Assessment of the influence of biochar on rumen and silage fermentation: A laboratory-scale experiment

Animal Feed Science and Technology 196 (2014) 22–31

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Assessment of the influence of biochar on rumen and silage fermentation: A laboratory-scale experiment R. Calvelo Pereira a,∗ , S. Muetzel b , M. Camps Arbestain a , P. Bishop a , K. Hina a,c , M. Hedley a a

Institute of Agriculture and Environment, Private Bag 11222, Massey University, Palmerston North 4442, New Zealand Ruminant Nutrition & Greenhouse Gas Mitigation, AgResearch Limited, Tennent Drive, Private Bag 11008, Palmerston North, New Zealand c Department of Environmental Sciences, Hafiz Hayat Campus, University of Gujrat, Gujrat, Pakistan b

a r t i c l e

i n f o

Article history: Received 12 November 2012 Received in revised form 27 June 2014 Accepted 30 June 2014 Keywords: Biochar Silage In vitro incubation Corn stover Pine

a b s t r a c t The addition of biochar – charcoal produced from pyrolysis of carbonaceous materials – to soil presents several challenges, mainly associated with its low bulk density, dustiness and the risk of loss when applied to hill pastures. Livestock could be an adequate vehicle for biochar delivery to New Zealand pastoral soils via dung pats; however, the potential effects of biochar on rumen metabolism need to be investigated. The objective of this study was to investigate the effect of biochar addition to grass before ensiling on the fermentation process and to test whether the addition of grass silage prepared with biochar or biochar directly to hay affected the in vitro rumen fermentation. The study included the use of different types of starting material (corn stover and pine wood chips), two pyrolysis temperatures (350 and 550 ◦ C), post-treatment (addition of different types of bio-oil at a ratio of 0.050 mL/g), and different doses of biochar. The use of biochar from either corn stover or pine pyrolysed at 550 ◦ C as silage ingredients at doses from 21 to 186 g biochar/kg dry matter had no negative effect on the final properties of the silage, and particularly on pH, NH4 + -N/total N, and acetic, N-butyric and l-lactic acid concentrations. The same silage mixtures with 84 and 186 g biochar/kg dry matter were in vitro incubated with buffered rumen fluid. There was a build-up in total volatile fatty acids (VFA) production (P<0.05) in the presence of biochar – increasing at high doses – irrespective of the type of starting material considered. This increase in VFA was also observed when biochar were added to hay before in vitro incubation, and was enhanced with low-temperature biochar. None of the mixtures of biochar and hay had any significant effect on methane emissions and ammonia released. There was no effect of starting material type or post-treatment on the in vitro incubations. The results obtained in this research demonstrate the lack of negative effect of biochar mixed with grass silage, or hay, on rumen chemistry during in vitro incubations. If large-scale studies including in vivo feeding of cattle with biochar confirm these findings, the use of cattle as a delivery system could become a novel solution to safely apply biochar to New Zealand pastoral soils. © 2014 Elsevier B.V. All rights reserved.

Abbreviations: ADF, acid detergent fibre; aNDF, neutral detergent fibre; BET equation, Brunauer, Emmett and Teller equation for surface area; CP/MAS C NMR, cross polarisation/magic-angle-spinning 13 C nuclear magnetic resonance spectroscopy; CS, corn stover; DM, dry matter; FID, flame ionisation detector; GC, gas chromatography; HHT, highest heating temperature; IPCC, Intergovernmental Panel on Climate Change; ME, metabolisable energy; mM, millimolar; NIRS, near infra-red reflectance spectrometry; OMD, organic matter digestibility; PI, pine; SEM, standard error of the mean; SIL, silage; SSS, starch and soluble sugars; VFA, volatile fatty acids. ∗ Corresponding author. Tel.: +64 6 356 9099x85790; fax: +64 6 3505632. E-mail addresses: [email protected], [email protected] (R. Calvelo Pereira). 13

http://dx.doi.org/10.1016/j.anifeedsci.2014.06.019 0377-8401/© 2014 Elsevier B.V. All rights reserved.

