Enhanced volatile fatty acids production from Napier grass

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Original Article 1

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Enhanced volatile fatty acids production from Napier grass 3

(Pennisetum purpureum) using micro-aerated anaerobic culture 4

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Chayanon Sawatdeenarunat1, Rotjapun Nirunsin2, and Sumate Chaiprapat3,* 6

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1 Asian Development College for Community Economy and Technology, Chiang Mai 8

Rajabhat University, Chiang Mai 50300, Thailand 9

2 Energy Engineering Program, School of Renewable Energy, Maejo University, 10

Chiang Mai 50290, Thailand 11

3 Department of Civil Engineering, Faculty of Engineering, Prince of Songkla University, 12

Songkla 90112, Thailand 13

*Corresponding Author, E-mail address: [email protected] 14

Manuscript

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Abstract 15

The laboratory-scale horizontal acidogenic bioreactor was conducted in semi-16

anaerobic and strict anaerobic conditions using Napier grass (Pennisetum purpureum) as the 17

sole substrate to determine the effects of micro-aeration on organic acids yield. During semi-18

anaerobic condition, total volatile fatty acids (VFAs) yield was significantly higher (α=0.05) 19

i.e. 3,524±191, and 2,831±89 mg/L for the micro-aerated, and anaerobic conditions, 20

respectively. The biomethane production was effectively suppressed during micro-aeration as 21

a result of methanogenesis inhibition by the controlled oxygenation. Improved hydrolysis by 22

facultative organisms was evidenced by the elevated soluble chemical oxygen demand 23

(SCOD) concentration which was readily available for organic acid production. 24

Hemicellulose in the biomass was efficiently degraded at 10.0% during micro-aerated period, 25

while other constituents i.e. cellulose and lignin remained in the digestate. Potential use of 26

these co-products of VFA-rich liquor and pretreated solid digestate for higher biofuels or 27

valuable biochemical compounds was discussed in the context of the bio-circular-green 28

economy model. 29

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Keywords: Micro-aeration; Lignocellulosic biomass; Volatile fatty acids; Digestate; 31

Anaerobic digestion 32

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1. Introduction 34

Anaerobic biorefinery is a relatively new concept in which anaerobic digestion serves 35

as a center piece to generate high-value products ranging from biofuels (hydrogen, ethanol, 36

and butanol) to many bio-based products (i.e. bioplastic and biochemicals) instead of a 37

conventional methane gas. Volatile Fatty Acids (VFAs), organic acids with carbon 2 to 5 38

atoms, can also be used as an excellent carbon source for denitrifying bacteria (replacing 39

methanol) for biological nutrient removal (Elefsiniotis & Wareham, 2007) or lipid-40

accumulating microalgae (e.g. Chlorella protothecoides and Cryptococcus albidus) that 41

contains high amounts of omega-3 fatty acids and exopolysaccharides (Chalima et al., 2017). 42

The main idea of this notion is to optimize the profit of an AD system by producing high-43

value bio-based products while serving the energy needs by producing biofuels in subsequent 44

stages. In addition, eliminating methanogenesis stage favors the production of VFAs as well 45

as shortens substrate residence time in reactors. Capital and operating costs can be cut down 46

leading to increased economic feasibility of the system. A variety of substrates including 47

agricultural residues (Zacharof & Lovitt, 2013) and vegetable wastes (Li et al., 2017) were 48

successfully used to produce VFAs by various strategies to inhibit VFAs conversion. The 49

shift in the generation as well as composition of VFA species was also found to respond to 50

operating parameters, i.e. pH, organic loading rate (OLR) and hydraulic retention time 51

(HRT), in connection with the different types of substrates digested (Bengtsson, Hallquist, 52

Werker, & Welander, 2008; Chen, Jiang, Yuan, Zhou, & Gu, 2007). This is worth further 53

investigations as it has implications on the values of the total end products. Since operating 54

digester at shorter HRT or high OLR favors acidogens to shift the final product to VFAs 55

instead of methane. Recently, aeration to the anaerobic digester has been suggested as a new 56

way to stimulate the growth of facultative microorganisms which could then enhance 57

hydrolysis and acidogenesis. Thus, the concept of micro-aeration to AD was applied to 58

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increase the excretion of extracellular enzymes to enhance hydrolysis-acidogenesis reaction 59

chain. Several researchers reported the enhanced hydrolysis of many organic materials 60

including lignocellulosic biomass) and sewage sludge (Johansen & Bakke, 2006; 61

Sawatdeenarunat, Sung, & Khanal, 2017) by micro-aeration. Zhu, Lü, Hao, He, and Shao 62

(2009) revealed that the acidogenesis efficiency correlated to the intensity and cycle of 63

aeration during anaerobic digestion of fresh vegetable and flower wastes. However, excessive 64

air dosage caused negative impacts on VFAs yield. Extended retention of the substrate with 65

suitable aeration could be key for continued VFAs production without acclimation of 66

methanogens to stresses such as low pH or OLR since methanogens are strict anaerobes. 67

