Development of methane-utilizing mixed cultures for the production of

Subscriber access provided by TRINITY COLL

Environmental Processes

Development of methane-utilizing mixed cultures for the production of polyhydroxyalkanoates (PHAs) from anaerobic digester sludge Ahmed Fergala, Ahmed Alsayed, Saif Khattab, Megan Ramirez, and Ahmed ElDyasti Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04142 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

Environmental Science & Technology

1

Development of methane-utilizing mixed cultures for the production of

2

polyhydroxyalkanoates (PHAs) from anaerobic digester sludge

3

Ahmed Fergalaa, Ahmed AlSayeda, Saif Khattabb, Megan Ramirezc, Ahmed Eldyastia*

4

aDepartment

5

Ontario, Canada M3J 1P3

6

bDepartment

7

Ontario, Canada M5B 2K3

8

cDepartment

9

Mexico.

of Civil Engineering, Lassonde School of Engineering, York University, Toronto,

of Chemical Engineering, Ryerson University, 350 Victoria Street, Toronto,

of Environmental Engineering, Universidad International, Cuernavaca, Morelos,

10 11 12

*Corresponding author:

13

Ahmed Eldyasti,

14

Assistant Professor, Department of Civil Engineering

15

Lassonde School of Engineering, York University

16

T: +1 (416) 736 2100 Ext. 31329

17

Email: [email protected]

1 ACS Paragon Plus Environment


18

Abstract

19

The fundamental components required for scaling up the production of biogas-based

20

biopolymers can be provided through a single process, i.e., anaerobic digestion (AD). In this

21

research, the possibility of enriching methane-utilizing mixed cultures from the AD process was

22

explored as well as their capability to accumulate polyhydroxyalkanoates (PHAs). For almost 70

23

days of operation in a fed-batch cyclic mode, the specific growth rate was 0.078±0.005 hr-1 and

24

the biomass yield was 0.7±0.08 mg-VSS/mg-CH4. Adjusting the nitrogen levels in AD centrate

25

resulted in results comparable to those obtained with a synthetic medium. The enriched culture

26

could accumulate up to 51±2 % PHB. On the other hand, when the culturing medium was

27

supplemented with valeric acid, the enriched bacteria were able to produce polyhydroxybutyrate-

28

co-valerate (PHBV) up to 52±6 % with an HV percentage of 33±5 %. Increasing the valeric acid

29

concentration in the culturing medium above 100 mg/l decreased the overall amount of PHBV by

30

60%, whereas the number of HV units incorporated was not affected. Changing the methane-

31

to-oxygen ratio (M/O) from 1:1 to 4:1 caused an almost 80 % decline in PHB accumulation. In

32

addition, M/O had a significant effect on the fraction composition of PHBV at different valeric

33

acid concentrations.

34

Keywords

35

Methanotrophs; Anaerobic digestion; Biogas; WRRF; PHA.

36 37


Page 2 of 39

Page 3 of 39

38


1. Introduction

39

Recently, the vision of wastewater treatment plants as facilities for the removal of pollutants has

40

changed. The water resources recovery facility concept (WRRF) is emerging, and different

41

applications for the biological processes involved in the wastewater treatment have been

42

employed for energy and value-added resource recovery (1). Anaerobic digestion (AD) of sludge

43

is not only an essential component in any modern wastewater treatment facility for stabilization

44

and the reduction of organics and pathogens but is also a key component to intensify resource

45

recovery from waste streams (2). AD sludge has been successfully employed for various

46

biotechnological applications such as production of single-cell proteins, biodiesel, dietary

47

supplements and biopolymers (3). Furthermore, AD results in the formation of methane or

48

hydrogen, which can both be considered as a valuable source for energy generation (2,4).

49

The production of biopolymers is one promising application for biogas generated from the

50

anaerobic digestion process (AD) through targeting mixed cultures dominated by bacterial

51

species with a high polyhydroxyalkanoate (PHA) accumulation capacity when subjected to feast-

52

famine or nutrient-limiting conditions (5,6). Containing up to 70% methane, biogas can be utilized

53

by a special type of bacteria called methanotrophs and is converted into polyhydroxybutyrate

54

(PHB) under nutrient-limited conditions (7). PHB can then be extracted from the bacterial cells

55

and processed to be an ideal substitute for conventional resource-consuming polymers in various

56

applications (8). Moreover, these products can be bio-degraded at the end of their useful lives (9).

57

It has been estimated that the feedstock contribution towards the cost of biopolymer production

58

can be reduced from 50% to 20% if methane is employed for biopolymer production (10), which

59

can overcome one of the major forces retarding the sustainability of biopolymer production.



60

Methanotrophs can be classified into different types based on their terminal electron acceptor,

61

that is, aerobic and anaerobic methanotrophs; or according to their carbon assimilation pathways,

62

namely, the ribulose monophosphate (RuMP), Serine or Calvin Benson Bassham (CBB)

63

pathways (11). Previous studies revealed that mainly aerobic methanotrophs have the capability of

64

undergoing the serine pathway for PHB accumulation; these are named type II methanotrophs

65

(12).

66

cultures is preventing the invasion of other non-PHB producing methanotrophs, mainly type I,

67

which tend to out-compete type II in mixed cultures owing to their higher growth rates and

68

methane affinity (13–15).

69

The nitrogen-driven selection for type II methanotrophs was the most successful approach on a

70

long-term basis (16,17). Using nitrogen gas or ammonium as nitrogen sources provides type II

71

methanotrophs with the required advantage to dominate in mixed cultures. Despite the existence

72

of the nitrogen-fixation genes in most of the type II species, unlike type I, the growth of type II

73

methanotrophs on nitrogen gas results in the slowest growth rate compared to other nitrogen

74

sources (18) due to the oxygen sensitivity of the enzymes involved in this process (19). Moreover,

75

using nitrogen gas introduces a third gaseous element to the cultivation process at the expense of

76

the energy input requirements. On the other hand, ammonium selection depends on the higher

77

ability of the type II organisms to handle the inhibition resulting from the co-oxidation of

78

ammonium by the methane monooxygenase enzyme (MMO) (20), which supports them in

79

out-competing type I in mixed cultures. However, in order to achieve an efficient ammonium-

80

based selection, ammonium concentration values should be adapted to eliminate any adverse

81

effects on the type II growth rate as well as the PHB accumulation capacity of the enriched

82

culture (16).

However, the main challenge to employing methanotrophs for PHB production in mixed


Page 4 of 39

Page 5 of 39


83

The possibility of obtaining methanotrophic mixed cultures with high PHB productivity from

84

wastewater treatment streams was mainly focused on WAS (21). In addition, previous studies

85

employing WAS mainly focused on deducing the operational conditions that would favor the

86

growth of type II methanotrophs and their PHB accumulation in mixed cultures without

87

considering the effect of the seed origin. Compared to waste activated sludge, anaerobic

88

digestion sludge can provide a better seed for the selection of type II over other non-

89

PHB-producing methanotrophs considering the higher ammonium concentrations found in the

90

digester resulting from ammonification during the anaerobic digestion process (2). Furthermore,

91

the ammonium-rich wastewater produced from anaerobic digestion (AD centrate) can replace the

92

traditional synthetic medium described for the growth of methanotrophs (15) to develop a

93

sustainable microbial culture and feedstock within the wastewater and waste stream process.

94

Interestingly, production of biopolymers by methanotrophs is not limited to only PHBs.

