1 Ameliorating tannery sludge dewaterability by disintegration of

2 Extracellular Biopolymers with FeCl3-MnSO4 /Oxalic Acid / Sodium

3 Percarbonate: Spectroscopic profiling of Dissolved Organic Matter in sludge

4 filtrate

5 K.M. Priyanka a M.P. Saravanakumar b*

6 a, b-Department of Environmental and Water Resources Engineering, School of Civil

7 Engineering, VIT, Vellore

8 * - Corresponding author

9 Ms.K.M. Priyanka (First Author)

10 Department of Environmental and Water Resources Engineering,

11 School of Civil Engineering,

12 VIT, Vellore-632014

13 Tamilnadu, India

14 priyanka.km2020@vitstudent.ac.in

15 Dr.M.P. Saravanakumar (Corresponding Author)

16 Professor,

17 Department of Environmental and Water Resources Engineering,

18 School of Civil Engineering,

19 VIT, Vellore-632014

20 Tamilnadu, India

1
21 mpsaravanakumar@vit.ac.in

22 Ameliorating tannery sludge dewaterability by disintegration of Extracellular

23 Biopolymers with FeCl3-MnSO4 /Oxalic Acid / Sodium Percarbonate:

24 Spectroscopic profiling of Dissolved Organic Matter in sludge filtrate

25 ABSTRACT

26 Effectual sludge dewatering techniques are strongly desired by industrial wastewater

27 treatment plants. In this work, we scrutinised the efficacy of various Fenton based

28 complex mediated processes like Ferric Chloride (FeCl3)/Oxalic Acid (OA)/Sodium

29 Percarbonate (SPC)(FOS), FS (Without OA), Manganese Sulphate (MnSO4)/ Oxalic

30 Acid (OA)/Sodium Percarbonate (SPC)(MOS) and MS (Without OA) for which

31 Capillary Suction Time (CST) was attained as follows: MS (204s) < MOS (180s) &

32 FS (180s) < FOS (174s) at pH 3. This implied the role of chelating agent (OA) in

33 improving sludge dewaterability by disintegrating Extracellular Polymeric Substances

34 (EPS) and releasing converted free water from the sludge flocs. FTIR results of sludge

35 samples revealed the higher release of aromaticity after treatment process. Presence of

36 aromatic carboxylate compounds were confirmed by GC-MS analysis. A detailed

37 inquisition on the Dissolved Organic Matter (DOM) of filtrate samples using 3DEEM

38 fluorescence methods like second derivative Accumulative Fluorescence Emission

39 (AFE), Fluorescence Regional Integration (FRI) and UV-Visible spectroscopic

40 method like multipeak Gaussian fitting were performed that signified Microbial

41 Soluble Products.

42 Keywords: Oxalate Fenton; Extracellular Polymeric Substances; Capillary Suction

43 Time; Spectroscopic characteristics; Fluorescence Regional Integration

44

2
45

46 1. Introduction

47 The leather sector is one of those that is increasing globally. Leather effluent

48 treatment plants are in high demand due to the significant water consumption, which

49 varies from 30,000 to 50,000 L for every tonne of processed pelts or hides and is
[1]
50 discharged as wastewater about 90% of the time . Meanwhile, a substantial volume

51 of sludge is turned out every year as a consequence of stringent treatment standards

[2]
52 for effluent .

53 Because of the difficulty in treating and disposing of sludge, increased sludge

54 generation poses a significant hazard to the environment. Sludge is an intricate

55 colloidal system in which tiny particles with a diameter of 1 µm create a consistent

[3]
56 suspension . Free water, hydration and interstitial water, vicinal water are several
[4][5].
57 water types found in sludge The term "bound water" usually refers to a mixture

58 of vicinal, hydration and interstitial fluids remain firmly attached to Extracellular

59 Polymeric Substances (EPS) and other sludge components by chemical or adherent

[6]
60 nature that makes it complicated to remove . In this regard, dewatering plays a

61 significant role in moisture reduction that also deduces volume of sludge and

62 subsequently saves cost in transportation for disposal of sludge.