With an ample information on batch studies, long-term stable operation with such micro-68

aeration in continuous anaerobic digestion of lignocellulosic biomass using horizontal 69

bioreactor to prevent scum formation is still lacked. 70

Therefore, the main purpose of this study is to evaluate the effect of micro-aeration on 71

the VFAs production from Napier grass using the continuous operation of horizontal 72

bioreactor. Changes in biogas composition and yields were monitored in relation to the 73

process outputs, i.e. biomass composition and organic acids generation and composition. 74

Finally, the benefits from acid production were assessed as a potential gain from our 75

proposed micro-aeration strategy. 76

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2. Materials and Methods 78

2.1 Substrate and inoculum 79

The 3-month-old Napier grass was harvested from the University of Hawai’i 80

Waimanalo Research Station (Waimanalo, HI, USA). The harvested biomass was proceeded 81

to the final size of 6 mm and moisture content less than 10%. Finally, the grass was kept in a 82

vacuum bag. The total solids (TS), volatile solids (VS), and fiber composition of the biomass, 83

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i.e. Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF), and Acid Detergent Lignin 84

(ADL), were analyzed. The characteristics of Napier grass used in this study were 91.0±1%, 85

83.0±1%, 39.5±0.9%TS, 25.3±1.6%TS, and 10.2±0.3%TS for TS, VS, cellulose, 86

hemicellulose, and lignin, respectively. The inoculum was anaerobically digested cattle 87

manure (ADCM) which was prepared in the laboratory using the 20-L anaerobic reactor fed 88

with cattle manure and operated at mesophilic conditions (33±2˚C) for over a year. The initial 89

seed sludge concentration of the reactors was 25,000 mg/L as volatile solids. 90

91

2.2 Reactor configuration 92

Two acrylic horizontal bioreactors with the radius of 0.15 m, and the length of 0.30 m 93

resulting the total volume of approximately 5 liters were used in this study as shown in Figure 94

1. Each reactor has a working volume of 2.7 L. The horizontally mechanical mixers were 95

installed to mix the reactor contents, move them to the outlet port, break down an 96

accumulated scum, and degas the liquid. The two reactors were operated as duplicate at 97

mesophilic condition (33±2˚C) where the average values were used in this paper. A silicone 98

rubber heating blanket (BriskHeat OH, USA) installed to maintain the reactor temperature. 99

The thermocouple type T (Omega Engineering, Inc., Connecticut, USA) and data locker 100

(Dataq Instruments, Inc., Ohio, USA) were used to monitor and control the reactor 101

temperature. The reactors were operated in semi-continuous mode. The mixing speed was 102

fixed at 90 rpm and turned on for 5 minutes in a 30-minute cycle. There are four air injection 103

ports installed at the bottom along the length of reactor. 104

105

Figure 1 Horizontal acid bioreactor used in the experiment 106

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2.3 Reactor operation 109

The bioreactors were started up at an initial OLR of 4 kgVS/m3.d for a month. The 110

feedstock was prepared according to section 2.1. The slurry of feed prepared at 7% was fed 111

thru the inlet port at the same time once a day. During operation, the reactor contents were 112

withdrawn at the outlet port and analyzed as detailed in section 2.4. During the reactor 113

operating period, the operating OLR was stepwise increased at an interval of 1 kgVS/m3.d 114

until reactor failure (high accumulation of solids) was observed at OLR 7 kgVS/m3.d. OLR 115

was then suddenly reduced to 6 kgVS/m3.d (HRT of 14 days) and continued until stable 116

operation was reached. Subsequently, the micro-aeration was applied thru fine diffusers for 1 117

min every 6 hours using an air pump (Super pond, WA, USA) at a constant flowrate of 450 118

mL/min. The appropriate air flow rate was fixed by the pump and the authors had run a 119

preliminary study at a few aeration frequencies. At higher frequency (i.e. every 2 hours), the 120

VFA production was not enhanced while the methane in the reactor’s headspace was diluted. 121

At lower frequency, no clear effect on the VFA production was detected. Thus, the micro-122

aeration frequency at 6 hours was selected for our experiment. The reactors were operated 123

under micro-aeration mode until stable which lasted 50 days. Effluents were collected for 124

analyses of both liquid and solid portions for comparison with those from anaerobic 125

condition. 126

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2.4 Analytical methods 128

A bench top pH meter (Accumet AB15, Fisher, Fairlawn, USA) was used to analyze 129

pH of the rector contents. Soluble chemical oxygen demand (SCOD), TS, and VS were 130

determined using the Standard Methods (APHA, 2005). NDF, ADF and ADL of the biomass 131

before and after AD were analyzed as described by (Madrid, 2004). Cellulose and 132

hemicellulose were calculated using the following equations: Hemicellulose = NDF – ADF 133