95

Introducing volatile fatty acids (VFAs) as a co-substrate to the culturing medium under nitrogen-

96

limited conditions results in the synthesis of other biopolymers from the PHA family (22). For

97

instance, addition of valeric acid supports the incorporation of hydroxyvalerate (HV) units and

98

the formation of polyhydroxybutyrate-co-valerate (PHBV) co-polymer (23,24). While the

99

applications of PHB can be limited by its stiffness, brittleness, high crystallinity and low thermal

100

stability resulting in difficulty processing it (25), PHBV has a lower melting temperature,

101

crystallinity, water permeability and enhanced mechanical properties (26), which broaden the

102

market for biopolymers produced from methanotrophs in various industrial, medical and

103

agricultural applications including food packaging, the production of water-resistant surfaces,

104

biodegradable implants, controlled drug delivery systems and fabrication of nanocomposites (27).

105

The properties of the produced co-polymer PHBV mainly depend on the HV fraction 5 ACS Paragon Plus Environment


106

incorporated in the produced biopolymer. Previous studies showed that the HV fraction is mainly

107

regulated by the co-substrate concentration in the culturing medium (16,23,28).

108

Therefore, the aim of this research is to evaluate the possibility of directing different products

109

from the AD process towards the production of biopolymers. First, the capability of establishing

110

methane-utilizing mixed cultures seeded from AD sludge with steady and consistent

111

performance is demonstrated in both the growth phase using an enhanced ammonium-driven

112

selection approach and in the PHB accumulation phase under nitrogen-limited conditions. In

113

addition, the effect of manipulating the AD centrate to support the enriched culture growth is

114

illustrated. Furthermore, the PHB accumulation capacity of the enriched culture is shown under

115

different operational conditions. Moreover, valeric acid addition is coupled with nitrogen-limited

116

conditions to induce PHBV production and to evaluate the environmental factors affecting the

117

biopolymer accumulation and the change in the fractions of the co-polymer composition to

118

develop an AD-dependent bioreactor for biopolymer production.

119

2. Materials and methods

120

2.1 Cultivation conditions

121

Sludge was collected from anaerobic digesters treating both primary sludge and waste activated

122

sludge at the Humber wastewater treatment plant located in Toronto, Canada, and used as a seed

123

for the cultivation of methanotrophs. All enrichments were cultivated in mineral salts medium

124

(MSM), (29) which had the following concentrations of ingredients (mg/l):1000 MgSO4.7H2O,

125

200 CaCl2.H2O, 272 KH2PO4, 610 K2HPO4, 4 Fe-EDTA and 1 ml/l Trace metal solution. The

126

trace metal solution had the following concentrations of ingredients (mg/l): 10 ZnSO4.7H2O, 3

127

MnCl2.4H2O, 30 H3BO3, 3 Na2MoO4.2H2O, 200 FeSO4.7H2O, 2 NiCl2.6H2O and 20 6 ACS Paragon Plus Environment

Page 6 of 39

Page 7 of 39


128

CoCl2.6H2O. Ammonium chloride (NH4Cl) was added as the sole nitrogen source to the MSM

129

medium with a concentration of 5 mmol (72 mg-N/l). Copper sulfate (CuSO4.5H2O) was added

130

from a stock solution to give a final concentration of 10 µmol.

131

Unless otherwise specified, all of the experiments for type II methanotrophs enrichment from

132

anaerobic digestion sludge were run in triplicate in a fed-batch mode using consecutive growth

133

cycles (16). Enrichments were incubated in 250-ml serum bottles capped with butyl rubber

134

stoppers. The liquid volume was 50 ml, and the headspace was filled with a methane and oxygen

135

(>99.9% purity, Praxair) mixture of volumetric ratio (1:1). All of the experiments were run at a

136

temperature of 25°C using table orbital shakers at a speed of 160 rpm. The pH was kept between

137

6 and 7 using a prepared solution of 10% NaOH. Samples were taken from the headspace

138

periodically to monitor the consumption of gases, and helium was added to restore atmospheric

139

pressure to eliminate any effect from the low methane solubility. The growth and ammonium

140

concentration were measured at the beginning and the end of the experiment by measuring the

141

optical density (OD600) and ammonium concentration as mg-N/l in the culturing medium.

142

2.2 Growth cycles

143

At the beginning of the experiment, anaerobic sludge was filtered through a 100-µm cell strainer

144

to remove undesired solids. After centrifuging, the filtered sludge was added to 50 ml of the

145

MSM. Air was removed from the headspace of the serum bottles using a vacuum pump and

146

replaced with 100 ml of O2 and 100 ml of CH4 every 24 hours. After 48 hours of cultivation, part

147

of the cultures was centrifuged (4000x) for 20 min. The collected biomass then was used in the

148

growth of the proceeding cycle while the rest of the biomass was tested for PHB accumulation.

149

To stimulate the growth of type II methanotrophs and to ensure their dominance during the 7 ACS Paragon Plus Environment


150

growth cycles, an ammonium-driven selection approach was employed (16) with some

151

modification (30). The amount of biomass returned from each growth cycle was adjusted to give

152

an initial OD600 of 0.37±0.08 (175 mg-VSS/l) and to initiate an inhibitory effect on type I

153

methanotrophs without affecting type II growth by maintaining a high N/M ratio and keeping the

154

C/N ratio at around 10±3 mg CH4/mg N. This would result in a high F/M ratio and eliminate any

155

inhibitory effect resulting from the competition between ammonium and methane for MMO

156

oxidation (31). By applying those conditions, the N/M, C/N and F/M ratios were 0.52±0.08 mg-

157

N/mg-VSS, 11.71±1.07 mg-CH4/mg-N and 5.39±0.94 mg-CH4/mg-N, respectively. Different

158

growth parameters were observed during each growth cycle, including the final biomass density,

159

specific growth rate (µ), biomass yield (Y), methane consumption, oxygen consumption and

160

methane utilization rate (q). The results from each cycle were compared with the previous cycle,

161

and steady-state was confirmed when the changes in the various parameters between cycles were

162

below 10%.

163

2.3 Methane biodegradation kinetics

164

Biomass was collected from cycles 8, 23 and 32 to estimate the maximum specific growth rate

165

(µmax), substrate half-saturation coefficient (Ks) and the maximum specific substrate utilization

166

rate (qmax) of the mixed cultures. The biomass collected from each cycle was divided among ten

167

250-ml serum bottles. Then, each duplicate was subjected to five different methane

168

concentrations by adding different methane volumes to the headspace of each bottle. The

169

volumes of methane were 25, 50, 100, 150 and 200 ml resulting in aqueous methane

170

concentrations of 1.87 ± 0.1, 2.5 ± 0.14, 6.34 ± 0.27, 9.73 ± 0.6 and 13.53 ± 0.53 mg-CH4/L,

171

respectively, by considering the value of the dimensionless Henry’s law constant (Hc) at 25oC

172

and 1 atm, which was 31.4 (32). The initial biomass density for all of the bottles was the same 8 ACS Paragon Plus Environment

Page 8 of 39

Page 9 of 39


173

(190 ± 20 mg-VSS/l). All other operational conditions were similar to the previous experiment

174

except for the duration where this experiment was running for 20 hours in a batch mode to

175

ensure the existence of the biomass in its exponential phase based on the observations from the

176

previous experiment. Methane, oxygen, and biomass density were measured at the beginning and

177

after the experiment for all cultures. The kinetic parameters were estimated using the equations

178

described by AlSayed et al., 2018 (32) where the Monod equation was used to describe the

179

microbial growth and the Lineweaver-Burk correlation was used to determine the maximum

180

specific growth rate (µmax) and the substrate half-saturation constant (Ks). Then, the maximum

181

specific substrate utilization rate (qmax) was estimated using µmax and the biomass yield.

