Effect of Microaerobic Fermentation in Preprocessing Fibrous Lignocellulosic Materials

Effect of Microaerobic Fermentation in PreprocessingFibrous Lignocellulosic Materials

Manar Arica Alattar & Terrence R. Green &

Jordan Henry & Vitalie Gulca & Mikias Tizazu &

Robby Bergstrom & Radu Popa

Received: 21 December 2011 /Accepted: 25 April 2012 /Published online: 26 May 2012# Springer Science+Business Media, LLC 2012

Abstract Amending soil with organic matter is common in agricultural and logging prac-tices. Such amendments have benefits to soil fertility and crop yields. These benefits may beincreased if material is preprocessed before introduction into soil. We analyzed the efficiencyof microaerobic fermentation (MF), also referred to as Bokashi, in preprocessing fibrouslignocellulosic (FLC) organic materials using varying produce amendments and leachatetreatments. Adding produce amendments increased leachate production and fermentationrates and decreased the biological oxygen demand of the leachate. Continuously drainingleachate without returning it to the fermentors led to acidification and decreased concen-trations of polysaccharides (PS) in leachates. PS fragmentation and the production of solublemetabolites and gases stabilized in fermentors in about 2–4 weeks. About 2 % of the carboncontent was lost as CO2. PS degradation rates, upon introduction of processed materials intosoil, were similar to unfermented FLC. Our results indicate that MF is insufficient foradequate preprocessing of FLC material.

Appl Biochem Biotechnol (2012) 167:909–917DOI 10.1007/s12010-012-9717-5

M. A. Alattar : J. Henry :M. Tizazu : R. Bergstrom : R. Popa (*)Biology Department, Portland State University, Portland, OR 97207, USAe-mail: [email protected]

M. A. Alattare-mail: [email protected]

J. Henrye-mail: [email protected]

M. Tizazue-mail: [email protected]

R. Bergstrome-mail: [email protected]

T. R. GreenTGA LLC, 13851 Verte Court, Lake Oswego, OR 97034, USAe-mail: [email protected]

V. GulcaState Agricultural University, Chișinău, Moldovae-mail: [email protected]

Keywords Lignocellulose . Microaerobic fermentation . Bokashi . Polysaccharides . Carbonrecycling . Soil fertilizers

Introduction

Adding fibrous lignocellulosic (FLC) materials into soil improves carbon (C) sequestrationand the recycling of macro- and micronutrients. It also improves soil properties, increasesdiversity and biomass of soil biota, suppresses weed growth, and stabilizes pH [1–5]. Themost thorough method of FLC preprocessing is composting, which recycles about 50 % ofthe initial organic C, decreases the risk of reintroducing plant pathogens, and may takebetween 6 and 12 months [6, 7]. The most common C sequestration and nutrient-recyclingpractices are tilling FLCs directly into soil or leaving them to decay on the surface, i.e.,no-till practices [3, 4, 8–10].

Lee et al. [9] compared greenhouse gas (GHG) emissions of composting and no-tilldisposal of logging residues and found that composting has lower GHG emissions. On theother hand, till/no-till practices are less energy intensive [11], but lesser integration of FLCinto soil, especially FLC from logging debris, can impede the incorporation of organics intothe soil ecosystem [3, 6, 8]. Furthermore, the lack of FLC preprocessing in till/no-tillpractices increases the risk of spreading plant pathogens [7].

Preprocessing of FLC materials by fermentation before tilling into soil, which is a methodused in non-intensive farming, may increase integration of FLC into soil without signifi-cantly increasing GHG emissions and processing costs [12–14]. Research indicates that thispreprocessing method may increase the availability of FLC nutrients in soil by initiating thehydrolysis of polysaccharidic polymers prior to their introduction into soil [15–17].This process is a form of microaerobic fermentation (MF), sometimes referred to asBokashi [16, 18]. MF occurs in enclosed fermentors, which remain under micro-aerobic conditions (about 0.5–2.0 % O2). With vegetal materials, such as food waste,leaves, and wheat bran, MF takes about 2–5 weeks [19, 20]. The fermentation periodrequired for FLC has not been reported. During fermentation, a compost leachate richin carboxylic acids, alcohols, and amines, is produced and continuously drained.Liquid, in the form of water or treated leachate, must be added to the systemperiodically to maintain moisture levels.