63 Considering EPS to be the crucial factor influencing sludge dewaterability, the main

64 constituents of EPS are humic substances, lipids, proteins, nucleic acids,

65 polysaccharides and many charged groups (such as hydroxyl, phosphoric, sulfhydryl

[7]
66 and phenolic) and apolar groups (such as aliphaticity and aromaticity) respectively .
[6] [8]
67 Various techniques such as acidification and ultrasound , flocculant , electric
[9] [10] [11]
68 field , biomass, Advanced Oxidation Process (AOP) , cationic

3
69 surfactant [12] , electro-Fenton [13] and hydrothermal treatment [10] have been implied

70 to enhance sludge dewaterability. Among these strategies, Advanced Oxidation

71 Processes (AOP’s) tend to be an effective and propitious technique in ameliorating

72 sludge dewaterability because it can efficiently accomplish two tasks, first is that

73 increasing water release from sludge cells and secondly, a higher proportion of bound
[14]
74 water turning into gravitational water to accelerate sludge dewatering . AOP’s

75 generate free radicals which possess strong oxidative properties and has higher
[15]
76 potential of degrading hydrated Extracellular Polymeric Substances (EPS’s) . In
[16] [17]
77 recent times , AOP involving Fenton oxidation, Fenton like oxidation,

78 persulfate oxidation and chlorine oxidation have been widely applied to ameliorate

79 sludge dewaterability. For instance, Zin Zhang et al from his study inferred that

80 OA/Fe2+-PDS process showed moisture reduction of 71% in terms of CST and

[16] 2+
81 breakdown of flocs of sludge EPS with better sludge dewaterability . Fe

82 −peroxydisulfate (PDS) in combination with citrate as a chelator promoted

[18]
83 dewatering of sludge and increased radical productivity . In Peng Yang’s study,

84 oxidation process based on organic acids chelated ferrous ion catalyzed NaClO was
[17]
85 employed that resulted in effective dewaterability

86 Recent studies have been focussing on Fe(III)Oxalate complex mediated Fenton

87 system for mitigating industrial wastewater pollution owing to two merits such as:

88 One is that the Fe (III)-oxalate complex has the ability to speed up the evolution of

89 Fe(II) ions as well as several different reactive oxygen species (ROS). The other is

90 that this system may degrade a range of organic pollutants at close to acidic pH

91 because suitable ligands are present. Superoxide radicals (O2) are created when
[19,20]
92 oxalate combines with dissolved oxygen in water which in turn evolves H2O2. .

93 Chelating agent is capable of aggravating both ferrous and ferric ion formation that

4
 94 leads to hydroxyl radical generation for consumption of target substances [21]. In

95 contrast to other inorganic acids, oxalic acid is thought to be a pollution-free

96 compound with strong complexing potential that can be quickly removed via
[22]
97 biodegradation. . This organic dicarboxylic acid, which is often made by oxidising

98 sucrose, is a naturally occurring compound in many plants and vegetables. Usage of

99 OA has also improved the stabilisation and resulted in higher affinity of heavy metals
[16]
100 due to its chelating capacity .

101 This Fe (III)/OA complex-mediated Fenton method known for its Insitu Chemical

102 Oxidation (ISCO), has gained appeal for its ability to degrade organic pollutants in
[19] [23] [24]
103 wastewater and water. . Current studies revealed that different versatile

104 oxidising agents like persulfate, percarbonate, perborate were extensively utilised in

105 multiple ISCO technologies. Unlike conventional Fenton process, Sodium

106 Percarbonate (SPC) is used as oxidising agent in this case because of its strong

107 oxidation potential and higher solubility than H2O2. Congcan Zhang as a result of his

108 study identified that SPC showed higher oxidising performance than other solid
[19]
109 oxidants generating O2 and OH radicals .

110 Dissolved Organic matter (DOM) is an extremely diverse mixture consisting of

111 soluble microbial products (SMP’s), fulvic and humic substances that is significant to

112 ecological processes. This DOM is considered to be a primary removal substance

113 which limits the application of wastewater (which includes sludge) reclamation and
[25]
114 reuse . Various adverse effects caused by DOM in wastewater reclamation process
[26]
115 increased demand for oxidants and promoted microbial growth of substrates .