182

2.3 AD centrate growth

183

After achieving a consistent performance in the growth cycles, the previously described synthetic

184

medium was replaced with AD centrate. The main characteristics of the centrate are summarized

185

in Table S1. Due to the high ammonium concentrations, the AD centrate was diluted with

186

deionized water in a series of batch experiments with concentrations ranging from 5% (v/v) to

187

100% with a final volume of 50 ml while the other operating parameters were similar to those of

188

previous experiments. Afterwards, the optimum AD centrate concentration was employed for 10

189

consecutive fed-batch cycles as described in section 2.2 to ensure the consistency of the behavior

190

observed during the batch tests.

191

2.4 PHB and PHBV accumulation

192

The PHB accumulation potential of the enriched culture was monitored by subjecting the

193

biomass remaining at the end of each growth cycle to nitrogen-limited conditions. The PHB

194

accumulation phase was run in in triplicate in a batch mode for 48 hours using 250-ml serum 9 ACS Paragon Plus Environment


195

bottles. The headspace of each bottle was evacuated and filled with 200 ml of methane and

196

oxygen with a volumetric ratio of 1:1. After a couple of cycles, the amount of biomass

197

transferred from the growth cycle to the PHB cycle reached a stable value and the initial OD600

198

of each cycle was 1.56±0.22 (720 mg-VSS/l) until the end of the experiments.

199

When the amount of PHB produced was above 40% and the change in PHB accumulation over

200

cycles was below 10% (cycle 11), the effect of other operational conditions was tested using

201

other triplicates in parallel to ensure the consistency of the PHB accumulation capacity of the

202

enriched methanotrophs and the uniformity of the results. The effect of the biomass density on

203

the PHB accumulation capacity of the enriched methanotrophs was tested by employing different

204

sets of experiments using triplicates having initial OD600 ranging from 0.5-3, which is equivalent

205

to 240-1500 mg-VSS/l. Then, the effect of the methane-to-oxygen ratio (M/O) on the PHB

206

accumulation was examined by changing the headspace gas composition from 1:1 (methane:

207

oxygen) to 1:2, 2:1 and 4:1. Finally, PHB accumulation over the 48-hour experiment duration

208

was monitored every 12 hours to estimate the PHB productivity of the enriched culture.

209

Similar experiments were run to test the PHBV accumulation capacity. The main difference was

210

the valeric acid addition as a co-substrate. Concentrations ranging from 50 to 2000 mg/l were

211

tested on the enriched methanotrophs while monitoring their effect on the amount of PHBV

212

accumulated and the percentage of HV units incorporated in the biopolymer. Upon addition of

213

valeric acid to the culturing medium, the pH dropped as expected. However, at high valeric acid

214

concentrations, the pH decreased below 4, which is below the tolerance known for type II

215

methanotrophs (7). Accordingly, the pH of the culturing medium was adjusted to between 6 and 7

216

using a prepared solution of 10% NaOH to eliminate any effect resulting from the pH drop


Page 10 of 39

Page 11 of 39


217

accompanying the valeric acid addition (23) and to study the effect of a single factor only (co-

218

substrate concentration). Experiments with different M/O ratios at different valeric acid

219

concentrations were conducted, and the PHBV productivity test was then performed as

220

previously described.

221

2.5 Microbial community analysis

222

Two samples were taken from the enrichment during the cultivation to ensure the dominance of

223

type II organisms in the mixed culture. The first sample was taken after 10 cycles of cultivation,

224

while the second sample was taken after 30 cycles. The DNA extraction and the amplification of

225

the V4 region of the 16S SSU rRNA were carried out using the Earth Microbiome Project

226

benchmarked protocols (http://www.earthmicrobiome.org/emp-standard-protocols/). The DNA

227

extraction protocol involved mechanical and enzymatic lysis followed by a phenol-chloroform

228

extraction and a clean-up step using a MoBio PowerMag soil DNA isolation kit as per the

229

manufacturer’s protocol (33).

230

Primers 515FB (5’-GTGYCAGCMGCCGCGGTAA-3’) and 806RB (5’-

231

GGACTACNVGGGTWTCTAAT-3’) were used, and the barcodes incorporated into the forward

232

primer enabled the usage of various reverse primer constructs to obtain longer amplicons and

233

removal of biases (34). Briefly, each 25-µl PCR mixture contained the following to amplify V4 of

234

the 16S rRNA gene by PCR: 13 µl of PCR-grade water (Sigma, USA), 10 µl of Platinum Hot

235

Start PCR Master Mix (2x) (ThermoFisher, USA), 1 µl of Template DNA, 0.5 ml of 515FB

236

primer (10 µM), and 0.5 ml of 806RB primer (10 µM). The reaction then was run for 35 cycles

237

(94°C for 2 min, 94°C for 30 s, 50°C for 30s, 72°C for 30 s), with a final polymerization step at

238

72°C for 10 min. The products were separated by electrophoresis in a 2% agarose gel and 11 ACS Paragon Plus Environment


239

visualized under a UV transilluminator light. The products corresponding to the amplified V4

240

(300-350 bp) were excised and purified using standard gel extraction kits (Qiagen, Netherland).

241

The products were then quantified with the Quant-iT PicoGreen dsDNA Assay Kit

242

(ThermoFisher, USA). Equal amounts of product from each sample (240 ng) were combined into

243

a single, sterile tube and then cleaned using MoBio UltraClean PCR clean-up kit. The final

244

concentration was measured using a nanodrop spectrophotometer, and the product quality was

245

checked by ensuring that the ratio of absorbances at 260/280 nm ranged from 1.8 to 2.

246

The resulting PCR products were sequenced using the Illumina MiSeq personal sequencer

247

(Illumina Incorporated, USA) at the McMaster Genomics Facility, Ontario, Canada. Cutadapt

248

was used to filter and trim adapter sequences and PCR primers from the raw reads with a

249

minimum quality score of 30 and a minimum read length of 100 bp (35). Sequence variants were

250

then resolved from the trimmed raw reads using DADA2, an accurate sample inference pipeline

251

from 16S amplicon data (36). The DNA sequence reads were filtered and trimmed based on the

252

quality of the reads, error rates were learned, and sequence variants were determined by

253

DADA2. Chimeras were removed, and taxonomy was assigned using the RDP classifier against

254

the SILVA database version 128. The raw sequencing data were deposited to the NCBI

255

Sequence Read Archive database (Project accession # PRJNA482955)

256

2.6 Analytical methods

257

Gas samples were withdrawn using a gas-tight syringe from the headspace of a bottle and then

258

injected into an SRI 8610C gas chromatograph (SRI Instruments, Torrance, USA) equipped with

259

a thermal conductivity detector (TCD) and molecular sieve column (Restek, Bellefonte, PA.) to

260

monitor methane, oxygen and nitrogen concentrations with a detection limit of 100 ppm. The


Page 12 of 39

Page 13 of 39


261

temperature program was as follows: injector, 60oC; oven, 80oC; and TCD, 80oC. Helium was

262

used as the carrier gas with a flowrate of 15 mL/min. External calibration curves were

263

constructed as follows: Three 250-ml serum bottles were evacuated. Each was filled with 50 ml

264

of water to mimic the conditions applied throughout the experiments. Then, about 200 ml of the

265

different gas mixes with known oxygen, methane and nitrogen compositions were injected into

266

the headspace of each bottle. Afterwards, the samples were injected into the GC and the peak

267

areas resulting from each bottle at different gas compositions were converted into concentrations

268

and masses. For bacterial growth monitoring, optical density OD600 was measured using a DR

269

3900 Benchtop Spectrophotometer (Hach Company, Loveland, Colorado, USA). Correlation

270

curves between OD600 and TSS, VSS and dry cell weight were developed. HACH methods and

271

testing kits were used to measure inorganic nitrogen (NH3-N, NO2-N, and NO3-N)

272

concentrations on the DR 3900 Benchtop Spectrophotometer (HACH Company, Loveland,

273

Colorado, USA).

