintenance of grassland biodiversity and ecosystem services (LIFE12 BIO/LV/001130) for access to biomass samples and data on sampling plots.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


*Article*