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and Cellulose = ADF – ADL. A milli gas counter (Ritter US LLC. NY, USA) and gas 134

chromatography with thermal conductivity detector (GC-TCD) (GC2014, Shimadzu, Japan) 135

equipped with a packed column (80/100 Hayesep D column, 2 m length 3.2 mm outer 136

diameter 2.1 mm inner diameter, Supelco, USA) were used to determine the produced 137

biogas, and biogas composition, respectively. The individual VFAs were analyzed using GC 138

with flame ionization detector (GC-FID) (GC2014, Shimadzu, Japan) using a capillary 139

column (ZB-Wax Plus column 30 m length 0.25 mm inner diameter 0.25 µm film 140

thickness, Phenomenex, USA). 141

142

2.5 Statistical analysis 143

The analysis of variance (ANOVA) and Tukey’s test for post-hoc analysis was used to 144

examine statistically significance with a threshold value α=0.05 using JMP statistical 145

software (JMP Pro 12.0.1, SAS Institute Inc., Cary, NC, USA). 146

147

3. Results and Discussion 148

3.1 VFAs yield during AD of Napier grass 149

The OLR was gradually increased by 1 kgVS/m3.d from 4 to 7 kgVS/m3.d within 150

approximately 100 days. The signs of system imbalance started to occur at OLR 7 151

kgVS/m3.d, i.e. the ratio of volatile fatty acids to alkalinity (VFA/ALK) over 1.0 and 152

lowering effluent pH value. It is noted that the maximum VFA/ALK of 0.8 is recommended 153

for stable anaerobic digester of various substrates (Khanal, 2008). During such period, 154

NaHCO3 was added to correct the buffering capacity and pH of these duplicate reactors. 155

After a few attempts, it was concluded that system failure at OLR 7 kgVS/m3.d was 156

permanent. The operating OLR returned to 6 kgVS/m3.d and the bioreactors were operated at 157

stable condition for more than a month before the data collection and analysis were 158

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performed. Ambient air was injected to the reactor content at day 57 into the operation at 159

OLR 6 kgVS/m3.d. After micro-aeraion (1 minute every 6 hours), the interesting changes in 160

VFAs concentrations appeared together with the fluctuating pH, as shown in Figure 2. 161

162

Figure 2 pH and VFAs composition in the effluent before and after micro-aeration at 163

the operating OLR of 6 kgVS/m3.d 164

165

At stable operating condition (OLR of 6 kgVS/m3.d), pH of the effluents was at 166

virtually the same level at 5.27±0.04 and 5.37±0.06 for anaerobic and micro-aerated 167

conditions, respectively. These values were a little lower than the recommend range for acid 168

bioreactor (i.e. 5.5-6.5) (Agler, Wrenn, Zinder, & Angenent, 2011) where methanogenic 169

growth is largely inhibited . The total VFA concentration (TVFA) was between 2,800 and 170

3,030 mg/L during day 42-57 (anaerobic period). However, after micro-aeration at day 57, 171

TVFA significantly decreased to between 2,500 and 2,600 mg/L during days 59 to 77. This 172

phenomenon clearly indicated an acclimation of the microorganisms toward micro-aerated 173

condition. The chain of reactions was disrupted, which caused harsher effect to the strict 174

anaerobes of methanogens and trickled up to the facultative hydrolytic and acidogenic 175

organisms in front end reactions. It took 18 days for the facultative organisms to adapt. After 176

which, there was a big leap of VFAs, indicating an enhanced growth of cellulolytic 177

organisms. It was clear that such increased hydrolysis was coupled with a suppressed 178

methanogenic activity. This phenomenon might be from the acclimation of the 179

microorganism toward micro-aerated condition (Yu et al., 2020). Continued operation of 180

micro-aeration scheme approximately 20 days has caused the significant shift in anaerobic 181

pathways in the horizontal bioreactors. The VFAs concentration sharply increased after day 182

77 by 43% from 2,600 to 3,730 mg/L within only two days. At stable condition, it was 183

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obvious that TVFA concentration of the micro-aerated operation was significantly higher 184

than that of anaerobic condition (i.e. 3,524±191 vs. 2,831±89 mg/L). It was due to the 185

biological conversion of organics by oxidative pathway of facultative organisms in the 186

culture. Such phenomenon was supported by our observation in the drop of methane and the 187

rise of carbon dioxide content in the biogas. Methane content in the produced biogas dropped 188

from 16.8±2.2% in anaerobic condition to 6.6±1.7% in micro-aerated condition. During this 189

period, methanogenic archaea was inhibited by the presence of molecular oxygen in the 190

environment in addition to the low pH environment they reside. 191

Our results agreed with the previous batch studies but at a lesser degree of VFA 192

increase. Studies of micro-aeration using lignocellulosic materials i.e. grass silage as 193

substrates reported 4-fold in VFAs production after micro-aeration (Jagadabhi, Kaparaju, & 194