274

PHB and PHBV were measured using the method developed by Braunegg et al. (37). Briefly, 10-

275

15 mg of lyophilized biomass was collected and 2 ml of acidified methanol (3% sulfuric acid)

276

and 2 ml of chloroform were added to a glass vial. After gentle mixing, the cocktail was heated

277

at 100 ºC for 3.5 hours and then left to cool down to room temperature. Afterwards, 1 ml of

278

deionized water was added, the mixture was vortexed for 1 minute, and then allowed to stand

279

until phase separation was achieved. The lower organic phase was tested for biopolymer

280

quantification using an SRI gas chromatograph equipped with a flame ionization detector (SRI

281

Instruments, Torrance, USA) and an MXT-wax column (Restek, Bellefonte, PA.). The

282

temperature program was as follows: 1 min 80 ºC, 10 ºC/min, 180 ºC for 4 min. The results were

283

compared to standard curves obtained using PHB and PHBV standards (Sigma Aldrich). Benzoic 13 ACS Paragon Plus Environment


284

acid was used as an internal standard to increase the accuracy and maintain a control for all

285

errors.

286

3. Results and discussion

287

3.1 Cultivation of methanotrophs from AD sludge

288

Filtered sludge obtained from AD was used for methanotrophs cultivation directly without

289

performing any additional adaptation procedures before starting the growth cycles. Then, an

290

increase in the OD600 was observed during the early cycles of the growth phase as shown in

291

Figure 1a. The conducted microbial analysis showed that Methylocystis was the main

292

methanotrophic genus detected, which belongs to type II (29), without any evidence for the

293

invasion of type I genera.

294

As shown in Figure 1b, a steady-state specific growth rate started after almost 5 cycles of

295

operation and the fluctuations were below 10% until the end of the experiments confirming the

296

suitability of the chosen operational conditions (F/M, N/M and C/N ratios) for type II selection

297

and growth. Similar behavior was also noticed for other growth parameters including biomass

298

yield, methane and oxygen uptake, substrate utilization rate and final biomass density as

299

illustrated in Figure 1b-1e. The average values for all kinetic and stoichiometric parameters are

300

summarized in Table 1. While the behavior of these parameters was stable for the enriched

301

culture in total, the abundance of the Methylocystis genus increased from 28±2% at cycle 10 to

302

reach 56±5% after 30 cycles of operation as shown in Figure S.1. This increase in the

303

Methylocystis genus in the mixed culture had a positive effect on the methane affinity and

304

biodegradation kinetics of the enriched culture. As shown in Table S.2, the substrate half-

305

saturation constant (Ks) decreased from 2.63±0.3 mg-CH4/l at cycle 8 to 1.82±0.2 mg-CH4/l at 14 ACS Paragon Plus Environment

Page 14 of 39

Page 15 of 39


306

cycle 23. The maximum specific methane utilization rates (qmax) at cycles 8 and 23 were

307

0.1±0.01 and 0.13±0.01 mg-CH4/mg-VSS-hr, respectively. On the other hand, the Ks and qmax

308

were 1.78±0.2 mg-CH4/l and 0.14±0.03 mg-CH4/mg-VSS-hr, respectively, at cycle 32. As this

309

study mainly focused on applying conditions to ensure the dominance of type II methanotrophs,

310

further studies should consider applying different molecular techniques to investigate the

311

relationships and co-operation between different members of the mixed culture. For instance, the

312

other two key genera detected at cycle 30 were Methylophilus and Hyphomicrobium with relative

313

abundances of 22 and 6.5% respectively. Both are facultative methylotrophs with the ability to

314

grow on methanotroph metabolites (17), and Hyphomicrobium can accumulate up to 59% PHB

315

(38).

316

Rhodobacter, and Pseudomonas have even higher PHB accumulation capacity than the

317

methanotrophs (17). Thus, optimizing conditions under which methanotrophs act as a biocatalyst

318

to support metabolites for other PHB-accumulating bacteria in addition to methanotrophs’ ability

319

can result in an improvement in the methane biodegradation and PHB productivity for the whole

320

mixed culture.

321

Different values for the kinetic and stoichiometric parameters for type II methanotrophs were

322

reported in previous studies according to different operational modes. Generally, the main

323

advantage noticed for AD enrichments compared to those from WAS was the shorter period

324

achieved to reach a stable performance. AD enrichments reached a steady state after 10 days of

325

cultivation while WAS enrichments needed between 24 and 40 days when ammonium was

326

employed as a nitrogen source (16,30) and up to 220 days under nitrogen-fixation conditions (17).

327

Considering full-scale application, this shorter adaptation (start-up) period could be one of the

328

factors affecting the choice of the type of sludge employed for methanotroph cultivation. In

In addition to Hyphomicrobium, other detected genera including Sphingomonas,



329

particular, when similar operational conditions were applied to AD and WAS enrichments, the

330

methane affinity, methane uptake rate and methane utilization rates were 1.5-fold higher in the

331

AD enrichments (30). In addition, considering the same cultivation period under the same

332

conditions, the abundance of type II organisms in the AD enrichments was double the WAS

333

enrichments after 30 cycles of operation. Moreover, the abundance of type II methanotrophs

334

never exceeded 30% in the WAS enrichments under the same operational conditions (30, 39).

335

Despite the relatively lower ammonium concentration adopted during these experiments, there

336

was no existence of type I methanotrophs throughout the cultivation of AD enrichments, in

337

contrast with the WAS enrichments in which a transition period took place and both types of

338

methanotrophs existed in the mixed culture and consequently affected the PHB productivity (39).

339

Collectively, AD enrichments can be more efficient for combining the need for methane

340

mitigation and recovery of value-added products.

341

In comparison with previous studies, the reported maximum specific growth rates (µmax) and

342

maximum utilization rates (qmax) ranged from 0.012 to 0.19 hr-1 and 0.021-0.26 mg-CH4/mg-

343

VSS-hr, respectively (16,18,23), while the biomass yields ranged from 0.3 to 0.9 mg-VSS/mg-CH4

344

(18,23,40–42).

345

studies. The attained results for the AD enrichments during this study were in the upper range of

346

these values and among the highest reported for different methane-utilizing mixed cultures. In

347

the present work, the maximum specific growth rate was 0.098±0.007 hr-1 and the maximum

348

methane utilization rate was 0.14±0.05 mg-CH4/mg-VSS-hr while the biomass yield was

349

0.7±0.08 mg-VSS/mg-CH4, which also confirms the potential of AD enrichments for methane

350

emissions valorization.