R. Calvelo Pereira et al. / Animal Feed Science and Technology 196 (2014) 22–31

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1. Introduction Biochar is a charcoal-rich product obtained when biomass (wood, manure, leaves, and organic wastes can be used as starting material) is pyrolysed (i.e., heated in a closed container with little, or no, available air). Biochar is intended to be applied to soil in order to achieve an agronomic and/or environmental benefit (Lehmann and Joseph, 2009; Jeffery et al., 2013). The pyrolysis of biomass to produce biochar converts a fraction of the C present in the original starting material into a much more persistent form (Lehmann, 2007) that can sequester C in soils for hundreds to thousands of years (Lehmann et al., 2008). Due to the increased concentration of atmospheric CO2 as consequence of human activities (IPCC, 2007), many studies have been carried out to explore the potential of biochar manufacture from waste biomass as a technique to reduce the flux of CO2 to atmosphere from wastes that otherwise would be readily decomposed (Lehmann et al., 2006, 2008; Kuzyakov et al., 2009; Nguyen and Lehmann, 2009; Woolf et al., 2010). Technologies for biochar application into agricultural soils have not been explored in detail (Blackwell et al., 2009; Cook and Sohi, 2010; Major et al., 2010). One of the challenging issues is the handling of high volumes of low bulk density starting materials (∼200 kg/m3 ) and biochar (∼100 kg/m3 ). When added to soil, the light, fine particulate biochar presents an air and water pollution risk. Cultivation of biochar into topsoil has therefore been considered the most viable method; however, this approach disturbs soil structure and creates dust and erosion problems (Blackwell et al., 2009; McHenry, 2009) and is not appropriate for permanent pastures. New approaches must therefore be developed to reduce the impact of biochar application on the environment. Additives are common ingredients in cattle feed. In fact, the use of activated charcoal is common with ruminants to prevent the absorption of toxicants (Garilio et al., 1995; Villalba et al., 2002), whereas activated charcoal mixed with wood vinegar is used to treat calves for several diseases (Watarai et al., 2008). Other additives, such as zeolites, are also included in cattle diets to control ammonium concentration (Papaioannou et al., 2005). Activated charcoal is manufactured by heating carbonaceous material at a high temperature and over long periods of time (i.e., high energy-demanding process), whereas biochar is produced by slow pyrolysis (Sohi et al., 2009), intended to be C-negative (Lehmann et al., 2006). These differences have implications for the properties of the substances. The impact of biochar on rumen fermentation can be related to the potential gas sorption capacity of biochar, as biochar usually combines a porous structure and large surface area (Lehmann and Joseph, 2009). Biochar mixed with either grass or silage may provide an ideal system to enable biochar to be incorporated into agronomic systems. Research is necessary to ensure that biochar has no negative effect on the quality of the silage and on rumen metabolism. To date, the potential benefit derived from charred material in cattle diet is being considered, but research in this area is still scarce (Hansen et al., 2012; Leng et al., 2012a, 2012b). In order to gain insight into how biochar affects rumen fermentation, different variables such as starting biomass type, pyrolysis temperature, dose rate, and any post-treatment of charred materials need to be investigated. The objective of this study was to investigate the effect of biochar addition to grass before ensiling on the fermentation process and to test whether a variety of biochar types (produced from different starting materials, pyrolysed at different temperatures, and including post-treatment), either added directly to hay or including grass silage previously mixed with biochar, influenced the in vitro rumen fermentation. 2. Materials and methods 2.1. Biochar production Biomass from pine wood chips (PI) and corn stover (CS) was pyrolysed independently in a self-purged, gas-fired, rotatingdrum kiln (inner volume of 5 L) to produce biochar and a fluid gas condensate that contained bio-oil. The pyrolysis was conducted at a mean rate of heating of 28 ◦ C/min up to a peak temperature of either 350 or 550 ◦ C and then allowed to cool. The light (aqueous), reduced (tar, insoluble) and full (mixed) fractions of the bio-oil produced from the pyrolysis of pine wood chips at 550 ◦ C were stored for further use. The different biochar substrates produced originally were identified as CS-350, CS-550, PI-350, and PI-550. Biochar subsamples from CS-550 and PI-550 were included in the ensiling experiment (see Section 2.2). Biochar subsamples from CS-350, CS-550, PI-350, and PI-550 were included in the in vitro incubations (see Section 2.3). Additionally, three subsamples of the PI-550 biochar were amended with light, reduced, or full bio-oils (0.050 mL oil/g biochar), identified as OL-550, OR-550 and OF-550 respectively, and included in one of the in vitro incubations (see Section 2.3). 2.2. Ensiling experiment To determine the effects of biochar type and dose on silage fermentation over time, a 2 × 4 factorial design with a control was used. Biochar produced at 550 ◦ C either from pine wood chips (PI-550) or from corn stover (CS-550) was added to grass silage at dose rates of 0 (control), 0.5, 1, 2, 5% on a wet weight basis, or 21, 42, 81 and 186 g biochar/kg dry matter (DM), respectively. The chemical properties of the biochar types used are presented in Table 1. Biochar subsamples were crushed by hand to pass through a 1-mm screen before being mixed with the herbage. The silage was made using perennial ryegrass. The ryegrass sward (40 cm height) was cut in mid-spring 2009, pooled and laid on a concrete pad to wilt, then chopped with a lawn mower to pieces ≤5 cm long. The dry matter content of the herbage at the time of harvest was 220 g/kg. The chemical

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Table 1 Main chemical characteristics of starting materials (pine wood chips and corn stover) and biochar types derived, obtained by slow pyrolysis at different highest heating temperatures (HHT: 350 and 550 ◦ C). Properties

pH Surface area (m2 /g) Composition (g/kg DM) C N Ash

Pine chips

nd nd 487 2.8 5.0

Biochar HHT (◦ C)

Corn stover

350 PI-350

550 PI-550

9.80 <5

9.80 235

nd nd

777 6.1 75

429 2.2 28

671 5.6 40

Biochar HHT (◦ C) 350 CS-350

550 CS-550

8.16 <5

9.89 12

651 7.4 100

743 7.8 108

nd, not determined; DM, dry matter.

characteristics of the herbage are reported in Table 2. Fifty grams of chopped herbage were mixed with the appropriate type and rate of biochar, packed and sealed into polythene bags (dimensions 20.5 × 28.2 cm), designed for vacuum-packing food. A commercial chamber vacuum-packing machine (VAC6650, Sunbeam, Auckland, New Zealand) was used for sealing the bags. An additional set of 9 bags containing 50 g of the same chopped herbage alone was included as control. The total number of experimental units was 81. These bags were incubated in a chamber at 25 ◦ C. 2.2.1. Ensiling experiment analyses During the ensiling period, three plastic bags per treatment were opened and destructively sampled after 31, 71 and 108 days of the start of the incubation. The content of each bag was analysed for dry matter content, pH and ammonium-N. The moisture content of the silage samples was determined after drying the samples at 65 ◦ C until constant weight was reached. pH was measured after shaking a suspension of 5 g silage in 25 mL of deionised water for 2 h. Five grams of fresh silage were extracted with 15 mL of 2 M KCl after shaking for 30 min in a horizontal shaker and then the soluble ammonium-N concentration was determined in the suspension using a Technicon autoanalyser (Technicon, Dublin). Acetic and n-butyric acids were measured following the methodology of Wronkowska et al. (2006). For that, samples were deproteinised using metaphosphoric acid. The supernatant was injected directly into a Carlo Erba 5380 (Carlo Erba, Italy) gas chromatograph (capillary column Alltech ATTM -1000, 15 m × 0.53 mm ID, 1.00 ␮m film) with hydrogen as the carrier gas, FID detector, and iso-caproic acid as an internal standard. l-Lactic acid content of the above supernatant was determined using a megaenzyme kit (Megazyme International Ireland Ltd, Wicklow, Ireland). The total content of C, H and N in the silage after 108 days of incubation was determined using a TruSpec CHNS analyser (LECO Corp. St Joseph, MO, USA). 2.3. In vitro incubation with rumen fluids 2.3.1. Incubation treatments Four different in vitro incubations were conducted in this study: Incubation I. The objective of this incubation was to determine the effect of the type of starting material and pyrolysis temperature on rumen fermentation. A 2 × 2 factorial design was used, including as factors: (i) type of starting material (corn stover and pine) and (ii) biochar temperature production (350 and 550 ◦ C). Hay (Table 2) was used as substrate for incubations and biochar subsamples were mixed with hay at 160 g biochar/kg DM. This hay was also used in Incubation II and Incubation IV (see below) to act as a standard. Table 2 Chemical composition of grass (ensiling experiment) and hay (incubation experiments) used in this study.

