Polyhydroxyalkanoate Biosynthesis from Paper Mill ...

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Mar 13, 2016 - Activated sludge versus aerated lagoon treatment of kraft mill effluents containing β-sitosterol and stigmasterol. Journal of Environmental ...
Water Air Soil Pollut (2016) 227:299 DOI 10.1007/s11270-016-2969-x

Polyhydroxyalkanoate Biosynthesis from Paper Mill Wastewater Treated by a Moving Bed Biofilm Reactor Rocío Baeza & Mayra Jarpa & Gladys Vidal

Received: 13 March 2016 / Accepted: 4 July 2016 # Springer International Publishing Switzerland 2016

Abstract One potential way for organic matter recovering contained in paper mill effluents can be obtaining polyhydroxyalkanoate (PHA). The aim of this work was to evaluate PHA biosynthesis from paper mill effluents by moving bed biofilm reactor (MBBR) under different operational strategies of the BOD5/nitrogen (N)/phosphorus (P) ratio. The operational strategies were evaluated in two phases. During phase I, organic loading rates (OLRs) were increased from 0.13 to 2.99 biological oxygen demand kg BOD 5 m −3 day −1 , and in phase II, kg BOD5 m−3 day−1 was increased from 0.81 to 2.83. In both phases, the BOD5/N/P ratios were 100:5:1 and 100:1:0.3. The maximum percentages of PHAaccumulating cells and organic matter removal were 85.10 and 95.60 % for phase I, both with a BOD5/N/P ratio of 100:5:1, while in phase II, PHA biosynthesis and organic removal were 89.41 and 97.10 % with 100:1:0.3 and 100:5:1, respectively.

Keywords Organic matter . Polyhydroxyalkanoate . Aerobic system . MBBR reactor

R. Baeza : M. Jarpa : G. Vidal (*) Engineering and Environmental Biotechnology Group, Environmental Science Faculty and Center EULA-Chile, Universidad de Concepción, P.O. Box 160-C, Concepción, Chile e-mail: [email protected]

1 Introduction Different productive sectors are moving to the management and resources recovery under the scenery of the circular economy (Jiang et al. 2012; Gameiro et al. 2015). Due to this, one potential way for organic matter (measured as chemical oxygen demand (COD) or biological oxygen demand (BOD5)) recovering contained in paper mill effluents can be obtaining polyhydroxyalkanoate (PHA). Mechanical pulping processes such as pressure groundwood (PGW) and thermomechanical pulping (TMP) are used to manufacture paper. Wastewater generated from these processes is characterized by organic matter, measured by chemical organic demand (COD), in concentrations ranging from 1000 to 5600 mg L−1 and biological oxygen demand (BOD5) between 400 and 1200 mg L−1 (Chamorro et al. 2005; Vidal et al. 2007; Xavier et al. 2009; Jarpa et al. 2012). Therefore, they need to be treated to reduce the impact on the aquatic ecosystem in which they will be discharged (Xavier et al. 2005; Chamorro et al. 2010). The moving bed biofilm reactor (MBBR) is one of the most widely used technologies for biologically treating paper mill wastewater. In the MBBR, biomass grows attached to an inert carrier with high area/volume (350 m2 m−3), forming biofilms and decreasing the probability of bulking (Odegaard 2006). Additionally, the MBBR works with organic loading rates (OLRs) between 2.5 and 3.0 kg COD m −3 day −1 and hydraulic retention times (HRT) of even less than 2 h. Under these operational conditions, BOD 5 removal efficiencies range