The goal of this work is to establish how various treatments during the MF of FLC affectfermentation endpoints, respiration/fermentation ratios, pH evolution, biological oxygendemand (BOD), C loss, polysaccharide (PS) hydrolysis, and PS degradation rate afterintroduction of fermented materials into soil.

Materials and Methods

MF treatments were set up in nine 20-l fermentors (Bokashicycle, LLC) as in Fig. 1. FLCconsisted of shredded poplar wood twigs. Produce amendments were composed of 50:50wet weight mixtures of Fuji apples and cabbage. Each fermentor was started with a 4-kgmixture of FLC and produce, 0.8 kg finely ground limestone (particle size010 mesh 90 %,100 mesh 49 %; composition020 % Ca, 11 % Mg, 50 % CaCO3, and 40 % MgCO3; Cacarbonate Eq.0102%; Oregon lime scale080; Greenacres Gypsum and Lime Co.), and 1.9 L tapwater. All organic materials were shredded twice with a wood chipper prior to MF processing.

910 Appl Biochem Biotechnol (2012) 167:909–917

The overall experimental design was as in Table 1. Fermentors A1, A2, and A3 contained4 kg wood chips and 0 kg produce (0 % w/w); B1, B2, and B3 contained 3.6 kg wood chipsand 0.4 kg produce (10 % w/w); and C1, C2, and C3 contained 2 kg wood chips and 2 kgproduce (50 % w/w).

Compost leachate was collected weekly. To each fermentor series amended with 0, 10,and 50 % produce, one of three leachate treatments was applied. In “Leachate el” treatments(A1, B1, and C1), leachates drawn from the fermentors were used for measurements anddiscarded. In “Leachate an” treatments (A2, B2, and C2), leachates were stored for 3 daysunder anaerobic conditions (∼95 % N2; ∼5 % H2), then returned to their original fermentors.In “Leachate ar” treatments (A3, B3, and C3), leachates were aerated continuously for3 days with an air pump and sparger, and returned to their fermentors. Fermentors were kept

Fig. 1 The basic scheme and functioning of an MF fermentor. Compost leachate is continuously drained.Liquid (water or treated leachate) is periodically added to maintain a high moisture level and to reintroducemineral nutrients. Conditions inside the fermentor are microaerobic, moist, and acidic

Table 1 Setup of MF fermentors based on starting abundance of produce relative to FLC and treatment ofleachate

Percentage produce Leachate el Leachate an Leachate ar

0 A1 A2 A3

10 B1 B2 B3

50 C1 C2 C3

“Leachate el,” leachate eliminated weekly; “Leachate an,” leachate stored in anaerobic conditions for 3 daysthen reintroduced into fermentor; “Leachate ar,” leachate aerated for 3 days, then reintroduced into fermentors

Appl Biochem Biotechnol (2012) 167:909–917 911

at room temperature. Their lids were opened weekly for sampling and measurements. Thevariables followed were temperature in the room and in the fermentor, atmospheric gases inthe headspace of the fermentor, water content of the top 2–5 cm of the solid residue, BOD ofthe solid residue and leachate, volatile organic acids (VOAs), alcohols, ketones, pH, andleachate production. Microbial activity was monitored based on the relationship between O2

and CO2 content in the headspace of fermentors and by tracking the formation of VOAs(acetic, propanoic, butyric, isovaleric, caproic, and valeric), alcohols (ethanol, i-propanol,n-propanol, i-butanol, n-butanol, and i-pentanol), acetone, and acetylacetone in the leachate.

For analysis of atmospheric gasses (O2, N2 CH4, and CO2), headspace gas was drawnthrough a stopper installed in the lid of each fermentor using a needle guidance sampler, andinjected in an SRI 310C gas chromatograph (GC) analyzer (SRI Instruments, CA, USA)equipped with thermal conductivity detector and two GC columns (3′-silica and a 6′-13X

Fig. 2 The effect of fermentate composition and leachate treatment on the pH of the leachate. Evolution of thepH in the fermentors: a leachate continuously eliminated; b leachate returned unaerated to the fermentors; andc leachate aerated for 3 days then returned to the fermentor. The labels (A1, A2, A3, B1, B2, … etc.) areexplained in Table 1

Fig. 3 The evolution of O2 in the headspace of the MF fermentors based on fermentate composition (a) andleachate treatment (b)


molecular sieve columns). VOAs, alcohols, and ketones were monitored by GC analysis of10-μl aliquots (with 50:1 split) using a Shimadzu GC-2010 with flame ionization detector ona Stabilwax-DA column (Restek, PA, USA) under the following conditions: 1.5 ml min−1

flow rate; 250 °C injector temperature; gradient temperature starting at 60 °C, 2 min iso,10 °C/min to 240 °C; and detector 260 °C. Calibrations and identifications were done bycomparing retention times and peak areas against standards using PeakSimple software(ver. 3.87).