The maximum values reported in those ranges were associated with pure culture


Page 16 of 39

Page 17 of 39


351

3.2 Growth of the enriched culture on AD centrate

352

As shown in Figure 2, applying AD centrate without dilution resulted in a complete inhibition of

353

the methane uptake by the enriched culture and consequently the growth of the culture. This

354

inhibition was mainly caused by the elevated ammonium concentration found in the centrate

355

(534±50 mg-N/l) while the concentration of phosphorus (120±15 mg-PO4/l) was in the range

356

required for methanotrophs (43,44). On the other hand, diluting the centrate with deionized water

357

initiated methane consumption and biomass growth and the highest growth rates were observed

358

in centrate concentrations between 5-25% v/v. It is noteworthy to mention that centrate

359

concentrations of 5% and 25% had almost the same specific growth rate (0.06±0.001 hr-1) and

360

biomass yields (0.62±0.066 mg-VSS/mg-CH4), and the maximum value for both growth

361

parameters was observed at 10%v/v with specific growth rates of 0.07±0.002 hr-1 and 0.75±0.212

362

mg-VSS/mg-CH4, respectively. However, the methane uptake rate at 5% (4.13±0.2 mg-CH4/hr)

363

was almost double the value obtained at 25% (2.14±0.1 mg-CH4/hr). This observation can be

364

explained by the release of competition between ammonium and methane for methane

365

monooxygenase enzyme but at the expense of the growth rate due to the decrease in the available

366

nitrogen source to be utilized for cell synthesis. Despite the lower methane uptake associated

367

with the increase in the centrate concentration, it is important to maintain this ammonium

368

inhibition to ensure the dominance of type II methanotrophs in the enriched culture (30).

369

Nonetheless, when the AD centrate was diluted to 10% (v/v) to give similar N/M, C/N and F/M

370

ratios to the synthetic medium experiment, almost 90% of the microbial activity was restored.

371

While this study mainly focused on the impact of the nitrogen source (ammonium) in the AD

372

centrate, further studies are required to elucidate the effects of other micronutrients such as

373

copper and iron and due to their importance in the regulation of the MMO enzyme activity (43,45) 17 ACS Paragon Plus Environment


374

.In addition, to study the effect of potential inhibitors to methane oxidation such as mercury,

375

zinc, chromium and cadmium (46,47) that can be found in the AD centrate(48). To validate the

376

previous findings, AD centrate at a concentration of 10% (v/v) was supplemented for the

377

enriched culture during 10 consecutive growth cycles where consistent performance for the

378

enriched culture was observed without any sign of inhibition, as summarized in Table 1.

379

3.3 PHB production by type II methanotrophs enriched from the AD process

380

The ability of PHB accumulation by the AD enrichments was induced by applying a nitrogen-

381

limited phase for 48 hours after the growth phase starting from the first cycle of operation. The

382

PHB accumulation phase had a behavior similar to that of the growth phase. During the first

383

couple of cycles, small amounts of PHB were accumulated with an average of 15±4%.

384

Afterwards, a significant improvement in the PHB accumulation capacity was achieved while

385

exhibiting a stable behavior, reaching 48±6% by the end of the experiments as shown in Figure

386

S.3. Furthermore, cultures supplemented with AD centrate during the growth phase were capable

387

of accumulating similar amounts of PHB and their production reached 46±4% under the

388

nitrogen-limited conditions.

389

The effect of manipulating different M/O on the PHB production was tested by changing the gas

390

composition in the headspace. As shown in Figure 3, the highest PHB accumulation was

391

observed at M/O ranging from 1:1 to 2:1, and the enriched methanotrophs could accumulate up

392

to 46±4% PHB in their cellular biomass. However, increasing the M/O mainly affected the PHB

393

yield. A decrease from 0.49±0.04 to 0.33±0.03 mg-PHB/mg-CH4 was observed when the M/O

394

increased from 1:1 to 2:1. A further increase in the M/O to 4:1 caused a severe drop in the PHB

395

accumulation capacity of the culture with only 10±2% PHB produced. While a high M/O is 18 ACS Paragon Plus Environment

Page 18 of 39

Page 19 of 39


396

considered as one of the favorable conditions for the growth of type II methanotrophs (49,50),

397

coupling lower oxygen concentrations placed additional stress on the type II methanotrophs and

398

on the energy-intensive PHB accumulation step in addition to nitrogen limitation. Their PHB

399

accumulation was consequently affected, which can also be confirmed by the lowest observed

400

yield of 0.13±0.02 mg-PHB/mg-CH4 at an M/O of 4:1. On the other hand, decreasing the M/O to

401

1:2 decreased the amount of accumulated PHB to 34±4%. The decrease in the PHB

402

accumulation can be mainly attributed to the decrease in the methane fraction in the headspace.

403

However, increased oxygen availability allowed the PHB yield to be less vulnerable to this

404

change and was maintained at 0.43±0.06 mg-PHB/mg-CH4.

405

Changing the biomass density can affect methanotrophs in two ways. First, previous studies

406

revealed that methanotrophic bioreactors are usually mass transfer limited at higher biomass

407

densities, which may cause a decline in the bacterial activity (51). Second, changing the biomass

408

density also alters the F/M ratio and consequently the growth of the enriched culture (52). As

409

shown in Figure 4a, increasing the biomass density (OD600) from 0.5 (238 mg-VSS/l) to 3 (1430

410

mg-VSS/l) decreased the F/M ratio from 6.29±0.6 mg-CH4/mg-VSS to 1.29±0.2 mg-CH4/mg-

411

VSS. However, as shown in Figure 4b, the enriched culture could maintain a stable amount of

412

PHB accumulation with an average value of 51±2% until the OD600 reached 2. In addition, the

413

PHB yield increased from 0.49±0.02 to 0.61±0.1 mg-PHB/mg-CH4 when the biomass density

414

increased from 0.5 to 2. A further increase in the OD600 to 3 resulted in a decrease in the amount

415

of biopolymer accumulated. At this value, only 10% PHB was produced by the enrichment,

416

while the yield was more tolerant to this change and had a value of 0.46±0.05 mg-PHB/mg-VSS.

417

Interestingly, both experiments OD600 = 3 and M/O ratio (1:2) had almost the same F/M ratios

418

and similar yields while the PHB accumulation in the M/O experiment was almost 3-fold the 19 ACS Paragon Plus Environment


419

amount attained for the OD600 = 3 experiments. This shows that the F/M ratio mainly affects the

420

yield but not the amount of PHB accumulated in the biomass. On the other hand, the negative

421

effect of methane solubility at increased biomass densities was mainly reflected in the rate of

422

methane utilized by the enriched culture. This rate slightly decreased from 0.51±0.01 to

423

0.43±0.006 mgCH4/mg-VSS-hr before OD600=1.5, followed by a sharp decrease, reaching

424

0.01±0.005 mgCH4/mg-VSS-hr when the optical density was 3 as illustrated in Figure 4a.

425

To assess the PHB productivity of the enriched type II methanotrophs from AD sludge, PHB

426

accumulation was measured every 12 hours during the 48 hours of nitrogen-limited cultivation.

427

The results show that the PHB accumulation increased with elapsed time during the experiment

428

and that the PHB reached its maximum after 48 hours as illustrated in Figure S.4. In addition,

429

the maximum productivity was achieved after 36-48 hours of cultivation under nitrogen-limited

430

conditions, reaching 9.5±0.8 mg-PHB/l-hr. Furthermore, increasing the cultivation period to

431

more than 48 hours did not show any noticeable increase in the PHB accumulation and therefore

432

would result in lower PHB productivity. Similar observations were also noticed in previous

433

studies in which the PHB accumulation reached the maximum value after 48 hours of cultivation

434

in nutrient-limited conditions followed by either a stability or a slight increase in the amount of

435

PHB produced by type II methanotrophs (17).