Dry matter (g/kg fresh weight) Composition (g/kg DM) Ash Crude protein NH4 + -N/total N Lipid Starch and soluble sugars aNDF ADF Lignin (sa) Organic matter digestibility Metabolisable energy (MJ/kg)

Grass

Hay

220

928

92.0 91.0 54.0 27.0 217 504 275 23.0 0.67 10.8

90.8 104.2 na 10.4 nd 613 347 84.2 nd nd

DM, dry matter; na, not applicable; nd, not determined; aNDF, neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash; ADF, acid detergent fibre expressed inclusive of residual ash.

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Incubation II. A second in vitro incubation was conducted to determine the effect of mixing bio-oil with biochar on rumen fermentation of hay. We included three treatments (OL-550, OR-550, and OF-550) created by mixing PI-550 biochar with three different fractions of bio-oil (light, reduced or full: OL, OR, and OF respectively; 0.050 mL oil/g biochar). Hay was also used, as in Incubation I. Incubation III. A third incubation experiment was set to test: (i) whether the biochar added to the ryegrass before ensiling had an effect on the in vitro rumen fermentation of grass silage and (ii) whether the biochar dose in the silage had an influence. A 2 × 2 factorial design with a control was used, including factors such as the type of starting material (corn stover, pine) and the dose rate (81 and 186 g biochar/kg DM). Grass included in the ensiling experiment (SIL; without biochar), and ensiled grass samples mixed with biochar (either from corn stover or from pine) at two levels (either 81 or 186 g biochar/kg DM) were used. Incubation IV. This incubation compared the effect of biochar addition to grass before ensiling with biochar addition to hay. We used: (i) an ensiled biochar sample (PI-SIL; 186 g biochar/kg DM) and (ii) hay mixed with the same biochar (PI-Hay; dose of PI-550 biochar was 186 g/kg DM). A sample of the freeze-dried grass used for silage preparation was included as a control. Due to a power cut no methane data are available and due to a lack of substrate the incubation could not be repeated. 2.3.2. In vitro incubation preparation Samples with 600 mg of substrate were weighed into 120-mL serum bottles. The bottles were pre-warmed 2 h before the incubation started. Artificial saliva (McDougall, 1948) was prepared the day before incubation and heated to 39 ◦ C in a water bath. One hour before incubation the buffer was flushed with CO2 and, 20 min before incubation started, Na2 S (100 mg/L) was added to the buffer to reduce the solution completely. Rumen fluid was manually collected from fistulated grazing cows directly from grazing in a pre-warmed insulated flask. The rumen fluid was then brought to the laboratory, filtered through one layer of cheese cloth and mixed at a ratio of 1:4 with reduced artificial saliva. The mixture was kept at 39 ◦ C, and stirred and continuously flushed with CO2 . A total of 60 mL of the mixture was dispensed into serum bottles containing the substrate under a stream of CO2 , and the bottles were closed with a butyl rubber stopper and placed in an incubator at 39 ◦ C for 24 h. In vitro incubations were repeated with rumen fluid from a second fistulated cow. 2.3.3. Gas analysis during in vitro incubation Gas analysis of Incubation I was done by manual sampling. After 2, 6, 12, and 24 h of incubation the gas volume was determined by releasing the gas into a calibrated syringe and a 1 mL aliquot was taken to determine the methane concentration of the gas. Gas samples of 200 ␮L were injected manually into a gas chromatograph (GC-2010, Shimadzu Corporation, Kyoto, Japan) fitted with a flame ionisation detector (FID). For the subsequent experiments (Incubations II, III, and IV) an automated system for gas injection was used where each incubation bottle was attached to a pressure sensor (40PC015G1A, Honeywell International Inc., Morristown, NJ, USA) and a solenoid valve. Gas volume was measured by the pressure in the bottles and logged every minute. A calibration curve for every sensor was used to convert pressure into gas volume. When the threshold pressure of 90 mbar was reached a solenoid valve was opened and the gas sample was injected into a GC. Gases were separated in a HP-Plot Molsieve column (length 35 m, ID 0.53 mm, Agilent Technologies Inc., Santa Clara, CA, USA) and methane was detected with an FID detector (250 ◦ C). The GC was run under isocratic conditions at 85 ◦ C, with N2 as a carrier gas. 2.3.4. Sampling and analyses at the end of the in vitro incubations At the end of each incubation experiment (i.e., after 24 h) an analytical subsample was collected from each bottle. For volatile fatty acids (VFA) and ammonia analysis, an aliquot of 2 mL of each subsample in duplicate was centrifuged at 21,000 × g for 10 min at room temperature. The known volume of the supernatant (900 ␮L) was transferred into a fresh vial containing 100 ␮l of internal standard [19.87 mM ethyl-butyric acid, and ortho-phosphoric acid (1:5, v/v)]. The samples were frozen overnight, and centrifuged (21,000 × g, 10 min at room temperature) the following day. A known volume of the supernatant (800 ␮l) was transferred into a crimp cap vial and samples for VFA were analysed in a gas chromatograph (HP 6890, Santa Clara, CA, USA) equipped with a capillary column (ZB FFAP &HK-G009-22) (Attwood et al., 1998). Part of the final supernatant was used for a colorimetric ammonium concentration assay (Weatherburn, 1967). 2.4. Analytical methods 2.4.1. Biochar characterisation 2.4.1.1. Elemental analysis. The total C, H and N content of both the biomass types and the biochar used in this study were determined using a TruSpec CHNS analyser (LECO Corp., St. Joseph, MO, USA). The ash content was determined by thermo gravimetric analysis on a TA instrument (Alphatech, SDT Q600 manufactured by TA Instruments, Newcastle, Australia). The samples were initially heated from room temperature to 900 ◦ C (at a rate of 5 ◦ C/min) under a N2 atmosphere and weight loss was recorded continuously; thereafter, an air current was provided and the ash content was determined as equal to the weight of the residue left after the oxidation step (Calvelo Pereira et al., 2011). 2.4.1.2. Solid-state cross polarisation/magic-angle-spinning 13 C nuclear magnetic resonance spectroscopy. Solid-state, cross polarisation/magic-angle-spinning 13 C nuclear magnetic resonance (CP/MAS 13 C NMR) spectroscopy was used to