Microbial respiration (R) was estimated based on changes in O2 concentration relative toan initial concentration of 21 % O2 (R0O2i−O2f, where O2i0percent initial O2 and O2f0percent final O2). Fermentation (F) was estimated based on the amount of CO2 notaccounted for by aerobic respiration assuming a stoichiometry of 1:1 between O2 respiredand CO2 produced, which is consistent with aerobic respiration of carbohydrates(F0ΔtotalCO2−ΔRCO2, whereΔtotalCO20change in CO2 between initial and final measure-ments and ΔRCO20CO2 production attributed to R).

PS (expressed in glucose equivalents) was measured using the phenol-sulfuric acidcarbohydrate assay [21, 22]. Changes in the fragmentation state of water-insoluble acid-soluble PS were tracked using the end-point fragmentation (EPF) index assay [23].

The pH of the leachate was measured with a Thermo Orion pH meter model 410. TheBOD of compost leachate and compost residue was measured using BOD bottles (Wheaton,Milville, NJ, USA) filled with air-saturated water at 20 °C. One hundred milligrams of driedand ground compost residue, or 20 μl of compost leachate, were inserted into each bottle.The bottles were capped and incubated at room temperature for 5 days. The soluble O2

content was measured with an Accumet Excel XL60 dual channel pH/ion/conductivity DO

Fig. 4 The evolution of CO2 in the headspace of the MF fermentors based on: a fermentate composition andb leachate treatment

Fig. 5 The fermentation/respiration ratios (F/R) for: a leachate treatments and b produce amendments. Thevalues given are based on total amounts of O2 consumed and CO2 produced during 5 weeks of experiment andare averages of the three fermentors from each series. Error bars represent±1 standard deviation


meter. BOD measurements were corrected based on O2 saturated controls, temperature, andatmospheric pressure. The same instrument was used to measure the temperature and salinityof the solutions. Atmospheric pressure was measured with a traceable pressure instrument(Fisher Scientific).

MF treated feedstock was amended into soil at a greenhouse facility located at PortlandState University (PSU). Soil had the general properties of a Helvetia silt loam [24]. Soilsamples (∼100 g per site) from the top ∼10 cm layer, were dried for 48 h at 60 °C and thenground to uniformity before weighing and analysis. Soil samples were amended with MFtreatment materials in a 1:1 ratio in the upper 15 cm layer and tracked for PS content andEPF index [23]. Soil pH was measured in aqueous slurries prepared from dried soil samplesin a ratio of 1:25 weight/deionized water.

Fig. 6 Evolution of: ethanol (diamond), acetic acid (triangle), and butyric acid (square) in the fermentors

Fig. 7 PS content in the leachate in relation to: a produce amendment and b leachate treatment. The labels(Leachate el, Leachate an, and Leachate ar) are explained in Table 1


Results and Discussion

The temperature of fermentors was between 20 and 24 °C for the duration of the experi-ments. Acidification occurred within the first 3 days; thereafter, leachates shifted towardneutral pH (Fig. 2). An increased abundance of produce relative to FLC resulted in moreacidic leachates. Elimination of leachates from the fermentors led to less acidic leachates.The sharp drop in pH, followed by a slight rise and subsequent stabilization at mildly acidicpH, indicates that sugars in many fermenters had not yet fully fermented after 35 days(Fig. 2). Aeration of leachates shifted the pH toward more neutral values (most likely due toturnover of carboxylic acids). The similar drop in pH to approximately pH 4 in all samples inthe first week indicates that pH inhibition is likely an important controller of microbialactivity. These results demonstrate that recycling leachates through MF fermentors, andconcomitant degradation of organic acids accumulating in the leachate, enhancesMF-mediated decomposition of organics.

We observed no CH4 production. In the first week, N2 in the headspace of the MFfermentors was inversely correlated with the amount of produce amendment. By the thirdweek, there was no measurable difference. O2 consumption decreased, leveling out at about2–3 weeks (Fig. 3), whereas CO2 increased, reaching a plateau by the second week offermentation (Fig. 4). O2 concentration in all of the fermentors fell to microaerobic con-ditions regardless of treatment.