Rintala, 2010). It could be due to the difference between continuous versus batch operation 195

where maximum increase was compromised by the constraints of reactor operations such as 196

hydraulic retention time and organic loading. Too long of the oxygenation to the digester 197

although at batch optimal oxygen dosage of 0.1 volume air per volume slurry per minute 198

caused a severe drop in the acetate produced after 60 hours of digestion time (Obeta 199

Ugwuanyi, Harvey, & NcNeil, 2005). This gap of hydrolysis-acidification improvement can 200

be closed once more studies embarking the optimized micro-aerated acidogenic operation is 201

carried out in greater details. There is still a wide opportunity for future studies on this micro-202

aeration acid reactor. 203

Analysis of the composition of VFA species produced during the anaerobic and 204

micro-aerated condition showed that acetic acid still dominated for more than 50% of the 205

total VFAs in both anaerobic and micro-aerated conditions. Other VFAs (i.e. propionic, 206

butyric, and valeric acids) observed during these operations were increased in accordance 207

with the acetic acid, but the concentrations of minor VFAs species i.e. iso-butyric, iso-valeric, 208

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and valeric acid were not significantly different. This was consistent across other operations 209

of digester configuration with grass biomass feedstock as well, such as the batch-type leach-210

bed reactor where acetic acid constituted for 40% of total VFAs in leachate (Jagadabhi et al., 211

2010). In such micro-aerated leach-bed operation, there did not appear to be acetogenic 212

inhibition since acetate was still produced at high level. Although inhibition of specific VFA 213

species to methanogens was widely reported by Zhao et al. (2020), to our knowledge, there is 214

no specific report on the VFA species on acidogenic activity other than product inhibition 215

(Sui et al., 2018). This study is worth further investigations. 216

217

3.2 Soluble chemical oxygen demand (SCOD) production 218

Typically, SCOD could indicate the soluble products from hydrolysis stage of AD. 219

The produced SCOD and VFAs during micro-aerated condition were 10 and 24% higher than 220

those of anaerobic condition, respectively. The SCOD and TVFA concentrations in the 221

reactor contents were illustrated in Table 1. According to the increase of SCOD and VFA, the 222

ratio of VFAs and SCOD of micro-aerated condition also rose 11% higher than that of strict 223

anaerobic condition. VFAs/SCOD ratio is indirectly used to determine the efficiency of 224

acidogenesis stage of AD systems. This parameter could be used to represent an efficiency of 225

acidogens to convert the hydrolyzed products (i.e. alcohol, sugars, amino acid, and fatty acids 226

among others) in the reactor contents to VFAs. 227

228

Table 1 Soluble chemical oxygen demand (SCOD) and volatile fatty acids (VFAs) 229

production during stable operations before and after micro-aeration 230

231

Enhanced hydrolysis during aerated AD of many organic substrates and bioreactor 232

configurations was reported by many researchers. The positive effect on hydrolysis of protein 233

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and carbohydrate during micro-aerated AD of primary sludge was presented by Johansen and 234

Bakke (2006). More than 50% increase in COD during hydrolysis was detected compared to 235

normal anaerobic condition. Under micro-aeration in thermophilic digester operation, an 236

increase in hydrolytic enzymes such as cellulase and protease production by the AD 237

microbial culture was found to be a factor to enhance acidogenesis (Jagadabhi, Kaparaju, & 238

Rintala, 2010). However, the air dosage plays a key role in the productivity of hydrolysis 239

reaction. An inappropriate aeration dosage (i.e. insufficient dosage) could also reduce the 240

hydrolysis efficiency compared to that of unaerated condition, as reported by Zhu et al. 241

(2009). The facultative microorganisms would be more active during micro-oxygenation as a 242

result of enhanced extracellular hydrolytic enzymes production (Bengtsson et al., 2008). 243

Some microorganisms are augmented during micro-areared condition depending on the flora 244

existed in the substrate; for example, higher microbes in Firmicutes phylum were the 245

dominent group of food waste and brown water digestion with some oxygenation (Zhu et al., 246

2009). However, too high of the oxygen would lead to the revival of aerobic organisms as 247

well. The balance of oxygenation rate to different kinds of substrates and reactor 248

configurations is of great interest for further development. 249

250

3.3 Biogas composition 251

Multiple reports indicated that aeration inhibited the strict anaerobic microorganisms 252

(i.e. methanogens) (Sui et al., 2018) and allowed the alternate production of other valuable 253

products (e.g. hydrogen and VFAs) particularly during acidogenesis stage of AD (Sołowski, 254