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characterise the carbon fraction of selected samples of biochar. All solid-state 13 C NMR measurements were conducted in a Bruker AMX 200 MHz spectrometer (Rheinstetten, Germany). Ground biochar subsamples were packed into a 7-mm i.d. diameter rotor and spun at speeds of 5 kHz in a dual resonance magnetic angle spinning (MAS) probe from Doty Scientific. The solid-state CP/MAS 13 C spectra were acquired with a 1 H 90◦ pulse for 5.5 ␮s, a cross polarisation contact time of 1000 ␮s, an acquisition time of 30 ms, and a relaxation time of 2 s and 5000 scans. A qualitative description of NMR spectra from selected biochar samples is presented. 2.4.1.3. Other analyses. Biochar pH was measured using the methodology of Ahmedna et al. (1997), which involved a 1% (wt/wt) suspension of biochar in deionised water. The suspension was heated in a water bath to about 90 ◦ C and stirred for 20 min to allow dissolution of the soluble biochar components. After cooling to room temperature, the pH of the biochar suspension was determined with a combined pH electrode (PHM83, Radiometer, Copenhagen). Biochar samples were outgassed at 250 ◦ C for 4 h prior to nitrogen gas adsorption analysis for specific surface area (Brunauer et al., 1938) was performed using a Micromeritics ASAP 2020 volumetric adsorption system. Values of the Brunauer, Emmett and Teller (BET) equation for surface area were obtained for selected biochar samples (Table 1). 2.4.2. Grass chemical composition analyses The pH, dry matter (DM), ash, crude protein, total and NH4 nitrogen, starch and soluble sugars (SSS), organic matter digestibility (OMD), and metabolisable energy (ME) of pasture used for silage was estimated by near infra-red reflectance spectrometry (NIRS). Calibrations for each component had been previously developed using NIRS after scanning finely ground pasture samples in the range of 400–2500 nm. These samples had previously been analysed for each of the above components by wet chemistry methods. A Bruker MPA NIR spectrophotometer (Ettlingen, Germany) was used to scan the samples and the resulting NIRS spectra were analysed using Optic user software (OPUS) v. 5.0 (Ettlingen, Germany). The resulting NIRS calibration against the wet chemistry data for each component typically had a correlation of 0.90. Neutral detergent fibre assayed with a heat stable amylase and expressed inclusive of residual ash (aNDF), acid detergent fibre expressed inclusive of residual ash (ADF) and lignin (sa) were determined using the Tecator Fibretec System (Leco Corporation, St. Joseph, MO, USA) following procedures of Robertson and Van Soest (1981). Dry matter content of the hay sample was determined by drying in an oven at 105 ◦ C for 24 h. The sample was analysed by Massey University Nutrition Laboratory (Palmerston North, NZ) for ash (AOAC 942.05), crude protein (Leco, total combustion method, AOAC 968.06), lipid (Soxtec extraction, AOAC 991.36), following AOAC (1990, 2000); finally, aNDF and ADF were determined by using the Tecator Fibretec System, as indicated above. 2.5. Statistical analyses Data from the ensiling experiment (pH, ammonium-N and VFA concentration) were statistically analysed using the GLM procedure of SPSS (IBM SPSS Statistics, version 20). The model included the fixed effect of the type of starting material used for pyrolysis (i.e., pine biochar and corn stover), the level of biochar addition [i.e., dose rate, 0 (control), 21, 41, 81 and 186 g biochar/kg DM], and the interaction of starting material type and level of addition. Each specified sampling time (31, 71, and 108 days after the start of the ensiling) was analysed individually, considering three replicates per treatment. If a significant (P<0.05) main effect was detected, difference between treatment means was tested using the least significant difference. Data from the in vitro incubations [total gas and CH4 production, ammonium concentration, total volatile fatty acids (VFA) concentration, and molar proportion of individual VFA] were analysed using the ANOVA procedure in Genstat v. 12.2. For Incubation I, the model included the fixed effect of the type of starting material (corn stover and pine), the temperature of biochar production (350 and 550 ◦ C), and the interaction of starting material and temperature. For Incubation II, the model included only the fixed effect of the treatment (i.e., three PI-550 biochar pre-mixed with light, reduced or full bio-oil mixed with hay). For Incubation III, the model included the fixed effect of the starting material type (i.e., corn stover and pine), the level of biochar addition (i.e., doses at 81 and 186 g biochar/kg DM) and the interaction between starting material and level of addition. Finally, for Incubation IV, the model included only the fixed effect of the different substrates, ensiled ryegrass with biochar (186 g biochar/kg DM), and hay mixed with the same biochar (186 g biochar/kg DM). The total number of measurements for the in vitro incubations was four for each factor considered, with the average of the duplicate bottles as analytical replicates and rumen fluid donor as the statistical replicate. No comparison was made across in vitro incubation runs. If a significant (P<0.05) main effect was detected, difference between treatment means was tested using the least significant difference. 3. Results 3.1. Characteristics of the biochar used for blending The biochar produced at 550 ◦ C (PI-550, CS-550) had a high C content (777 and 743 g/kg, respectively) and a low N content (6.1 and 7.8 g/kg, respectively) (Table 1). The C was highly aromatic, as obtained from the NMR spectra (data not shown), with a prominent peak at 120–130 ppm; this was attributed to C- and H-substituted aromatic C. The ash content was lower in the

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Table 3 Average (n = 3) pH, NH4 + -N concentration, NH4 + -N/total N ratio, acetic and l-lactic acid concentration of from grass ensiled with increasing doses of biochar produced from corn stover and pine produced at 550 ◦ C. Sampling day

Biochar level (g/kg DM)

Property

0

21

42

81

186

Corn stover

Pine

3.98 0.12 0.01 54.1 41.6

3.92 0.11 0.01 49.7 45.2

3.92 0.12 0.01 50.5 47.5

3.92 0.12 0.01 47.6 52.8

3.99a 0.10a 0.01a 37.4a 51.2a

3.94 0.11 0.01 45.0 48.8

3.94 0.12 0.01 50.8 46.8

0.01 <0.01 <0.01 2.00 1.14

3.83 0.86a 0.05 54.9a 49.8

3.82 3.83 3.85 3.89 0.82ab 0.81ab 0.75bc 0.65c 0.05 0.05 0.04 0.05 49.1ab 55.2ab 46.0bc 38.2c 47.5 46.5 48.3 45.8