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between 85 and 99 %, while COD ranges between 24 and 85 % (Villamar et al. 2009). However, several tons of excess sludge are generated during MBBR operation due to BOD5 removal, with approximately 1.8–3.5 t of sludge per ton of BOD5 (Jarpa et al. 2012). Because of this, special value was given to BOD5 under specific treatment condition like biosolid enrichment by PHA biosynthesis (Pozo et al. 2011). PHAs are polyesters that accumulate as intracellular granules from carbon and energy sources under limited nutrient concentration (e.g., nitrogen and phosphorous) (Pozo et al. 2011). The main substrate for PHA biosynthesis is volatile fatty acids (VFA) like acetic, propionic, and butyric acids, among others (Jarpa et al. 2012; Bengtsson et al. 2008). PHAs have physical and thermal characteristics that allow their use as bioplastics, and they are considered a biodegradable alternative to conventional plastics (Keshavarz & Roy 2010). Previous studies show that PHAs can feasibly be produced using pure cultures and substrates. However, due to the high cost of sterilizing equipment and the substrate, PHA production is very costly (Jiang et al., 2012). PHA biosynthesis from mixed cultures and renewable carbon sources like BOD5 obtained from industrial wastewater has emerged as a very important alternative (Pozo et al. 2011, 2012; Jiang et al. 2012). Bengtsson et al. (2008) reported 48 % PHA biosynthesis for dry weight of sludge in an activated sludge system using paper mill wastewater and mixed cultures. In contrast, MBBR systems have reported a range between 85 and 95 % of PHA-accumulating cells from kraft mill and paper mill effluent (Jarpa et al. 2012; Pozo et al. 2012). The goal of this study was to evaluate PHA biosynthesis from paper mill wastewater under different operational strategies of the BOD5/nitrogen (N)/phosphorus (P) ratio by MBBR.

2 Materials and Methods 2.1 Raw Wastewater Wastewater was obtained after primary treatment from a paper mill that processes Pinus radiata with PGW and TMP processes. The wastewater was stored in darkness at 4 °C ± 1. PGW and TMP wastewaters were used as influent for the MBBR operation (Navia et al. 2003). 2.2 Inoculum MBBR phase I, operating with PGW influent, was inoculated in two stages: the sludge concentrations were 5.91 g volatile suspended solids (VSS) L−1 and 7.40 g VSS L−1 for the first (1–74 days) and second stages (75– 300 days), respectively. MBBR phase II, operating with TMP influent, was inoculated with 5.21 g VSS L−1. The biomass used as inoculum was collected from activated sludge from a municipal wastewater treatment plant. 2.3 Moving Bed Biofilm Reactor The MBBR, with a usable volume of 0.85 L, was filled with 200 inert polyethylene assisting moving bed (AMB)type carriers with a density, specific surface area, and porosity of 98 kg m−3, 850 m2 m−3, and 85 %, respectively. The MBBR was operated in two phases. In phase I, the MBBR was fed with PGW influent (300 days) and in phase II with TMP influent (167 days) at a temperature of 19.1 ± 2.1 °C and with a dissolved oxygen concentration between 6.0 and 7.6 mg L−1. Table 1 shows the MBBR operational conditions. The operational strategy in phase I was to increase the OLR from 0.13 to 2.99 kg BOD5 m−3 day−1 for two BOD5/N/P ratios. The first BOD5/N/P ratio of 100:5:1 was between 1 and 270 days, and the second was 100:1:0.3 (271 to 300 days).

Table 1 MBBR operational conditions Parameter

Phase I

BOD5/N/P

100:5:1

Phase II 100:1:0.3

100:5:1

100:1:0.3

Period (days)

1–99

100–179

180–225

226–270

271–300

1–73

74–109

110–170

HRT (h)

47.8 ± 0.77

12.20 ± 0.20

6.06 ± 0.05

3.06 ± 0.05

3.08 ± 0.01

48.16 ± 0.54

14.35 ± 0.79

14.07 ± 0.82

OLR (kg BOD5 m−3 day−1)

0.13

0.56

2.36

2.99

2.99

0.81

2.67

2.83

COD BOD5−1 influent

1.54 ± 0.07

1.35 ± 0.14

1.55 ± 0.02

2.53 ± 0.00

2.48 ± 0.00

1.51 ± 0.22

1.63 ± 0.05

1.61 ± 0.01

COD BOD5−1 effluent

2.22 ± 0.03

2.19 ± 0.01

3.24 ± 0.04

2.70 ± 0.04

1.24 ± 0.02

5.00 ± 0.2

4.76 ± 0.02

2.71 ± 0.07

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During phase II, the operational strategy was to increase the OLR from 0.81 to 2.83 kg BOD5 m−3 day−1 with BOD5/N/P ratios of 100:5:1 for an OLR of 0.81 and 2.67 kg BOD5 m−3 day−1 and for an OLR of 2.83 kg BOD5 m−3 day−1, the BOD5/N/P ratio was 100:1:0.3. MBBR performance was evaluated for BOD5, COD, and total phenolic compounds in the influent and effluent for the operation period according to Eq. 1: Rð%Þ ¼

Qi ⋅C i −Qo ⋅C o ⋅100 Qi ⋅C i

ð1Þ

where R (%) is the percentage removed, Q is the flow rate (L day −1), C is the parameter concentration (mg L−1), and sub-indices Bi^ and Bo^ are inflow and outflow, respectively. MBBR biomass and the percentage of PHA-accumulating cells were monitored (Jarpa et al. 2012; Pozo et al. 2012).