Konkol, & Cenian, 2020). Our results show that methane yield under the anaerobic condition 255

was significantly lower (α=0.05) than that of the micro-aerated condition. However, no 256

significant difference of CO2 yield was observed among the two operating conditions. This 257

phenomenon was explained by the inability of methanogenic species to synthesize superoxide 258

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dismutase which is the enzyme responsible for mitigating oxygen ion and radical toxicity 259

(Botheju & Bakke, 2011). Many methanogen cells such as those of Methanococcus voltae 260

and Methanococcus vannielii could be severely damaged when exposed to oxygen (Kiener & 261

Leisinger, 1983). The average CH4 and CO2 yields during the operations are illustrated in 262

Table 1.Analogues to this study, Botheju and Bakke (2011) revealed that in the batch 263

experiment, the aerated inoculum could result in three times longer lag phase of 264

methanogenation compared with that of the unaerated inoculum. The relation between the 265

methane production and VFAs yield versus oxygenation was critical in the scheme of organic 266

acid producing reactor as the substrate type and loading rate to the reactor must be balanced 267

with the oxygenation for targeted optical operation (Eryildiz, Lukitawesa, & Taherzadeh, 268

2020). The level of methanogenic inhibition and hydrolytic-acidogenic augmentation will 269

play an important role in valuation of the products from the process. In addition, it is noted 270

that CH4 content in the produced biogas was low (in a range of 15-25%) as a synergic result 271

of air dilution and methanogenic inhibition. For CO2 generation, by introducing the 272

appropriate oxygen dosage, rather than converting organic matter via aerobic respiration to 273

produce CO2, the facultative microorganisms selectively kept metabolic pathway via 274

fermentation to produce VFAs. However, oxygen overdosing posed a threat of increased CO2 275

production, which has lowest value, as the metabolic function of facultative organisms had 276

shifted to aerobic respiration (Botheju & Bakke, 2011). Besides, the more CO2 production 277

means the loss of carbon source for synthesizing organic acid molecules and waste of energy 278

for over oxygenation. Thus, the appropriate reactor operation must preserve VFAs as the 279

main product from the AD of Napier grass while minimizing all other competing products at 280

the same time. Further studies on such issues are under investigations in the valuation 281

improvement of biocircular research. 282

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3.4 Fiber composition 284

Cellulose, hemicellulose (structural carbohydrates) and acid detergent lignin (ADL) of 285

Napier grass before and after AD from both micro-aerated and anaerobic conditions are 286

presented in Figure 3. The changing in cellulose, hemicellulose, and ADL would dictate the 287

appropriate conversion and utilization of the digestate. Hemicellulose contents of the 288

digestates from anaerobic and micro-aerated conditions were 26±1 and 27±1%TS, 289

respectively. These values from both conditions were significantly lower than that of raw 290

Napier grass (i.e. 30±0%TS). The hemicellulose removal in anaerobic operation was found to 291

be 13.3% compared to 10.0% during the micro-aerated one. Reversely, cellulose and lignin 292

still persisted in the digestate with negligible degradation. These constituents could be further 293

utilized to produce solid biofuel via thermochemical processes such as hydrothermal 294

carbonization (Funke & Ziegler, 2010) and torrefaction (Rentizelas & Li, 2016), or other 295

biobased products (Sawatdeenarunat et al., 2016). Hemicellulose was leached off the biomass 296

well even in mild acidic condition as similarly observed during anaerobic fermentation of 297

palm fiber (Saritpongteeraka, Chaiprapat, Boonsawang, & Sung, 2015) and Napier grass 298

(Sawatdeenarunat et al., 2017). Operating in the acidic condition functioned as a mild acid 299

pretreatment to increase the accessibility of the hydrolytic enzymes by weakening the 300

recalcitrant structure of lignocellulosic biomass. Typical compositions of a plant cell wall are 301

mainly lignocellulosic materials namely cellulose, hemicellulose, and lignin. However, 5-302

10% of a plant cell wall could consist of other nonstructural components such as protein and 303

lipid (Zeng et al., 2017). Thus, the total percentages of lignocellulosic compositions in Figure 304

3 were not equal to 100%. 305

306

Figure 3 Fiber composition in Napier grass biomass during anaerobic and micro-307

aerated operations 308

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3.5 Value chain perspective 309

To optimize the benefit of the system, the downstream purification processes are 310

required to obtain only VFAs as a product. Many physical and chemical techniques have 311

been reported for purifying the anaerobic mixtures (Zacharof & Lovitt, 2013). The individual 312

VFAs could be used as initial substrates to produce many specific high-value biobased 313

products. For example, acetic acid, the major produced acid in this study, could be used to 314

produce a food additive and favoring agents in some food industries. Propionic acid could 315

also be potentially served as food and feed preservative compounds and used to produce 316

vitamin E (Yang, Wang, Lin, & Yang, 2018). The well-known biodegradable plastics; 317

polyhydroxyalkanoates (PHA), and polyhydroxybutyrate (PHB) could specifically be 318

produced from acetic acid and butyric acid. The market size and price of the three main 319