3.85 0.78 0.05 47.5 50.7a

3.83 0.77 0.05 49.1 44.3b

3.76a 1.23a 0.07 71.3a 50.2

3.76a 1.11b 0.07 56.0b 45.6

3.76b 1.06b 0.07 50.2 44.0b

Day 31 pH NH4 + -N (g/kg DM) NH4 + -N/total N Acetic acid (g/kg DM) l-Lactic acid (g/kg DM) Day 71 pH NH4 + -N (g/kg DM) NH4 + -N/total N Acetic acid (g/kg DM) l-lactic acid (g/kg DM) Day 128 pH NH4 + -N (g/kg DM) NH4 + -N/total N Acetic acid (g/kg DM) l-lactic acid (g/kg DM)

3.76a 1.06b 0.07 45.7b 44.3

Starting material

3.76a 1.03b 0.06 45.8b 45.4

3.82b 3.79a 1.00bc 1.12a 0.07 0.07 37.0bc 52.5 45.0 48.5a

SEM

P value Biochar level

Starting material

Interaction

0.576 0.341 0.424 0.057 0.112

0.945 0.638 0.476 0.860 0.774

0.344 0.420 0.498 0.996 0.974

0.01 0.02 <0.01 1.55 1.32

0.171 0.004 0.380 0.003 0.624

0.361 0.597 0.896 0.722 0.040

0.970 0.103 0.889 0.814 0.090

0.01 0.02 <0.01 2.56 0.91

0.010 0.002 0.309 <0.001 0.063

0.003 0.011 0.240 0.202 0.007

0.071 0.022 0.105 0.079 0.033

Means within a row, and within a category, with different superscript letters differ (P<0.05); DM, dry matter; SEM, standard error of the mean.

PI-550 (75 g/kg) than in the CS-550 (108 g/kg), although the pH values were almost identical (9.80 and 9.89, respectively). The surface areas obtained from N2 isotherms obtained at 77 K and by applying the BET equation were 235 and 12 m2 /g, for PI-550 and CS-550, respectively. Carbon contents for PI-350 and CS-350 biochar substrates were 671 and 651 g/kg respectively, considerably lower than their corresponding high-temperature chars (Table 1). The N content was low, with values of 5.6 and 7.4 g/kg for PI-350 and CS-350, respectively. The nature of the C fractions in those biochar produced at 350 ◦ C was different from that in biochar produced at 550 ◦ C. PI-350 and CS-350 had a greater proportion of biochar C in alkyl form (peak between 5 and 95 ppm) with a prominent peak at 80–90 ppm, which represents alkyl-C groups bound to oxygen. Most of these oxygen groups were absent after heating to 550 ◦ C (data not shown). The spectra thus reflect the presence of a less stable C in the 350 ◦ C biochar. Low-temperature chars had a small surface area (<5 m2 /g), regardless of the starting material considered.

3.2. Changes in silage characteristics during fermentation: ensiling experiment After 1 month of ensiling, the average pH value of the silage control samples was 3.98; after 2 months it decreased to 3.83 (Table 3). The pH values of the silage samples prepared with biochar followed the same pattern, with a decreasing trend with time, and final values were ∼3.80. We found that, at day 108, the highest dose of biochar increased (P<0.05) silage pH with respect to control; such increase was detected only with biochar produced from corn stover (final average pH value of silage prepared with CS-550 biochar: 3.90; data not shown) and was attributed to the alkalinity of the material (see above). Dry matter content tended to increase with time, independently of the treatment considered (data not shown); there was a positive correlation (r = 0.778; P=0.014) between average dry matter content and average pH at the end of the experiment (day 108). Ammonium-N concentrations (on ensiled grass mass basis) increased with time as a result of protein degradation (Table 3). This increase was especially evident after the second month of incubation, but continued to increase during the third month. The type of starting material had no overall effect on silage NH4 + -N concentrations, regardless of the dose used. When NH4 + -N concentrations were expressed on ensiled grass + biochar mass basis, there was an increase with time in concentration for PI-550 (P<0.01) and CS-550 (P<0.05) treatments, respectively (data not shown). The NH4 + -N/total N ratio of samples at the end of the ensiling ranged between 0.06 and 0.07 (Table 3). It should be noted that initial NH4 + -N/total N ratio of the fresh grass was 0.05 (Table 2). Average acetic acid concentration in the silage control samples and in the low dose of CS-550 biochar (21 g biochar/kg DM) increased sharply in the last month of ensiling, with values even above 55 g/kg (DM basis) (Table 3). This could indicate some problems with fermentation. This pattern was not observed in the rest of treatments (Table 3). The addition of the biochar to the silage decreased (P<0.001) the content of acetic acid at day 108, this decrease being greater at the highest doses of biochar. l-Lactic acid concentration in the silage control samples stabilised after 1 month of ensiling, rising to a value of 50 g/kg DM (Table 3). In the silage samples prepared with biochar, l-lactic acid concentrations remained relatively stable with time. N-butyric was below detection limit (data not shown).