5890 Series II, with a flame ionization detector (FID)). Samples were membrane filtered through 0.45-μm pore size filters. PHA biosynthesis was determined by flow cytometry (FACSCalibur system) and spectrofluorimetry. Prior to determination, the microbial sludge was deflocculated (tetrasodium pyrophosphate 0.01 %, 2-dodecoxyethanol 0.01 %) (Pozo et al. 2011). Samples were measured using optical density at 600 nm (UV-vis spectrophotometer, TU1810 Split Beam) for standardization (0.8–1.0). The sludge samples were washed with distilled water and stained with Nile red (25 μmol L−1 in DMSO). In the case of flow cytometry, the results were based on the analysis of 30,000 events. For spectrofluorimetry, an excitation wavelength at 550 nm and emission wavelength between 530 and 650 nm of fluorescence intensity were used (Pozo et al. 2011).

2.4 Analytical Methods

3 Results and Discussion

Physicochemical parameters like chemical oxygen demand (COD), biological oxygen demand (BOD5), VSS, total nitrogen (TN), total phosphorus (TP), and sludge volume index (SVI) were measured following Standard Method (APHA-AWWA-WEF 1998). Total phenolic compounds were measured in liquid samples by spectrophotometry at a wavelength of 215 nm (UV215) (quartz cuvettes 1 × 1 cm). Volatile fatty acids (VFA) were measured by gas chromatography (Hewlett Packard Model

3.1 Raw Wastewater Characteristics Table 2 shows the physicochemical characterization of raw wastewater from the PGW and TMP pulp processes. PGW wastewater shows average values of organic matter of 839.00 ± 12.80 and 441.03 ± 26.55 mg L−1 for COD and BOD5, respectively. The TMP influent had COD and BOD 5 concentrations of 2.97 ± 0.62 and 1.67 ± 0.39 g L−1, respectively. Total phenolic compounds were

Table 2 Physicochemical characterization of the raw wastewater Parameter

Unit

PGW influent

pH −1

TMP influent

Range

Averagea

Range

Averagea

6.33–7.67

6.83 ± 0.60

6.46–7.04

6.77 ± 0.22

COD

mg L

825.21–857.40

839.00 ± 12.80

2190.00–3380.00

2970.00 ± 620.00

BOD5

mg L−1

405.02–484.11

441.03 ± 26.55

1600.00–2160.00

1670.00 ± 395.00

Total nitrogen

mg L−1

0.50–0.60

0.53 ± 0.05

9.52–10.00

9.76 ± 0.34

Total phosphate

mg L−1

0.80–1.41

1.01 ± 0.21

5.40–5.50

5.45 ± 0.07

Total phenolic compounds

g L−1

0.80–2.36

1.75 ± 0.74

7.12–10.67

8.89 ± 1.16

Color

Abs

0.02–0.06

0.05 ± 0.01

0.50–0.51

0.51 ± 0.01

Acetic acid

mg L−1

20.00–45.10

34.83 ± 9.72

ND

ND

Propionic acid

mg L−1

30.21–40.40

35.01 ± 5.47

ND

ND

Butiric acid

mg L−1

10.02–20.10

16.66 ± 5.16

ND

ND

ND not determined a

Values of six average determinations

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PGW wastewater were 34.83, 35.01, and 16.16 mg L−1 for acetic, propionic, and butyric acids, respectively.