More CO2 was initially produced in fermentors with larger produce/FLC ratios. Based onthe O2 and CO2 results, considerable amounts of biodegradable materials were still presentafter 5 weeks. Little variation occurred in fermentation associated with different leachatetreatments, whereas fermenters with the highest produce amendments had the highestfermentation levels (Fig. 5).

The fermentation/respiration ratio comparing the 50 and 10 % produce treatments of FLCwas about 3:1, markedly skewed from an expected ratio of 5:1, assuming fermentation wasunimpaired with increasing proportions of produce. This is consistent with earlier observa-tions related to pH trends that suggest incomplete fermentation of produce, and shows thatproduce amendments did not help accelerate the fermentation of FLC materials.

Of the VOAs measured, only acetic, propanoic, and butyric acid were produced insignificant amounts. No significant differences were seen between produce treatments inthe case of propanoic and butyric acid. Acetic acid production increased initially, and thendecreased after week 1 (Fig. 6). Total acid production (measured as the sum of all VOAsdetected) increased over time, but never above 0.01 % (data not shown). Ethanol and lesseramounts of n-propanol were also produced in the fermentors. Ethanol production was

Fig. 8 Evolution of BOD in leachate relative to: a produce amendments and b leachate treatment. The labels(Leachate el, Leachate an, and Leachate ar) are explained in Table 1


highest in FLC material amended with 50 % produce (Fig. 6). For any given produceabundance, the leachate treatment did not appear to influence alcohol production.

PS measured in the leachate in all fermentors decreased over time. In fermentors amendedwith 50 % produce, PS was initially high. Over a period of about 1 week, it decreased tovalues similar to those in other produce treatments (Fig. 7a). PS was lower in leachates whenleachate was eliminated relative to that of treatments where leachate was recycled (Fig. 7b).Total PS was unaffected in leachates by recycling of the leachates stored under aerobic oranaerobic conditions. The PS degradation profile in the FLC materials amended with 50 %produce (Fig. 7a) is likely attributable to degradation of small molecular weight PS andstarch originating from produce rather than cellulosic polymers from FLC.

In all untreated leachates, the BOD increased after the first week and decreased progres-sively thereafter. Leachate BOD was inversely correlated with the amount of produceamended with FLC (Fig. 8a). The same trend was observed in leachate BOD for the variousleachate treatments.

In fermentors containing FLC amended with produce, no significant difference inleachate BOD was seen among the three leachate treatments applied in this study (Table 1and Fig. 8b). The BOD of the residue remained within the range of 0.1 and 0.2 mg/L O2

throughout the course of the study. In soil, all of the MF-processed residues led to EPFindices and PS values significantly higher than the unamended control soil, but similar tounfermented control FLC material (Fig. 9). These results lead us to conclude that MFpreprocessing of FLC does not significantly accelerate its degradation.

Conclusions

Previous studies have shown that preprocessing of vegetal and agricultural material by MFincreases the availability of nutrients in soil by initiating the hydrolysis of PS prior to itsintroduction into soil [15–17]. The preprocessing by MF of FLC amended with produce for5 weeks increased fermentation rates and decreased the BOD of the fermented residues.Draining leachate from the fermentor without returning it increased acidification anddecreased the free PS content of leachate subsequently recovered from fermentors. Gasproduction and BOD results indicate that amending FLC materials with produce did notenhance the degradation of FLC materials. Increasing the amount of produce appears tocause inhibition of the fermentation process that biases microbial degradation towards smallmore easily digestible PS over the less accessible FLC materials. Produce amendment, or

Fig. 9 PS degradation in soil in relation to: a produce amendment and b leachate treatment with FLC materialrelative to unfermented FLC material (Wood C) and control soil (Soil C). PS evolution in soil was similar inall treatments


leachate treatments, did not influence the degradation of PS in soil amended with MF-processed materials relative to unfermented controls. Preprocessing FLC by MF thereforeappears no more efficient than direct tilling of FLC into soil regarding its assimilation backinto soil. Our results demonstrate that 2–5 week MF preprocessing (when fermentationendpoints have been reached with, or without, leachate recirculation or produce amend-ments) is not sufficient in advancing the degradation of FLC material for amending into soil.

Acknowledgments This work was supported by a sustainability initiative grant from the Miller Foundationat PSU, by a grant from the Graduate School at PSU and by Bokashicycle LLC.

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Effect of Microaerobic Fermentation in Preprocessing Fibrous Lignocellulosic Materials

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