116 Hence, the adverse variations in sludge dewatering due to DOM needs to be

117 addressed. Several post processing techniques are available for extracting EEM data

118 information which includes Fluorescence Regional Integration (FRI). By integrating

5
119 the volumes beneath five designated EEM zones, fluorescence regional integration

120 (FRI) could quickly offer quantitative information concerning the relative abundance

121 of defined fractions of DOM.

122 Conclusively, though role of chelating agents in increasing dewaterability is been

[17] [16]
123 investigated in sewage and domestic sludge , , no studies with chelating agents

124 for improving tannery sludge dewaterability has been done that instigated for our

125 study. Since Manganese (Mn) is considered as easily oxidizable metal with suitable

126 ligands, comparison has been made with two different transition metals in this

127 complex system.

128 Therefore, the primary goal of this study is to explore the impact of two transition

129 metals (Fe and Mn) Oxalate mediated Fenton system on improving tannery sludge

130 dewaterability. At the same time, outcome of these processes on biopolymers and

131 heavy metals prior and after treatment were examined. Further detailed investigation

132 was carried out to monitor the variations in DOM using UV-Vis and 3D-EEM

133 fluorescence spectroscopic analysis. Finally, DAS derived multi-peaks Gaussian

134 fitting approach, second derivative based on Accumulative Fluorescence spectra

135 (AFE) and Fluorescence Regional Integration (FRI) were performed for better

136 understanding of DOM substances in filtrate samples.

137 2. Materials and Methods

138 Tannery sludge samples from primary clarifier were collected from Common Effluent

139 Treatment plant (CETP) in the vicinity of Vellore, India. These sludge samples were

140 fetched and stored at 4 °C in refrigerator in order to preserve its characteristics.

141 2.1 Reagents and Chemicals

6
142 Ferric chloride (FeCl3), Manganese sulphate (MnSO4), Oxalic acid (C2H2O4), Sodium

143 Percarbonate [Na2CO3.1.5 H2O2] (SPC), Sulphuric acid (H2SO4), Sodium hydroxide

144 pellets (NaOH), Dichloromethane (DCM), n-pentane, p-benzoquinone and Tert-butyl

145 alcohol (TBA) employed in this study has been purchased from SD Fine Chemical

146 Limited, Mumbai.

147 2.2 Instrumental Characterisation of raw and treated sludge/filtrate

148 Fourier Transform Infrared spectrum (FT- IR, Model: IR Affinity -1, Shimadzu) was

149 employed in analysing the presence of different functional group in both raw and

150 treated sludge samples. Determination of Dissolved Organic Carbon (DOC) in sludge

151 samples was performed using TOC analyser (TOC-LCPH FA, E-200). Concentration

152 of heavy metals in raw and treated filtrate samples was evaluated by Inductively

153 Coupled Plasma Optical Emission Spectral analysis (ICP-OES, Avio 200). 3D-EEM

154 Excitation Emission Matrix (EEM, Hitachi Fluorescence Spectrophotometer F-7000)

155 spectroscopic analysis was used to examine the fluorescence peak intensities of DOM

156 present in filtrate samples before and after treatment. UV-Vis (UV-VIS SPEC, Hach

157 DR-6000) spectroscopic analysis was also used to investigate the variations in DOM

158 of filtrate before and after treatment. GC-MS analysis (GC-MSD, Agilent 5977B) was

159 employed to identify the recalcitrant organic compounds present in sludge samples.

160 2.3 Optimisation of parameters and sludge dewaterability experiments

161 Five beakers containing 250ml of raw sludge samples were taken to conduct the

162 optimisation and sludge dewaterability experiments. pH, dosages of C2H2O4(OA),

163 Na2CO3·1.5H2O2 (SPC), FeCl3 and MnSO₄ were the varying parameters which

164 influences the Capillary Suction Time (CST) of raw and treated samples. In order to

165 find the optimal dosage and pH, experiments were carried out using Jar Test

7
166 Apparatus. First, pH was modified to desired optimisation levels for Fe-SPC(FS), Mn-

167 SPC(MS), Fe-OA-SPC(FOS) and Mn-OA-SPC(MOS) processes. Then required FeCl3

168 and MnSO4 was added in the respective beakers which was then pertained to fast

169 rotation of 200rpm for 1 min. Consequently, required dosages of C2H2O4 (OA),

170 Na2CO3·1.5H2O2 (SPC) has been added and subjected to rapid mixing of 200 rpm for

171 1min. Finally, slow mixing of 100rpm was carried out for ten minutes.