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Table 4 Average (n = 2) total gas production (GP), methane (CH4 ) production, ammonium (NH4 + ) concentration, total volatile fatty acids (VFA) concentration, and molar proportion of individual VFA obtained from hay and hay mixed with 160 g biochar/kg DM, using biochar produced from different starting material (from corn stover or pine) and produced at different material (either 350 ◦ C or 550 ◦ C) after 24 h of in vitro incubation (Incubation I). Hay

Corn stover ◦

GP (mL/g DM) CH4 (mL/g DM) NH4 + (mM) Total VFA (mM) VFA (mol/100 mol) Acetate Propionate Butyrate

136 29.3 3.7 75.1 64.7 22.9 9.5

Pine ◦

SEM

◦

◦

P value

350 C

550 C

350 C

550 C

Starting material

Temperature

Interaction

138a 29.7 4.0 76.9a

133b 28.9 3.9 76.5a

139a 30.9 3.8 77.7a

135b 28.6 3.3 73.3b

1.00 0.40 0.36 0.67

0.416 0.514 0.473 0.071

0.049 0.074 0.614 0.002

0.954 0.304 0.792 0.005

64.4 23.2 9.5

64.9 22.8 9.5

64.9 22.7 9.5

63.9 23.6 9.7

0.36 0.25 0.06

0.648 0.583 0.339

0.579 0.462 0.319

0.158 0.091 0.095

Means with different superscript letters indicate differences (P<0.05); SEM, standard error of the mean; DM, dry matter, mM, millimolar. Table 5 Average (n = 2) total gas production (GP), methane (CH4 ) production, ammonium (NH4 + ) concentration, total volatile fatty acids (VFA) concentration, and molar proportion of individual VFA obtained from hay and hay mixed with different biochar containing bio-oil (either full, reduced or light, OF-550, OR-550 and OL-550 respectively) at a dose of 160 g biochar/kg DM after 24 h of in vitro incubation (Incubation II). Treatment

GP (mL/g DM) CH4 (mL/g DM) NH4 + (mM) Total VFA (mM) VFA (mol/100 mol) Acetate Propionate Butyrate

SEM

P value

161 24.0 14.9 70.1

2.98 0.79 0.84 0.22

0.592 0.920 0.987 0.202

68.8 20.6 7.8

0.17 0.12 0.12

0.539 0.539 0.972

Hay

OF-550

OR-550

OL-550

157 24.3 14.7 68.8

158 23.6 15.7 69.5

148 22.5 15.2 69.2

68.0 21.1 8.0

68.3 20.9 8.0

68.4 21.0 7.9

Means with different superscript letters indicate differences (P<0.05) among treatments; SEM, standard error of the mean; DM, dry matter, mM, millimolar.

3.3. In vitro incubation of biochar added to hay or silage with rumen fluids The results for Incubation I are shown in Table 4. There was no difference in total gas production and the proportion of methane released from any treatment. However, the low-temperature biochar produced greater (P<0.05) amounts of gas than high-temperature biochar. Total VFA production was different (P=0.020) between treatments, but none of the biochar treatments were different from the standard (hay). Low-temperature biochar produced slightly more (P=0.002) VFA than high-temperature biochar substrates. The proportion of the major fatty acids was the same for all the biochar tested, as was the release of ammonia after 24 h of incubation. No effect of the type of starting material on in vitro incubation was observed. A summary of the results obtained for Incubation II is shown in Table 5. Results indicate that none of the biochar samples pre-mixed with bio-oil decreased methane emissions in vitro. However, they did not have a negative effect on the overall fermentation since total gas production, VFA production, and the concentration of ammonia were the same, despite the differences observed with the control (hay). The results obtained for Incubation III are reported in Table 6. The variability in gas production was relatively high in this dataset (due to differences between rumen fluid donor animals) and, therefore, differences in gas production between Table 6 Average (n = 2) total gas production (GP), methane (CH4 ) production, ammonium (NH4 + ) concentration, total volatile fatty acids (VFA) concentration, and molar proportion of individual VFA obtained from grass included in the ensiling experiment (SIL; without biochar), and ensiled grass samples mixed with biochar (either from corn stover or from pine) at two levels (either 81 or 186 g biochar/kg DM) after 24 h of in vitro incubation (Incubation III). SIL

GP (mL/g DM) CH4 (mL/g DM) NH4 + (mM) Total VFA (mM) VFA (mol/100 mol) Acetate Propionate Butyrate

Corn stover

Pine

SEM

P value

81

186

81

186

Starting material

Level

Interaction

174 26.5 14.3 80.9

189 27.7 14.7 86.7a

188 27.4 16.1 88.5a

172 25.3 14.7 82.2ab

191 27.8 14.0 95.5c

4.64 0.67 0.46 1.78

0.414 0.619 0.216 0.399

0.297 0.607 0.641 <.001

0.200 0.504 0.227 0.001

56.5 30.0 9.9

57.1a 29.6a 10.0

59.4b 27.3b 9.8

58.9b 28.4b 9.4

0.51 0.35 0.38

0.836 0.170 0.519

0.023 <.001 0.839

0.646 0.237 0.986

57.3a 29.7a 9.5

Means with different superscript letters indicate differences (P<0.05); SEM, standard error of the mean; DM, dry matter, mM, millimolar.

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Table 7 Average (n = 2) total gas production (GP), methane (CH4 ) production, ammonium (NH4 + ) concentration, total volatile fatty acids (VFA) concentration, and molar proportion of individual VFA obtained from freeze-dried grass used for silage preparation (Grass), the same grass mixed with biochar (from pine) at a dose of 186 g biochar/kg DM and ensiled (PI-SIL), and a sample of hay mixed with the same biochar at a dose of 186 g biochar/kg DM (PI-Hay) after 24 h of in vitro incubation (Incubation IV). Treatment

GP (mL/g DM) NH4 + (mM) Total VFA (mM) VFA (mol/100 mol) Acetate Propionate Butyrate

SEM

P-value

218 31.4b 98.9

5.15 0.83 1.57

0.166 0.034 0.810

59.1b 24.0 11.9b

0.98 0.43 0.51

0.006 0.106 0.006

Grass

PI-SIL

PI-Hay

234 28.6a 98.4

210 32.8b 101.3

63.6a 22.2 9.5a

59.0b 24.0 11.8b

Means with different superscript letters indicate differences (P<0.05) among treatments; SEM, standard error of the mean; DM, dry matter, mM, millimolar.

the incubated substrates were non-significant, although nearly all the biochar-amended samples had a numerically higher gas production. Total VFA production increased (P<0.001) as the level of biochar in the silage increased. The proportion of methane, however, was the same for all treatments, with a very low standard error of the measurements. The same results were obtained for the composition of the VFA, where acetate and butyrate proportions did not differ between incubations. Nonetheless, acetate tended to increase (P=0.023) with increasing level of biochar, with a concomitant decreasing trend (P<0.001) for propionate. No difference in ammonia release was detected in these incubations. The type of starting material had no effect in the variables studied (Table 6). Results from the in vitro incubations suggested that biochar had a positive effect on substrate digestibility when added before ensiling, compared with a direct addition of biochar to hay; therefore, Incubation IV was carried out to verify this. Unlike the previous incubation, Incubation IV showed no difference between ensiled and added biochar for either gas or total VFA production (Table 7). The differences in ammonia concentration were due to differences in the grass sample compared with the silage, as were the differences in the proportion of individual VFA.