VFAs are presented in Table 2. It should be noted that the dominant individual VFAs species 320

during AD of Napier grass in this study are acetic acid and propionic acid. By their large 321

market size globally and production potential from the abundant biomass, the technical 322

appropriateness of the extraction technologies and their associated costs will only be a limit 323

and plays an important role in this bio-based business. In addition, the values of the produced 324

individual acids per ton of dry Napier grass obtained in this study are showed in Table 2. 325

Acetic acid showed the highest worth of 310 USD/dry metric ton of grass following by 326

butyric acid and propionic acid. For the supply side of the feedstock, other biomasses (i.e. 327

cheap cultivated crops, agricultural residues, or wastes) can be used as starting material for 328

the acid producing reactor and more research to convert these different organic masses to 329

high value compounds are underway. 330

331

Table 2 The volatile fatty acids market size and price 332

333

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4. Conclusion 334

The VFAs yields in the horizontal bioreactors operation using Napier grass feedstock 335

were enhanced by micro-aeration. The significant increasing of VFAs concentrations at the 336

operating OLR of 6 kgVS/m3.d were observed. The strict anaerobe methanogens were 337

hindered by micro-aeration while augmenting hydrolytic and acidogenic organisms. The 338

VFAs produced from micro-aerated AD system are the primary substrate to synthesize the 339

high-value bio-based products in the bio-circular-green technology platform to enhance the 340

economic viability of anaerobic digestion systems. 341

342

5. Acknowledgements 343

This study was financially supported by the Sun Grant Western Regional Center at 344

Oregon State University through a grant provided by the United States Department of 345

Agriculture and National Institute of Food and Agriculture, under proposal number 2012-346

03373. 347

348

6. References 349

350

Agler, M. T., Wrenn, B. a, Zinder, S. H., & Angenent, L. T. (2011). Waste to bioproduct 351

conversion with undefined mixed cultures: the carboxylate platform. Trends in 352

Biotechnology, 29(2), 70–78. https://doi.org/10.1016/j.tibtech.2010.11.006 353

APHA. (2005). Standard methods for the examination of water and wastewater (twenty-fir). 354

American Public Health Association/American Water Works Association/Water 355

Environment Federation. 356

Bengtsson, S., Hallquist, J., Werker, A., & Welander, T. (2008). Acidogenic fermentation of 357

industrial wastewaters: Effects of chemostat retention time and pH on volatile fatty acids 358

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

16

production. Biochemical Engineering Journal, 40(3), 492–499. 359

https://doi.org/10.1016/j.bej.2008.02.004 360

Botheju, D., & Bakke, R. (2011). Oxygen Effects in Anaerobic Digestion – A Review. The 361

Open Waste Management Journal, 4, 1–19. https://doi.org/10.4173/mic.2010.2.2 362

Chalima, A., Oliver, L., De Castro, L. F., Karnaouri, A., Dietrich, T., & Topakas, E. (2017). 363

Utilization of volatile fatty acids from microalgae for the production of high added value 364

compounds. In Fermentation (Vol. 3, Issue 4, p. 54). MDPI AG. 365

https://doi.org/10.3390/fermentation3040054 366

Chen, Y., Jiang, S., Yuan, H., Zhou, Q., & Gu, G. (2007). Hydrolysis and acidification of 367

waste activated sludge at different pHs. Water Research, 41(3), 683–689. 368

https://doi.org/10.1016/j.watres.2006.07.030 369

Elefsiniotis, P., & Wareham, D. G. (2007). Utilization patterns of volatile fatty acids in the 370

denitrification reaction. Enzyme and Microbial Technology, 41(1–2), 92–97. 371

https://doi.org/10.1016/j.enzmictec.2006.12.006 372

Eryildiz, B., Lukitawesa, & Taherzadeh, M. J. (2020). Effect of pH, substrate loading, 373

oxygen, and methanogens inhibitors on volatile fatty acid (VFA) production from citrus 374

waste by anaerobic digestion. Bioresource Technology, 302, 122800. 375

https://doi.org/10.1016/j.biortech.2020.122800 376

Funke, A., & Ziegler, F. (2010). Hydrothermal carbonization of biomass: A summary and 377

discussion of chemical mecha- nisms for process engineering. Biofuels, Bioproducts and 378

Biorefining, 4, 160–177. https://doi.org/10.1002/bbb.198 379

Jagadabhi, P. S., Kaparaju, P., & Rintala, J. (2010). Effect of micro-aeration and leachate 380

replacement on COD solubilization and VFA production during mono-digestion of 381

grass-silage in one-stage leach-bed reactors. Bioresource Technology, 101, 2818–2824. 382

https://doi.org/10.1016/j.biortech.2009.10.083 383

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

17

Johansen, J.-E., & Bakke, R. (2006). Enhancing hydrolysis with microaeration. Water 384