436

3.4 PHBV production by type II methanotrophs enriched from the AD process

437

Valeric acid was introduced to the nitrogen-limited cultivating phase to induce the accumulation

438

of PHBV. Valeric acid was chosen as a model for volatile fatty acids (VFAs) as the maximum

439

amount of PHBV accumulated by pure type II strains was achieved by using valeric acid

440

compared with propionic acid or the salts of either acid (23,28). PHBV is mainly formed by the


Page 20 of 39

Page 21 of 39


441

polymerization of two units: 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). While

442

methane assimilation secures the formation of the first one only, the main role of the added co-

443

substrate is to provide precursors for the HB units (3-hydroxybutyryl-CoA) or HV units (3-

444

hydroxybutyryl-CoA) for incorporation in the produced biopolymer. Apparently, valeric acid

445

regulates the second of these (23,24,28). As shown in Figure 5, increasing the valeric acid

446

concentration from 50 to 100 mg/l increased the amount of PHBV accumulated from 34±3% to

447

47±4% while the percentage of the HV units increased from 25±2% to 35±3%. A further

448

increase in the valeric acid concentration decreased the amount of PHBV accumulated; a severe

449

decline in the accumulation was noticed at 2000 mg/l with only 15% PHBV being produced.

450

Generally, the addition of valeric acid introduced a second energy-demanding step in addition to

451

the methane-to-methanol oxidation. Although these requirements are fulfilled through the

452

methane oxidation pathway (at the expense of increased oxygen requirements), increasing the

453

valeric acid concentration resulted in higher energy demands, and the amount of CH4 oxidized

454

for energy generation increased the substrate partitioning coefficient for energy (fe), reaching

455

0.86 at 2000 mg/l compared to 0.4 at 50 mg/l. This increase was not only reflected in the amount

456

of biopolymer accumulated but also in the noticeable decrease in the observed PHBV yield. The

457

observed yield declined from 0.68±0.03 mg-PHBV/mg-CH4 to 0.17±0.01 mg-PHBV/mg-CH4 at

458

concentrations of 50 and 2000 mg/l, respectively. Despite the drop noticed for the overall PHBV

459

production, the percentage of the incorporated HV units was not affected and remained at 49±3%

460

for concentrations above 100 mg/l as shown in Figure 5. Similar behavior was also observed for

461

the strain Methylocystis parvus OBBP with the maximum observed PHBV accumulation at a

462

sodium valerate concentration of 100 mg/l (54%) followed by a decline in the biopolymer

463

production and a stability in the incorporation of HV units (24). In addition, the maximum PHBV



464

accumulation for Methylocystis hirsuta was 53.8% when the concentration of valeric acid was

465

130 mg/l (23).

466

The aim of manipulating different M/O ratios during PHBV production was not only to study the

467

effect on the amount of the accumulated biopolymer but also to discuss the capability of

468

manipulating a factor other than the valeric acid concentration that would change the fractions of

469

the PHBV without decreasing the overall production for a tuned product. As shown in Figure

470

6a, when the methane-to-oxygen ratio was 1:2 and the valeric acid concentration was 100 mg/l,

471

the amount of PHBV accumulated by the enriched methanotrophs was 44±3% with almost the

472

same amount accumulated when this ratio was 1:1 at a valeric acid concentration of 100 mg/l.

473

However, the HV units increased from 34±1% to 39±2% by changing the M/O to 1:2, which can

474

imply the increased availability in oxygen and more favorable conditions for the energy-

475

intensive valeric acid uptake by type II methanotrophs during PHBV accumulation (24). A further

476

increase in the M/O ratio to 1:3 resulted in a decline in the amount of PHBV, reaching 25±3%

477

while the HV incorporation was maintained at 38±2%. These observations show that the

478

presence of conditions favoring valeric acid uptake such as high acid concentration or oxygen

479

availability will direct methane oxidation to serve the synthesis of HV units until it reaches its

480

maximum even if it affects the overall PHBV accumulation. On the other hand, increasing M/O

481

to 2:1 was expected to result in a decrease in the HV monomers in the accumulated biopolymer.

482

However, due to the increased availability of methane, the percentage of the incorporated HV

483

units was almost constant at 35±1% while the overall PHBV accumulation slightly increased

484

from 43±2% to 47±3% as shown in Figure 6b.


Page 22 of 39

Page 23 of 39


485

To elaborate on the previous observations, the same M/O ratio was tested at valeric acid

486

concentrations of 50 and 200 mg/l. While at both of these concentrations the overall amount of

487

PHBV followed a trend similar to that observed in the previous experiment at 100 mg/l shown in

488

(Figure 6a), the incorporation of the HV units had a different behavior. At 50 mg/l, increasing

489

the M/O to 2:1 decreased the HV fraction from 20±3% to 9±2%, while almost no change in this

490

fraction was observed at 200 mg/l and the HV units represented 36±2% of the accumulated

491

biopolymer as shown in Figure 6b. On the other hand, an M/O of 1:2 increased the HV fraction

492

at both concentrations. However, at 50 mg/l, changing the M/O from 1:2 to 1:3 did not affect the

493

PHBV composition and the maximum HV fraction was 29±2%. In contrast, at 200 mg/l, further

494

increasing the oxygen ratio to 1:3 elevated the HV content to 58±4%, which is higher than the

495

fraction observed in the previous experiment run at a valeric acid concentration of 2000 mg/l.

496

This result supports the significance of oxygen on the PHBV composition taking into

497

consideration that the PHBV accumulation under both conditions was almost the same (14%).

498

The PHBV productivity profile throughout the 48-hour duration of the experiment had a

499

different behavior compared to that observed for PHB. The amount of the produced PHBV by

500

the enriched methanotrophs and the percentage of the incorporated HV units were increasing and

501

reached their maxima at the end of the 48-hour experiment with values of 52±6% and 33±5% of

502

PHBV and HV, respectively. In contrast, the rate of HV incorporation reached its maximum after

503

24-36 hours of operation followed by a decrease during the subsequent period, which introduces

504

another method for controlling the characteristics of the accumulated biopolymer by controlling

505

the exposure time to nitrogen-limited conditions. On the other hand, the overall productivity of

506

the PHBV had the highest value between 12-24 hours (10±2 mg-PHBV/l-hr) followed by a slight



507

decrease during the following periods of the experiment (7±1.9 mg-PHBV/l-hr) as shown in

508

Figure S.5.

509

4. Acknowledgements

510

The authors gratefully acknowledge the Natural Science and Engineering Research Council of

511

Canada (NSERC), the Ontario (Canada) Center of Excellence (OCE) Seed Fund, Dr. Gerald

512

Audette and Rana Salem, Biology Department, Faculty of Science, York University, the Surette

513

Laboratory for Microbiome and Polymicrobial Research at McMaster University and the City of

514

Toronto, ON, Canada, for their endless support and interest at every stage of this research

515

project.


Page 24 of 39

Page 25 of 39


517

5. References

518 519 520

1.

Coats ER, Wilson PI. Toward Nucleating the Concept of the Water Resource Recovery Facility (WRRF): Perspective from the Principal Actors. Environ Sci Technol. 2017 Apr 18;51(8):4158–64.

521 522

2.

Appels L, Baeyens J, Degrève J, Dewil R. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog Energy Combust Sci. 2008 Dec 1;34(6):755–81.

523 524

3.

Tyagi VK, Lo S-L. Sludge: A waste or renewable source for energy and resources recovery? Renew Sustain Energy Rev. 2013 Sep 1;25:708–28.

525 526

4.

Elbeshbishy E, Dhar BR, Nakhla G, Lee H-S. A critical review on inhibition of dark biohydrogen fermentation. Renew Sustain Energy Rev. 2017 Nov 1;79:656–68.