4. Discussion In order to investigate the effect of biochar addition to grass before ensiling on the fermentation process and to test whether biochar either added directly to hay or including grass silage previously mixed with biochar influenced the in vitro rumen fermentation, we used a variety of biochar types. Aspects considered for selecting the biochar in this study were: (i) the use of biomass available in New Zealand in large quantities (e.g., pine) as starting material; (ii) the effect of starting material (e.g., corn stover and pine) on the silage quality and on enteric fermentation; (iii) the influence of biochar with different C stabilities (e.g., biochar produced at 350 and 550 ◦ C) on enteric fermentation; (iv) the effect of modified biochar (e.g., containing bio-oil) on rumen fermentation; (v) the feasibility of adding the biochar before or after ensiling; and (vi) biochar dose rate. Selected biochar produced from contrasted starting materials, mixed with ryegrass, apparently had no negative impact on the quality of the silage after 3 months of ensiling. However, the ryegrass used in this study had low protein content; as the spring of 2009 was rainy, cloudy and cold, conditions were not favourable for the production of high-quality silage (Henderson, 1993). The use of biochar as a silage ingredient showed some influence in the final properties of the mixtures, and these differences were more evident as the dose of the biochar increased. Unfortunately, the silage samples could not be analysed using NIRS to track in detail silage quality changes (Cozzolino et al., 2006), as the presence of biochar required the development of new calibration curves. The concentration of acetic acid in the control silage and in grass–biochar mixtures including low doses of biochar was above a desirable 40 g/kg DM (Kung and Shaver, 2001). These values, however, tended to decrease with increasing doses of biochar, and finally fell below 40 g/kg DM in the silage prepared at the highest level of biochar addition. Therefore, increasing the dose of biochar in the silage seemed to enhance the silage quality. Besides, concentrations of l-lactic acid at the end of the ensiling were not affected by the presence of biochar. Acceptable values of l-lactic acid are in the range of 40–70 g/kg DM basis, and at least 65–70% of the total acid present in the silage (Kung and Shaver, 2001). All silages, except that with pine biochar added at the highest dose, had an l-lactic acid concentration above 40 g/kg. However, percentage of l-lactic acid from the total acid present in the silage was below 65%, due to the high concentration of acetic acid in all samples, including the control (grass) without biochar. Only the silage prepared with grass mixed with biochar produced from corn stover at the highest dose showed an adequate relative proportion of VFA. Biochar addition to silage and hay do not have a specific effect on methane production in the rumen. A net reduction in methane emission (due to sorption) could have been anticipated because of the relatively high specific surface area of those biochar produced at 550 ◦ C (Table 1), as previously reported by Leng et al. (2012a), but this was not the case in this study. Leng et al. (2012a) produced a biochar from rice husks at a very high temperature (∼900 ◦ C) and both differences in starting material and highest heating temperature can influence sorptive capacity of biochar (Lehmann and Joseph, 2009).