172 After jar test experiments based on optimum pH and dosages attained, Capillary

173 Suction Time (CST) (in seconds) was measured for both raw and treated sludge

174 samples. The uttermost important factor to assess sludge dewaterability is Capillary

175 Suction Time (CST). CST was measured by deploying a tube of 6cm height over

176 Whatman Chromatography Filter Sheet #17 marked with two circles of 5cm and 3cm

177 diameter respectively. When the sludge samples were siphoned into the tube, time

178 taken for the filtrate to reach from 3 to 5cm diameter was recorded as Capillary
[27]
179 Suction Time (CST) . Finally, the derived optimum pH and three consecutive CST

180 values were utilised for experimental analysis. In addition, basic properties of tannery

181 sludge samples were analysed and is depicted in Table S1.

182 2.4 Extraction Protocol of EPS from sludge samples

183 In general, Extracellular Polymeric Substances (EPS) are made of polysaccharides,

184 lipids, humic substances, proteins and fulvic acids. EPS has a capacity to hold the

185 water molecules within its polymeric matrix which significantly influences sludge

186 dewaterability. They are classified as soluble-EPS, tightly bound-EPS, loosely bound-

187 EPS based on their structure and properties. These EPS content in sludge needs to be

188 extracted to investigate the different polymeric substances present in sludge and their

189 significant impact on sludge dewatering. EPS extraction protocol was followed based

8
190 on previous studies [28], [29]. Using method of modified heat extraction, separation of

191 different EPS fractions was carried out in the raw and treated sludge. Initially, a 50ml

192 sludge sample was centrifuged for 5 minutes at 4000g and then the supernatant was

193 recovered as soluble EPS (S-EPS). Those leftover pellets were resuspended in 50ml

194 of 70°C warmed 0.05wt% NaCl solution. Following that, the sludge mixture was

195 sheared for 1 minute with a mini-type vortex mixer (VM-370, INTLLAB), followed

196 by 10 minutes of centrifugation at 4000g, and the supernatant was loosely bound EPS

197 (LB-EPS). Following that, the pellets were dissolved in a NaCl solution of

198 0.05%(70°C) and boiled for 30 minutes in a hot plate at 600°C. After centrifuging the

199 mixture for 15 minutes at a rate of 4000g, the remaining supernatant can be recovered

200 as the tightly bound EPS (TB-EPS). Finally, the protein and polysaccharide contents

201 in various EPS fractions were estimated using modified Lowry and Anthrone reagent

202 method.

203 2.5 UV-Vis analysis coupled with Gaussian multipeak fitting approach

204 In order to perform the Gaussian multipeak fitting approach, the absorption spectra

205 obtained from UV-Vis spectroscopy was used to derive the Differential Absorption

206 Spectrum i.e. (A (λ, i)-A (λ, ref)) and the obtained spectra was then smoothened. The

207 smoothened curve was then evaluated using Gaussian function with multiple

208 iterations performed in Origin version 9.95 Software.

209 2.6 AFE coupled via second derivative spectroscopy

210 The sum of the intensities amidst the wavelength axes were determined to ascertain

211 the corresponding bands. The emission spectrum is made up of the summation of the
[30]
212 intensities along the excitation wavelengths that is elucidated as Accumulative

213 Fluorescence Emission (AFE) spectrum. All AFE spectra were then subjected to

9
214 obtain second derivative for identification of fluorescent organic matter present in

215 sludge samples.