Science & Technology, 53(8), 43. https://doi.org/10.2166/wst.2006.234 385

Khanal, S. K. (2008). Anaerobic biotechnology for bioenergy production: principles and 386

applications. John Wiley & Sons, Inc. 387

Kiener, A., & Leisinger, T. (1983). Oxygen Sensitivity of Methanogenic Bacteria. Systematic 388

and Applied Microbiology, 4(3), 305–312. https://doi.org/10.1016/S0723-389

2020(83)80017-4 390

Li, Y., Hua, D., Mu, H., Xu, H., Jin, F., & Zhang, X. (2017). Conversion of vegetable wastes 391

to organic acids in leaching bed reactor: Performance and bacterial community analysis. 392

Journal of Bioscience and Bioengineering, 124(2), 195–203. 393

https://doi.org/10.1016/J.JBIOSC.2017.02.020 394

Madrid, F. (2004). Methods in Agricultural Chemical Analysis. Journal of Environment 395

Quality, 33(4), 1580-a. https://doi.org/10.2134/jeq2004.1580a 396

Obeta Ugwuanyi, J., Harvey, L. M., & McNeil, B. (2005). Effect of aeration rate and waste 397

load on evolution of volatile fatty acids and waste stabilization during thermophilic 398

aerobic digestion of a model high strength agricultural waste. Bioresource Technology, 399

96(6), 721–730. https://doi.org/10.1016/j.biortech.2004.06.028 400

Rentizelas, A. A., & Li, J. (2016). Techno-economic and carbon emissions analysis of 401

biomass torrefaction downstream in international bioenergy supply chains for co-firing. 402

Energy, 114, 129–142. https://doi.org/10.1016/j.energy.2016.07.159 403

Saritpongteeraka, K., Chaiprapat, S., Boonsawang, P., & Sung, S. (2015). Solid state co-404

fermentation as pretreatment of lignocellulosic palm empty fruit bunch for organic acid 405

recovery and fiber property improvement. International Biodeterioration and 406

Biodegradation, 100, 172–180. https://doi.org/10.1016/j.ibiod.2015.03.001 407

Sawatdeenarunat, C., Nguyen, D., Surendra, K. C., Shrestha, S., Rajendran, K., Oechsner, H., 408

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

18

Xie, L., & Khanal, S. K. (2016). Anaerobic biorefinery: Current status, challenges, and 409

opportunities. Bioresource Technology, 215, 304–313. 410


Sawatdeenarunat, C., Sung, S., & Khanal, S. K. (2017). Enhanced volatile fatty acids 412

production during anaerobic digestion of lignocellulosic biomass via micro-oxygenation. 413

Bioresource Technology, 237, 139–145. https://doi.org/10.1016/j.biortech.2017.02.029 414

Sołowski, G., Konkol, I., & Cenian, A. (2020). Methane and hydrogen production from 415

cotton waste by dark fermentation under anaerobic and micro-aerobic conditions. 416

Biomass and Bioenergy, 138, 105576. https://doi.org/10.1016/j.biombioe.2020.105576 417

Sui, Q., Meng, X., Wang, R., Zhang, J., Yu, D., Chen, M., Wang, Y., & Wei, Y. (2018). 418

Effects of endogenous inhibitors on the evolution of antibiotic resistance genes during 419

high solid anaerobic digestion of swine manure. Bioresource Technology, 270, 328–336. 420


Wang, K., Yin, J., Shen, D., & Li, N. (2014). Anaerobic digestion of food waste for volatile 422

fatty acids (VFAs) production with different types of inoculum: Effect of pH. 423

Bioresource Technology, 161, 395–401. https://doi.org/10.1016/j.biortech.2014.03.088 424

Yang, H., Wang, Z., Lin, M., & Yang, S. T. (2018). Propionic acid production from soy 425

molasses by Propionibacterium acidipropionici: Fermentation kinetics and economic 426

analysis. Bioresource Technology, 250(October), 1–9. 427


Yu, N., Guo, B., Zhang, Y., Zhang, L., Zhou, Y., & Liu, Y. (2020). Different micro-aeration 429

rates facilitate production of different end-products from source-diverted blackwater. 430

Water Research, 177, 115783. https://doi.org/10.1016/j.watres.2020.115783 431

Zacharof, M. P., & Lovitt, R. W. (2013). Complex effluent streams as a potential source of 432

volatile fatty acids. Waste and Biomass Valorization, 4(3), 557–581. 433

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

19

https://doi.org/10.1007/s12649-013-9202-6 434

Zeng, Y., Himmel, M. E., & Ding, S. Y. (2017). Visualizing chemical functionality in plant 435

cell walls Mike Himmel. Biotechnology for Biofuels, 10(1), 1–16. 436

https://doi.org/10.1186/s13068-017-0953-3 437

Zhao, W., Jeanne Huang, J., Hua, B., Huang, Z., Droste, R. L., Chen, L., Wang, B., Yang, C., 438