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It is interesting that the contrasted biochar studied here appears to be inert to rumen fermentation process and did not negatively influence any of the fermentation parameters considered. Some mixtures showed a decreased proportion of propionate with respect to the total VFA production, which could indicate an increase in the amount of hydrogen produced during rumen fermentation (Moss et al., 2000). These changes, however, were too small to influence the emission of methane. The observed increase in the total VFA concentration from grass–biochar mixtures ensiled for 3 months incubated in vitro was related with the level of biochar added. This suggested that, although no general effect on methane was observed, the in vitro rumen fermentation of the feed including biochar was actually increased when the biochar was ensiled before incubation rather than recently added to the silage. However, when similar additions were tested using ensiled mixtures and fresh mixtures using added biochar, the effect was not repeatable and therefore might have been due to the preparation of the previously used silages. However, the experimental design chosen did not clearly distinguish the effect of forage type (silage and hay) and that of biochar addition method (direct mixing and ensiling). In this sense, more specific research is needed to clarify this point. 5. Conclusions The addition of biochar produced from different starting materials and mixed at increasing dose rates (21–186 g biochar/kg DM) with grass before ensiling did not have any negative effect on the fermentation process of the silage. Moreover, biochar produced from the same starting materials, at similar temperatures, with several pre- and post-treatments, and added at different dose rates (up to 186 g biochar/kg DM) did not have any negative effect on in vitro incubation with rumen fluids. Further research will be needed to address: (i) the effects of biochar on in vivo rumen fermentation; (ii) the extent to which this measure is safe in terms of animal safety and performance; and (iii) the fate of biochar once delivered to the soil and its agronomic and environmental impact. If large-scale studies on feeding cattle with biochar prove the safety and adequacy of this delivery system, this could become an option for biochar application to pastoral soils. Conflict of interest The authors declare no conflict of interest. Acknowledgements The authors are very grateful for financial support from the Ministry of Agriculture and Forestry of New Zealand (project reference CONT-20453-SLMACC-MAU). The authors thank Fliss Jackson, from the Nutrition laboratory of Massey University, for technical support in the analysis of the silage samples, and Adolfo Alvarez for assessment in the preparation of the silage. S.M. would like to thank German Molano and Holly Kjestrup for their assistance in the in vitro incubations and sample analysis. References Ahmedna, M., Johns, M.M., Clarke, S.J., Marshall, W.E., Rao, R.M., 1997. Potential of agricultural by-product-based activated carbons for use in raw sugar decolourisation. J. Sci. Food Agric. 75, 117–124. AOAC, 1990. Official Method of Analysis, 15th ed. Association of Official Analytical Chemists, Washington, DC, USA. AOAC, 2000. Official Method of Analysis, vol. I., 17th ed. Association of Official Analytical Chemists, MD, USA. Attwood, G.T., Klieve, A.V., Ouwerkerk, D., Patel, B.K.C., 1998. Ammonia-hyperproducing bacteria from New Zealand ruminants. Appl. Environ. Microbiol. 64, 1796–1804. Blackwell, P., Reithmuller, G., Collins, M., 2009. Biochar application to soil. In: Lehmann, J., Joseph, S. (Eds.), Biochar for Environmental Management: Science and Technology. Earthscan, London, United Kingdom, pp. 207–226. Brunauer, S., Emmett, P.H., Teller, E., 1938. Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 60, 309–319. Calvelo Pereira, R., Kaal, J., Camps Arbestain, M., Pardo Lorenzo, R., Aitkenhead, W., Hedley, M., Macías, F., Hindmarsh, J., Maciá-Agulló, J.A., 2011. Contribution to characterisation of biochar to estimate the labile fraction of carbon. Org. Geochem. 42, 1331–1342. Cook, J., Sohi, S., 2010. Application to soil. In: Shackley, S., Sohi, S. (Eds.), An Assessment of the Benefits and Issues Associated with the Application of Biochar to Soil. UK Biochar Research Centre, Edimburg, UK, p. 132 p. Cozzolino, D., Fassio, A., Fernández, E., Restaino, E., La Manna, A., 2006. Measurement of chemical composition in wet whole maize silage by visible and near infrared reflectance spectroscopy. Anim. Feed Sci. Technol. 129, 329–336. Garilio, E., Pradhan, R., Tobioka, H., 1995. Effects of activated charcoal on growth, ruminal characteristics, blood profiles and feed digestibility in growing sheep. Proc. Sch. Agric. Kyushu Tokai Univ. 14, 57–64. Hansen, H.H., Storm, I.M.L.D., Sell, A.M., 2012. Effect of biochar on in vitro rumen methane production. Acta Agric. Scand. A: Anim. Sci. 62, 305–309. Henderson, N., 1993. Silage additives. Anim. Feed Sci. Technol. 45, 35–56. IPCC, 2007. Climate Change 2007: The Physical Science Basis. Intergovernmental Panel on Climate Change, Geneva, pp. 21. Jeffery, S., Bezemer, T.M., Cornelissen, G., Kuyper, T.W., Lehmann, J., Mommer, L., Sohi, S.P., van de Voorde, T.F.J., Wardle, D.A., Van Groenigen, J.W., 2013. The way forward in biochar research: targeting trade-offs between the potential wins. GCB Bioenergy, http://dx.doi.org/10.1111/gcbb.12132 Kung, L., Shaver, R., 2001. Interpretation and use of silage fermentation analysis reports. Focus Forage 3, 1–5. Kuzyakov, Y., Subbotina, I., Chen, H., Bogomolova, I., Xu, X., 2009. Black carbon decomposition and incorporation into soil microbial biomass estimated by 14 C labeling. Soil Biol. Biochem. 41, 210–219. Lehmann, J., 2007. A handful of carbon. Nature 447, 143–144. Lehmann, J., Gaunt, J., Rondon, M., 2006. Bio-char sequestration in terrestrial ecosystems – a review. Mitig. Adapt. Strateg. Glob. Chang. 11, 395–419. Lehmann, J., Joseph, S., 2009. Biochar for Environmental Management: Science and Technology. Earthscan, London. Lehmann, J., Skjemstad, J., Sohi, S., Carter, J., Barson, M., Falloon, P., Coleman, K., Woodbury, P., Krull, E., 2008. Australian climate – carbon cycle feedback reduced by soil black carbon. Nature Geosci. 1, 832–835.

R. Calvelo Pereira et al. / Animal Feed Science and Technology 196 (2014) 22–31

31

Leng, R.A., Inthapanya, S., Preston, T.R., 2012a. Biochar lowers net methane production from rumen fluid in vitro. Livest. Res. Rural Dev. 24, 103. Leng, R.A., Preston, T.R., Inthapanya, S., 2012b. Biochar reduces enteric methane and improves growth and feed conversion in local Yellow cattle fed cassava root chips and fresh cassava foliage. Livest. Res. Rural Dev. 24, 199. Major, J., Lehmann, J., Rondon, M., Goodale, C., 2010. Fate of soil-applied black carbon: downward migration, leaching and soil respiration. Global Change Biol. 16, 1366–1379. McDougall, E., 1948. Studies on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochem. J. 43, 99–109. McHenry, M.P., 2009. Agricultural bio-char production, renewable energy generation and farm carbon sequestration in Western Australia: certainty, uncertainty and risk. Agric. Ecosyst. Environ. 129, 1–7. Moss, A.R., Jouany, J.-P., Newbold, J., 2000. Methane production by ruminants: its contribution to global warming. Ann. Zootech. 49, 231–253. Nguyen, B.T., Lehmann, J., 2009. Black carbon decomposition under varying water regimes. Org. Geochem. 40, 846–853. Papaioannou, D., Katsoulos, P.D., Panousis, N., Karatzias, H., 2005. The role of natural and synthetic zeolites as feed additives on the prevention and/or the treatment of certain farm animal diseases: a review. Microporous Mesoporous Mater. 84, 161–170. Robertson, J.B., Van Soest, P.J., 1981. The detergent system of analysis and its application to human foods. In: James, W.P.T., Theander, O. (Eds.), The Analysis of Dietary Fiber in Food. Marcel Dekker, Inc, New York, pp. 123–158. Sohi, S., Lopez-Capel, E., Krull, E., Bol, R., 2009. Biochar, climate change and soil: a review to guide future research. In: CSIRO Land and Water Science Report 05/09, p. 64. Villalba, J.J., Provenza, F.D., Banner, R.E., 2002. Influence of macronutrients and activated charcoal on intake of sagebrush by sheep and goats. J. Anim Sci. 80, 2099–2109. Watarai, S., Tana, Koiwa, M., 2008. Feeding activated charcoal from bark containing wood vinegar liquid (Nekka-Rich) is effective as treatment for cryptosporidiosis in calves. J. Dairy Sci. 91, 1458–1463. Weatherburn, M.W., 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39, 971–974. Woolf, D., Amonette, J.E., Street-Perrott, F.A., Lehmann, J., Joseph, S., 2010. Sustainable biochar to mitigate global climate change. Nat. Commun. 1, 56. Wronkowska, M., Soral-Smietana, M., Krupa, U., Biedrzycka, E., 2006. In vitro fermentation of new modified starch preparations-changes of microstructure and bacterial end-products. Enzyme Microb. Technol. 40, 93–99.