216

217 3 Results and discussions

218 3.1 Significance of operational factors on sludge dewaterability during

219 FS/MS/FOS/MOS processes

220 3.1.1 Variations in FS/MS//FOS/MOS processes and EPS organics due to pH

221 Several oxidation processes have greater efficacy in acidic or near neutral pH range

222 which includes Fenton mediated processes. Sulphuric acid and sodium hydroxide was

223 utilised to adjust the pH of the sludge before treatment. The impact of pH on sludge

224 dewaterability has been investigated by varying in the range of 3 to 11 as indicated in

225 Fig 1. Initial pH was noted to be in the range of 7 to 8.5 in raw sludge sample.

226
227 Fig 1. Effect of pH on CST

10
228 From Fig 1, it is observed that in FS and MS processes, minimum CST value of

229 around 260secs and 207secs was obtained at pH 3 and thereafter CST values

230 increased eventually when pH values were adjusted. Similarly, as shown in Fig 1, in

231 FOS and MOS processes which conducts similar to simpler Fenton process, higher

232 dewaterability (FOS-210 secs and MOS-180 secs) was observed in the pH value of 3.

233 The catalytic capacity of ferric iron is disrupted at pH more than 4 that resulted in

234 precipitation of ferric oxyhydroxide (FeOOH) (Eqn (3)). Unlike other, in MOS

235 process from Fig 1, even after there is a rise in pH from 5 to 12, a steady increase in

236 CST pattern was noticed which might be due to the stability of Mn ions to promote

237 more destruction of EPS and role of Oxalic acid in generating more of hydroxyl (OH•)

238 and superoxide (O2 •) radicals. Acidification was proven to improve sludge dewatering

239 owing to its effects on Extracellular Polymeric Substances (EPS) present in sludge.

240 One effect is that acidic pH was more prone to deteriorate sludge flocs thereby

241 destructing EPS and releasing organic matters along with excess bound water. The

242 second outcome paved way for suppression of liberated EPS due to protonation that

243 includes protein like substances mainly. The initial suppressed EPS was released into

244 the outer surface of sludge when the protonation effect was reduced by an increase in

245 pH, and the soluble EPS's protein content steadily increased, influencing the sludge
[31]
246 dewaterability . These effects mentioned above were found to be in consistence in

247 case of acidification (i.e. pH - 3) from our study as shown in Fig 2a. Moreover, under

248 acidic conditions, negatively charged sols, particularly EPS that contains a lot of

249 functional groups of negative charge tend to be neutralised by the protons in the
[32]
250 supernatant that effectively compresses EPS . Therefore, acidic pH was preferable

251 for higher sludge dewaterability in all the treated processes.

11
252 3.2 Mechanism insights into improved sludge dewaterability resulted from FS, MS,

253 FOS &MOS processes

254 It is proposed that all four of the advanced oxidation processes involves oxidation

255 mechanism. In first two processes, FS and MS, SPC gets dissociated into Na2CO3 and
[33]
256 H2O2 when entering water thereby implicating certain degree of oxidation (Eqn

257 (1)). On the other hand, when Fe (III) reacts with H2O2, Fe (III) catalyses H2O2 to

258 form OH•. radical that is represented below in (Eqn (2)). Similarly, on H2O2 and Mn

259 (II) interaction, OH•. radicals are liberated as a result of Mn (II) catalysis, while Mn

260 (II) has been oxidised to Mn (III) simultaneously (Eqn (4)). Therefore, the hydroxyl

261 radicals generated from these processes aided in oxidation of organic contents and

262 helped in improving sludge dewaterability.

263 N a2 C O3 .1.5 H 2 O2 → N a2 C O3+1.5 H 2 O2 (1)

−¿+ HO • ¿
264 Fe ( III ) + H 2 O2 → Fe ( III ) +O H (2)

2 +¿3+¿ → FeOOH ¿
265 Fe (3)

−¿+HO • ¿
266 Mn ( II ) + H 2 O2 → Mn ( III ) +O H (4)

267 In case of FOS and MOS processes, on reaction of Fe and Mn oxalate complex, more

268 hydroxyl radicals are generated that is dissociated to form an intermediate product

269 (C2O4 -) (Eqn 5). This product tends to form superoxide radicals (O2•-) as shown in
[20]
270 (Eqn (6)) that finally produces H2O2 (Eqn (7)) . At desirable concentration of

271 oxalate ions, stronger oxidation and efficient sludge dewaterability was observed.