& Yang, S. (2020). A new strategy to recover from volatile fatty acid inhibition in 439

anaerobic digestion by photosynthetic bacteria. Bioresource Technology, 311, 123501. 440

https://doi.org/10.1016/j.biortech.2020.123501 441

Zhu, M., Lü, F., Hao, L.-P., He, P.-J., & Shao, L.-M. (2009). Regulating the hydrolysis of 442

organic wastes by micro-aeration and effluent recirculation. Waste Management (New 443

York, N.Y.), 29(7), 2042–2050. https://doi.org/10.1016/j.wasman.2008.12.023 444

445

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Point-by-point response to the reviewer’s and editor’s comments

Reviewer 1

Manuscript 'Enhanced volatile fatty acids production from Napier grass (Pennisetum purpureum)

using micro-aerated anaerobic culture' has some points to be corrected and revised as listed below;

Comment 1: The condition for micro-aeration and anaerobic conditions were not clearly explain.

Please state in detail how to carry out the experiment in each condition and for how long each

condition was held.

Response 1: The details of reactor configuration, and reactor operation during micro-aeration

period are given in more details as presented in lines 93-95 and 117-126, respectively.

Comment 2: Figure 2 is too difficult to distinguish which line is which. I recommend to use color

for each acid type and/or put label at the line in the graph.

Response 2: Thank you for the comment. The lines and markers in figure 2 are colored.

Comment 3: Figure 3, the percentage composition in total was not equal to 100% please recheck

the number. Otherwise, report in mass is appreciable.

Response 3: The additional explanation of Figure 3 regarding the summation of fiber composition

is added between line 301 and 305. There will be composition other than cellulose, hemicellulose

and lignin in any biomass. This should justify the existence of other composition in the biomass.

Comment 4: Table 2 should be in the introduction part since it is fact which you collect it from

literature (not your own experimental result).

Response to reviewers

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Response 4: Thank you for the comment. The additional information on the value of individual

acids calculated from the experimental results of this study is added to Table 2 to strengthen section

3.5. In addition, the more discussion is also added between line 324 and 327, thus, Table 2 is kept

in section 3.5.

Reviewer 2

Comment: The manuscript 'Enhanced volatile fatty acids production from Napier grass

(Pennisetum purpureum) using micro-aerated anaerobic culture' has been revised

carefully according to the reviewers comments. I recommend to be published as it is.

Response: Noted with thanks.

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Figure 1 Horizontal acid bioreactor

Figure

Figure 2 pH and VFAs composition in the effluent before and after micro-aeration at the

operating OLR of 6 kgVS/m3.d

0

1

2

3

4

5

6

0

500

1000

1500

2000

2500

3000

3500

4000

4500

40 60 80 100

pH

Conce

ntr

atio

n (m

g/L

)

Operting day

Acetic acid Propionic acid

Isobutyric acid Butyric acid

Isovaleric acid Valeric acid

Total volatile fatty acids pH

Anaerobic Micro-aeration

Figure 3 Fiber composition in Napier grass biomass during anaerobic

and micro-aeration operations

0

5

10

15

20

25

30

35

40

45

50

Raw Napier

grass

Anaerobic Micro-

aeration

Conce

ntr

atio

n (

%T

S)

Operating condition

Hemicellulose Cellulose Acid detergent lignin

Table 1 Soluble chemical oxygen demand (SCOD) and volatile fatty acids (VFAs)

production during stable operations before and after micro-aeration

Condition SCOD

(mg/L)

VFAs

(mg/L)

VFAs/SCOD CH4 yield

(NmL/g VSadded)

CO2 yield

(NmL/g VSadded)

Anaerobic 8017±1039 2831±89 0.36±0.04 13.7±1.8* 21.7±3.0

Micro-aeration 8839±482* 3524±191* 0.40±0.03* 8.5±1.8 20.8±2.9

Note: Values are presented in average ± SD (n =10)

* significantly different at α=0.05

Table

Table 2 The individual volatile fatty acids market size and price, and the estimated value of organic acid yield from the experiment

Volatile fatty acid Global market size

(metric ton/year)

Unit price *

(USD/metric ton)

Market value

(Million USD/year)

Estimated value in this study**

(USD/ dry metric ton of grass)

Acetic acid 3,500,000 400-800 1400-2800 310

Propionic 180,000 1500-1650 270-297 157

Butyric 30,000 2000-2500 60-75 174

Note: * Unit price from Zacharof and Lovitt (2013)

** Calculated based on each organic acid yield per ton of Napier grass from the experimental data

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Enhanced volatile fatty acids production from Napier grass