527 528 529

5.

Korkakaki E, van Loosdrecht MCM, Kleerebezem R. Impact of phosphate limitation on PHA production in a feast-famine process. Water Res. 2017 Dec 1;126(Supplement C):472–80.

530 531 532

6.

Kinyua MN, Miller MW, Wett B, Murthy S, Chandran K, Bott CB. Polyhydroxyalkanoates, triacylglycerides and glycogen in a high rate activated sludge A-stage system. Chem Eng J. 2017 May 15;316(Supplement C):350–60.

533 534

7.

Strong PJ, Xie S, Clarke WP. Methane as a Resource: Can the Methanotrophs Add Value? Environ Sci Technol. 2015 Apr 7;49(7):4001–18.

535 536 537

8.

Khosravi-Darani K, Mokhtari Z-B, Amai T, Tanaka K. Microbial production of poly(hydroxybutyrate) from C₁ carbon sources. Appl Microbiol Biotechnol. 2013 Feb;97(4):1407–24.

538 539 540

9.

Rostkowski KH, Criddle CS, Lepech MD. Cradle-to-Gate Life Cycle Assessment for a Cradle-to-Cradle Cycle: Biogas-to-Bioplastic (and Back). Environ Sci Technol. 2012 Sep 18;46(18):9822–9.

541 542 543 544

10. Levett I, Birkett G, Davies N, Bell A, Langford A, Laycock B, Lant P, Pratt S. Technoeconomic assessment of poly-3-hydroxybutyrate (PHB) production from methane—The case for thermophilic bioprocessing. J Environ Chem Eng. 2016 Dec 1;4(4, Part A):3724– 33.

545

11. Hanson RS, Hanson TE. Methanotrophic bacteria. Microbiol Rev. 1996 Jun;60(2):439–71.

546 547 548

12. Pieja AJ, Rostkowski KH, Criddle CS. Distribution and selection of poly-3hydroxybutyrate production capacity in methanotrophic proteobacteria. Microb Ecol. 2011 Oct;62(3):564–73.

549 550 551

13. Chidambarampadmavathy K, Karthikeyan OP, Huerlimann R, Maes GE, Heimann K. Response of mixed methanotrophic consortia to different methane to oxygen ratios. Waste Manag. 2017 Mar 1;61(Supplement C):220–8. 25 ACS Paragon Plus Environment


552 553 554

14. López JC, Quijano G, Pérez R, Muñoz R. Assessing the influence of CH4 concentration during culture enrichment on the biodegradation kinetics and population structure. J Environ Manage. 2014 Dec 15;146(Supplement C):116–23.

555 556

15. López JC, Merchán L, Lebrero R, Muñoz R. Feast-famine biofilter operation for methane mitigation. J Clean Prod. 2018 Jan 1;170(Supplement C):108–18.

557 558 559 560

16. Myung J, Galega WM, Van Nostrand JD, Yuan T, Zhou J, Criddle CS. Long-term cultivation of a stable Methylocystis-dominated methanotrophic enrichment enabling tailored production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Bioresour Technol. 2015 Dec;198:811–8.

561 562 563

17. Zhang T, Wang X, Zhou J, Zhang Y. Enrichments of methanotrophic–heterotrophic cultures with high poly-β-hydroxybutyrate (PHB) accumulation capacities. J Environ Sci. 2018 Mar 1;65:133–43.

564 565 566

18. Rostkowski KH, Pfluger AR, Criddle CS. Stoichiometry and kinetics of the PHB-producing Type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP. Bioresour Technol. 2013 Mar;132:71–7.

567 568

19. Murrell JC, Dalton H. Nitrogen Fixation in Obligate Methanotrophs. Microbiology. 1983;129(11):3481–6.

569 570 571

20. Criddle CS, Sundstrom ER. Intermittent application of reduced nitrogen sources for selection of PHB producing methanotrophs [Internet]. 2015 [cited 2015 Oct 11]. Available from: http://www.freepatentsonline.com/y2015/0159185.html

572 573 574

21. Myung J, Wang Z, Yuan T, Zhang P, Van Nostrand JD, Zhou J, Criddle CS. Production of Nitrous Oxide from Nitrite in Stable Type II Methanotrophic Enrichments. Environ Sci Technol. 2015 Sep 15;49(18):10969–75.

575 576 577

22. Myung J, Flanagan JCA, Waymouth RM, Criddle CS. Expanding the range of polyhydroxyalkanoates synthesized by methanotrophic bacteria through the utilization of omega-hydroxyalkanoate co-substrates. AMB Express. 2017 Jun 5;7:118.

578 579 580

23. López JC, Arnáiz E, Merchán L, Lebrero R, Muñoz R. Biogas-based polyhydroxyalkanoates production by Methylocystis hirsuta: A step further in anaerobic digestion biorefineries. Chem Eng J. 2018 Feb 1;333(Supplement C):529–36.

581 582 583

24. Myung J, Flanagan JCA, Waymouth RM, Criddle CS. Methane or methanol-oxidation dependent synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by obligate type II methanotrophs. Process Biochem. 2016 May 1;51(5):561–7.

584

25. Lee SY. Bacterial polyhydroxyalkanoates. Biotechnol Bioeng. 1996 Jan 5;49(1):1–14.

585 586 587

26. Strong PJ, Laycock B, Mahamud SNS, Jensen PD, Lant PA, Tyson G, Pratt S. The Opportunity for High-Performance Biomaterials from Methane. Microorganisms. 2016 Feb 3;4(1):11. 26 ACS Paragon Plus Environment

Page 26 of 39

Page 27 of 39


588 589

27. Philip S, Keshavarz T, Roy I. Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J Chem Technol Biotechnol. 2007 Mar 1;82(3):233–47.

590 591 592

28. Cal AJ, Sikkema WD, Ponce MI, Franqui-Villanueva D, Riiff TJ, Orts WJ, Pieja AJ, Lee CC. Methanotrophic production of polyhydroxybutyrate-co-hydroxyvalerate with high hydroxyvalerate content. Int J Biol Macromol. 2016 Jun 1;87(Supplement C):302–7.

593 594 595 596

29. Bowman J. The Methanotrophs — The Families Methylococcaceae and Methylocystaceae. In: Dr MDP, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes [Internet]. Springer New York; 2006 [cited 2017 Mar 28]. p. 266–89. Available from: http://link.springer.com/referenceworkentry/10.1007/0-387-30745-1_15

597 598 599

30. Fergala A, AlSayed A, Eldyasti A. Factors affecting the selection of PHB accumulating methanotrophs from waste activated sludge while utilizing ammonium as their nitrogen source. J Chem Technol Biotechnol. 2018 May 93(5):1359–69.

600 601 602

31. Veillette M, Viens P, Ramirez AA, Brzezinski R, Heitz M. Effect of ammonium concentration on microbial population and performance of a biofilter treating air polluted with methane. Chem Eng J. 2011 Jul 15;171(3):1114–23.

603 604

32. AlSayed A, Fergala A, Khattab S, Eldyasti A. Kinetics of type I methanotrophs mixed culture enriched from waste activated sludge. Biochem Eng J. 2018 Apr 15;132:60–7.

605 606 607 608

33. Stearns JC, Davidson CJ, McKeon S, Whelan FJ, Fontes ME, Schryvers AB, Bowdish DM, Kellner JD, Surette MG. Culture and molecular-based profiles show shifts in bacterial communities of the upper respiratory tract that occur with age. ISME J. 2015 May;9(5):1246–59.