−¿ ¿
2−¿+OH •+ H O ¿
O2 → Fe ( III ) / Mn( C 2 O4 ) ¿
272 Fe ( II ) / Mn ( C 2 O4 ) 2−¿+H 2
(5)

¿
−¿+O2 → 2 CO 2+O2 ¿ ¿
273 C 2 O4 (6)

12
 •−¿
O2 +¿→ H 2 O2+O 2 ¿
274 •
+H ¿ (7)
HO 2

275 When hydroxyl (OH•) and superoxide (O2•-) radicals are generated during this

276 oxidation, disintegration of sludge flocs takes place furthermore the excess water

277 bounded in the cells are released which could attribute to better sludge dewaterability

278 in FOS and MOS processes. Moreover, degeneration of EPS has attributed to this

279 which originated due to oxidation process involved in these mechanisms. Results

280 obtained from zeta potential analysis revealed that raw sludge showed a surface

281 charge of -18.8mv. On the other hand, FS, MS, FOS and MOS processes unveiled

282 surface charges of -1.1mv, -0.6mv, -1.5mv and -1.5mv respectively. It was

283 proclaimed that a decrease in electrostatic attraction between sludge particles

284 corresponds to an increase in zeta potential. These surface charges were attributed to

285 decomposition of EPS in which negative charge by OH• and O2•_ oxidation enhanced

286 dewaterability of sludge. Further, the increase in organic compounds in FS and MS

287 was observed depicts the role of OH• in releasing them. As shown in Fig S 3a-b the

288 protein and polysaccharide content were increased both in soluble and loosely bound

289 EPS. On the other hand, the amount of tightly bound compounds was decreased after

290 treatment, indicating the breakdown of TB-EPS that turns into soluble compounds.

291 Similarly, in oxalate mediated process released more amount of soluble organic

292 compounds than FS and MS process (without oxalate). This could be due to the

293 formation of superoxide radical in addition to OH• because of the presence of Fe-

294 OA/Mn-OA complex (Eqn 5-7), which signifies the role of chelating agent. Wang

295 stated that TB-EPS resulted in reduction of charge and thereby improved
[34]
296 dewaterability . This is evident from Fig S3a that degradation of TB-EPS lead to

297 better dewaterability. Therefore, these four processes mentioned above were

13
298 investigated for effects on sludge dewatering which remained auxiliary to each other,

299 out of which processes supported by Oxalic acid showed higher potency compared to

300 other treatment systems.

301 3.3 Physicochemical variations in sludge organics

302 To examine the different physical and chemical characteristics in samples of both

303 untreated and treated sludge samples, distinctive instrumental studies were performed.

304 3.3.1 3D-EEM Fluorescence Spectral Variation of raw and treated filtrate

305 In addition to various approaches in UV-Vis spectroscopic technique(S1.3), results

306 obtained from 3D-EEM fluorescence spectroscopy (Fig 2a-e) and FRI (Fig 2f) were

307 found to be in consistence with the UV-Vis results. The fluorescence EEM spectra

308 shown below was separated into five areas reflecting distinct components of DOM

309 such as: Region I comprising of aromatic proteins with smaller molecular sizes,

310 Region II containing aromatic proteins with larger molecular sizes, Region III having

311 substances resembling fulvic acid, Region IV including proteins, polypeptides, and

312 substances resembling amino acids, and Region V has humic acid-like substances)
[35]
313 . Fig 2a-e shows that the contribution of DOM in raw and various treated filtrate

314 samples is varied in terms of intensity of fluorescent peaks. It is notified that strongest

315 fluorescence intensity is seen in region IV representing more of tryptophan & protein

316 like, tyrosine & protein like and biological compounds with aromaticity (Fig 2a). On

317 the other side, no significant peak intensity was found in Region I, III and V

318 corresponding to humic and fulvic acid regions in raw filtrate. Yu et al from his study
[36]
319 found that fluorescent organic EPS components alter sludge dewaterability .

320 Considering this statement, protein and polysaccharide content (S-EPS) (Fig 2a-b)

14
321 was higher in treated filtrate samples than raw filtrate due to the fact that complex

322 organic molecules are fragmented into

323

15
324 Fig 2. 3D-EEM spectra of filtrate samples a) Raw b) FS c) MS d) FOS e) MOS f) FRI

325 distribution of filtrate samples

326 simpler soluble forms during oxidation which contributes to the moisture reduction

327 and enrichment of CST. In this regard, FOS process showed higher EPS content than

328 other processes that is evident in EEM results also (Fig 2d-e). It is also understood
[37]
329 that protein is said to have more of water absorbing capacity that when oxidised

• •
330 and disintegrated due to O2 - and OH radicals leads to better sludge dewaterability.

331 Next to FOS, MOS process showed good results in release of bound compounds that

332 might be due to role of radicals involved in the corresponding oxidation processes

333 (Fig S3 a-b). In the last, FS and MS process was found to be weak in release of

334 organic compounds even though they resulted in moderate CST with other two

335 processes. Xin Zhang reported from his study that the release of more bound water

336 and the subsequent major rise in sludge particle size were both caused by oxalic acid,
[16]
337 which was crucial in the breakdown of sludge flocs. . This is the due reason for

338 FOS and MOS being more capable of releasing organic substances than FS and MS

339 processes. Overall, it was found that the EPS content of raw filtrate sample was lesser

340 than that of treated filtrate, indicating that the oxidative ability of radicals played a

341 major role in the treated processes by which organic components were broken down

342 and complex organic matter was emancipated. From Fig 2d of 3D EEM spectra of

343 treated filtrate samples, FOS treated filtrate had Em peak of 330 - 380 nm at Region

344 IV revealing proteinic fluorophore with some microbial soluble products (MSP’s)

345 which is not more prevalent in case of MOS process(Fig 2e) and humic like

346 substances at Region V. FS treated filtrate exhibited lower peak intensity of

347 fluorescent substances than FOS process which could be due to the effect of chelating

16
348 agent in release of organic matter(Fig 2b,d). On the contrary, MOS process unveiled

349 higher humified nature with larger peak intensity at region V (Em -380 to 600nm)

350 (Fig 2e). From Fig 2c, it is evident that overall fluorescent intensity is found to be

351 higher in MS process than MOS process (Fig 2e). MS filtrate proliferated more of

352 fulvic, humic like substances and few protein components with aromaticity. In recent
[38] [39]
353 studies conducted by Zhong and Huo et al, after Fenton treatment there was

354 a considerable reduction in the fluorescence signals of humic like peaks and few

355 fulvic like peaks vanished. This was found to be in accordance with our results as

356 mentioned above. Fluorescence Regional Integration (FRI) technique was adopted for

357 profound understanding of organic matter in different filtrate samples. Based on the

358 five regions in EEM spectra, the area underneath each region of EEM spectra was
[40]
359 calculated . The projected excitation -emission area of each region was derived

360 for each sample which is denoted as (Φi, n). Subsequently, fractional projected area

361 and multiplication factor (MF) was calculated for each filtrate sample having five

362 fluorescent regions. Finally, percentage of fluorescent response distribution has been

363 estimated from total regional area which is epicted below in Fig 2f. From the results,

364 it is seen that higher percentage of fluorescent distribution was found in Region V

365 (humic substances) of all treated filtrate samples. In addition, Pi value for raw sample

366 in Region V was comparatively lesser indicating the release of more humic substances

367 in treated filtrate samples. The obtained quantitative result remained consistent with

368 the EEM spectra shown in Fig 2a-e. Raw filtrate sample tends to have very lower P

369 (II, n) value. On the other side, higher percentage of fluorescent response distribution

370 was noted in MOS filtrate sample corresponding to Region II having aromatic

371 proteins. From Fig 2f it could be found that percentages of Region IV for Raw and

372 MS filtrate were relatively same showing 38.64% and 32.87% respectively. On the

17
373 contrary, FS and FOS showed P(IV, n) values of 14.2% and 13.9% with MOS having

374 lower percentage of 9.69%. From Fig 2e, it is clear that MOS had higher fluorescent

375 intensity peaks in Region III (fulvic) and Region V(humic). However, P (i, n) values

376 for combined Regions (III) and Region (V) is found to be as lower as 11.9% and

377 58.5% respectively. Thus, MOS might have more of humified components than fulvic

378 substances.