609 610 611 612

34. Walters W, Hyde ER, Berg-Lyons D, Ackermann G, Humphrey G, Parada A, Gilbert JA, Jansson JK, Caporaso JG, Fuhrman JA, Apprill A, Knight R. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys. mSystems. 2016 Feb 25;1(1):e00009-15.

613 614

35. Martin M. cutadapt removes adapter sequences from sequencing reads [Internet]. 2018 [cited 2018 Jul 5]. Available from: https://github.com/marcelm/cutadapt

615 616 617

36. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016 Jul;13(7):581–3.

618 619 620

37. Braunegg G, Sonnleitner B, Lafferty RM. A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Eur J Appl Microbiol Biotechnol. 1978 Mar 1;6(1):29–37.

621 622

38. Zhao S, Fan C, Hu X, Chen J, Feng H. The microbial production of polyhydroxybutyrate from methanol. Appl Biochem Biotechnol. 1993 Sep 1;39–40(1):191–9.



623 624 625

39. Fergala A, AlSayed A, Eldyasti A. Behavior of type II methanotrophic bacteria enriched from activated sludge process while utilizing ammonium as a nitrogen source. Int Biodeterior Biodegrad. 2018 May 1;130:8–16.

626 627 628

40. García-Pérez T, López JC, Passos F, Lebrero R, Revah S, Muñoz R. Simultaneous methane abatement and PHB production by Methylocystis hirsuta in a novel gas-recycling bubble column bioreactor. Chem Eng J. 2018 Feb 15;334(Supplement C):691–7.

629 630 631

41. van der Ha D, Nachtergaele L, Kerckhof F-M, Rameiyanti D, Bossier P, Verstraete W, et al. Conversion of Biogas to Bioproducts by Algae and Methane Oxidizing Bacteria. Environ Sci Technol. 2012 Dec 18;46(24):13425–31.

632 633 634

42. Pfluger AR, Wu W-M, Pieja AJ, Wan J, Rostkowski KH, Criddle CS. Selection of Type I and Type II methanotrophic proteobacteria in a fluidized bed reactor under non-sterile conditions. Bioresour Technol. 2011 Nov 1;102(21):9919–26.

635 636 637

43. Karthikeyan OP, Chidambarampadmavathy K, Cirés S, Heimann K. Review of Sustainable Methane Mitigation and Biopolymer Production. Crit Rev Environ Sci Technol. 2015 Aug 3;45(15):1579–610.

638 639 640

44. Veraart AJ, Steenbergh AK, Ho A, Kim SY, Bodelier PLE. Beyond nitrogen: The importance of phosphorus for CH4 oxidation in soils and sediments. Geoderma. 2015 Dec 1;259–260:337–46.

641 642 643

45. AlSayed A, Fergala A, Eldyasti A. Sustainable biogas mitigation and value-added resources recovery using methanotrophs intergrated into wastewater treatment plants. Rev Environ Sci Biotechnol. 2018 Jun 1;17(2):351–93.

644 645

46. Bowman JP, Sly LI, Hayward AC. Patterns of tolerance to heavy metals among methaneutilizing bacteria. Lett Appl Microbiol. 1990 Feb 1;10(2):85–7.

646 647 648

47. Mohanty SR, Bharati K, Deepa N, Rao VR, Adhya TK. Influence of Heavy Metals on Methane Oxidation in Tropical Rice Soils. Ecotoxicol Environ Saf. 2000 Nov 1;47(3):277– 84.

649 650

48. HUMBER WASTEWATER TREATMENT PLANT 2017 Annual Report. Annu Rep. 2017;81.

651 652 653

49. Knief C. Diversity and Habitat Preferences of Cultivated and Uncultivated Aerobic Methanotrophic Bacteria Evaluated Based on pmoA as Molecular Marker. Front Microbiol. 2015 Dec; 6:1346.

654 655 656

50. Graham DW, Chaudhary JA, Hanson RS, Arnold RG. Factors affecting competition between type I and type II methanotrophs in two-organism, continuous-flow reactors. Microb Ecol. 1993 Jan 1;25(1):1–17.


Page 28 of 39

Page 29 of 39


657 658 659

51. Han B, Su T, Wu H, Gou Z, Xing X-H, Jiang H, Chen Y, Li X, Murrell JC. Paraffin oil as a “methane vector” for rapid and high cell density cultivation of Methylosinus trichosporium OB3b. Appl Microbiol Biotechnol. 2009 Jun;83(4):669–77.

660 661 662

52. AlSayed A, Fergala A, Eldyasti A. Influence of biomass density and food to microorganisms ratio on the mixed culture type I methanotrophs enriched from activated sludge. J Environ Sci. 2018 Aug 1;70:87–96.



664 665 666 667 668

6. List of Figures

669 670

Figure 2: Effect of centrate dilution on the specific growth rate, biomass yield and gas uptake rates. The error bars represent the standard deviation for triplicate enrichments.

671

Figure 3: Effect of the methane-to-oxygen ratio on the PHB accumulation and yield

672 673 674

Figure 4: Observed a) F/M and methane utilization b) PHB accumulation, concentration and yield at different biomass densities. The error bars represent the standard deviation for triplicate enrichments.

675 676

Figure 5: PHBV accumulation, HV unit incorporation and yield at different valerate concentrations. The error bars represent the standard deviation for triplicate enrichments.

677 678 679

Figure 6: Effect of the methane-to-oxygen ratio on a) PHBV accumulation and b) the HV fraction at different valeric acid concentrations. The error bars represent the standard deviation for triplicate enrichments.

Figure 1: The average a) final biomass density b) specific growth rate c) biomass yield d) methane utilization rate and e) O/M consumption ratio observed for AD enrichments during cycle operation. The error bars represent the standard deviation for triplicate enrichments.

680


Page 30 of 39

Page 31 of 39

682


Figure 1.



684 685

Figure 2.

686 687 688 689 690 691 692 693 694


Page 32 of 39

Page 33 of 39

695


Figure 3

696 697



699 700

Figure 4.

701

702 703 704 705


Page 34 of 39

Page 35 of 39

706


Figure 5.

707



709 710

Figure 6.

a

711 712 713

714 715 716 717 718 36 ACS Paragon Plus Environment

Page 36 of 39

Page 37 of 39


719 720

7. List of Tables

721 722

Table 1: Average values for different kinetic and stoichiometric parameters observed for the enriched culture during the cycle operation after reaching steady state Parameter

MSM

AD centrate 10%(v/v)

Specific growth rate (hr-1)

0.078±0.005

0.06±0.002

Biomass yield (mg-VSS/mg-CH4)

0.7±0.08

0.6±0.11

Final biomass density (mg-VSS/l)

1099±80

984±19

Specific substrate utilization rate (mg-CH4/mg-VSS-hr)

0.11±0.02

0.09±0.01

O/M (mg-O2/mg-CH4)

2.86±0.12

2.97±0.22

fe

0.44±0.06

0.48±0.09

fs

0.56±0.06

0.52±0.09

723



725

Highlights

726 727



AD sludge provides a shorter start up period to achieve a stable methanotrophic culture

728



Methylocystis genus dominated the enriched culture while maintaining high µmax and qmax

729



AD centrate successfully maintained the selection of PHB-accumulating methanotrophs

730



The cultivated methanotrophs accumulated up to 52±6% PHA in their cellular biomass

731



Methane to oxygen ratio affected the PHBV monomer composition

732


Page 38 of 39

Page 39 of 39


734 735

Graphical Abstract

736


Development of methane-utilizing mixed cultures for the production of

Recommend Documents