379 The fluorescent substances of DOM could be precisely matched by the AFE

380 spectroscopy that can be derived by adding up the intensities alongside the excitation
[41,42]
381 wavelengths . According to second derivative spectroscopy, the sheer area at a

382 particular fluorescence band is thought to be analogous to the relative concentration of

[43]
383 the pertinent luminescent peaks in DOM . AFE along with second derivative

384 spectrum was imposed in our study to understand in depth about the DOM substances

385 present in raw and treated samples which is depicted in Fig 3a-e below.

18
386

387 (a) (b)

388

19
389 (c) (d)

390

391 (e)

392 Fig 3: AFE with second derivative spectroscopy a) Raw b) FS c) FOS d) MS e) MOS

393 It is evident that AFE spectrum has one strong sharpened peak (Raw-345nm, FS-

394 370nm, FOS-360nm, MS-400nm, MOS-470nm) in both raw and treated filtrate

395 samples. From Fig 3a of raw filtrate, one strong and sharpened shoulder was spotted

396 at 345nm indicating presence of microbial soluble products. On the other hand, one

397 weak shoulder at 420nm was observed confirming the presence of humic acid which

398 matched the peak of raw EEM spectrum (Fig 2a). The strong sharp-edged fluorescent

399 peak at 370nm in FS process (Fig 3b) is attributed to the soluble products like proteins

400 and polysaccharides and was also exhibited in Fig 2b. The small weak shoulder seen

401 at 455nm concerned with humic and fulvic like substances overlapped the concurrent

402 fluorescence spectra of FS process (Fig 2b). The sharp peak corresponding to 360nm

403 associated with microbial products is seen in FOS process of AFE (Fig 3c) and also in

20
404 3D-EEM spectrum (Fig 2d). Humic like substances also appeared at a weaker peak

405 intensity of 450nm in FOS process. The higher fluorescent intensity peak at 400nm

406 and broad weak shoulder at 460nm in MS process (Fig 3d) signified the presence of

407 fulvic and humic like substances which corresponded with both EEM (Fig 2c) and

408 AFE spectra. From Fig 3e of MOS process the peak pertaining to humic like and

409 fulvic like components was observed at 470nm with more of humic substances. The

410 deep weakened shoulder from Fig 3e exhibiting more of microbial associated by-

411 products having tyrosine like and tryptophan like compounds was seen at 390nm

412 which was also found in EEM spectra. Thus, in comparison of 3D-EEM

413 (experimental result), FRI and AFE along with second derivative spectrum (analytical

414 result), it is inferred that MOS process tends to have more of humic like compounds

415 and FOS process exhibiting more of microbial soluble products (MSP’s) was found to

416 have higher aromaticity than other processes.

417

418 4. Conclusion

419 From this study, it is suggested that of all the four processes, FOS and MOS was

420 demonstrated to be effective for improving tannery sludge dewaterability in terms of

421 CST (174-180 secs) than FS and MS processes. Though FS and MS showed relatively

422 equal efficiency in dewaterability of sludge, degradation of EPS substances was found

423 to be higher when Oxalic acid was included in the process. Results obtained from

424 TOC analysis and EPS contents were found to be in consistent which resulted in

425 better dewaterability of sludge. Additionally, stabilisation of metals like Fe,Cr,Mn

426 was enhanced due to the chelating capacity of OA. Due to the considerable release of

427 metals to filtrate after treatment, it is suggested that the sludge could be utilised as raw

21
428 material for processing industries like cement. The reduction in spectral intensity at

429 1085cm-1 indicates the oxidation of biopolymers due to scavenging radicals such as

430 OH• and O2•_. From various spectroscopic analysis such as UV-Vis involving DAS

431 coupled with Gaussian multipeak fitting and 3D-EEM Fluorescence along with FRI &

432 AFE having second derivative spectrum ensures the presence of aromaticity

433 specifically FOS process having higher intensity.

434 Acknowledgment

435 We wish to express our sincere thanks to VIT University, Vellore for providing us

436 with essential materials, lab facilities, infrastructure, and instruments to perform this

437 study.

438

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