higher in TMP than in PGW wastewater (8.89 versus 1.75 mg L−1). The kraft mill wastewater reported total phenolic compounds ranging between 240.0 and 720.0 mg L−1 (Jarpa et al. 2012; Chamorro et al. 2010). The BOD5/COD ratios were 0.52 and 0.56 for PGW and TMP, indicating that both wastewaters are highly biodegradable. Previous studies of kraft mill wastewater have reported BOD5/COD ratios between 0.20 and 0.33 due to the high concentration of recalcitrant compounds present as aromatic compounds with high molecular weight (>10,000 Da) (Chamorro et al. 2010; Vidal et al. 2001). The concentration of nutrients of TN and TP obtained for PGW wastewater was 0.53 and 1.01 mg L−1, respectively, while TMP wastewater presented concentrations of 9.76 and 5.45 mg L−1 for TN and TP, respectively. The BOD5/N/P ratio of 100:5:1 was lower than that recommended by Diez et al. (2002) for optimal organic matter removal. Finally, volatile fatty acid concentrations in

Figure 1 shows PHA biosynthesis measured as fluorescence intensity for different OLRs and BOD5/N/P ratios for TMP influent. Gorenflo et al. (1999) and Wu et al. (2003) found correlations between fluorescence intensity and PHA biosynthesis, because the former indicates the presence of PHA in the cells. Figure 1a shows that the PHA biosynthesis from TMP influent in phase I was, on average, 64.14 Abs when the MBBR reactor was operated with an OLR of 2.36 kg BOD5 m−3 day−1 and a nutrient ratio of 100:5:1. These values decreased to 42.99 when the BOD5/N/P ratio decreased to 100:1:0.3. On the other hand, for phase II, the PHA biosynthesis was higher than the phase mentioned before and the PHA increased an average of 113.21 Abs,

Fluorescence intensity (absorbance)

BOD5 :N: P

100:5:1

100:1:0.3

220

(a)

200 180 160 140 120 100 80 60 40 20 0 100

PHA accumulating bacterial cell (%)

Fig. 1 PHA biosynthesis from TMP influent by MBBR reactor. ) and phase II Phase I ( ). a Fluorescence intensity ( (absorbance) and b PHAaccumulating bacteria (%)

3.2 PHA Biosynthesis

(b)

90 80 70 60 50 40 30

0.5

1.0

1.5

2.0

OLR (kg BOD5 m-3 d-1)

2.5

3.0

3.0

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when the OLR was 0.81 kg BOD5 m−3 day−1 and the nutrient ratio was 100:5:1. However, PHA biosynthesis decreased to 30.15 Abs when the OLR was increased to 2.83 kg BOD5 m−3 day−1 and the nutrient ratio decreased to 100:1:0.3. On the other hand, Chua et al. (2003) noted that there have been few studies of operational conditions such as C/N ratio, pH, dissolved oxygen concentration, and substrate concentration in PHA biosynthesis. On the other hand, Pozo et al. (2011) increased PHA biosynthesis from 66.90 to 271.00 Abs for the treatment of kraft mill effluents with inoculum from paper and kraft mills, respectively, by decreasing the C/N/P ratio in batch systems from 100:5:1 to 100:1:0.2. Chua et al. (2003) concluded that pH control is not critical in PHA biosynthesis, but pH can affect wastewater treatment. Additionally, under anaerobic conditions, carbon substrates and PHA are synthesized by polyphosphate-accumulating organisms. On the other hand, under aerobic conditions, microorganisms use the stored PHA for growing and maintenance. Finally, PHA biosynthesis was confirmed by the percentage of PHAaccumulating cells under different OLRs and BOD5/N/P ratios, as shown in Fig. 1b. During phase I, a maximum value of PHA-accumulating cells (85.1 %) was achieved under an OLR of 2.99 kg BOD5 m−3 day−1 and a nutrient ratio of 100:5:1. This percentage decreased to 78.75 % when the BOD5/N/P ratio was reduced to 100:1:0.3. This behavior is due to flocculent sludge characteristics (35–

3.3 Biomass Evolution Figure 2 shows the behavior of the biomass by the SVI in the MBBR under different F M−1 conditions. The optimal settling sludge conditions measured by SVI are

-3

kg BOD5 m .d-1

0.13

0.56

2.36

2.99

2.99

0.81

2.67

2.83

250 225 200 175 SVI (mL g-1 VSS)

Fig. 2 Biomass behavior. SVI and F M−1 at OLR (kg BOD5 m−3 day−1) 0.13 (■), 0.56 (●), 2.36 (▲), 2.99 (▼), 2.99 (♦), 0.81 (◁), 2.67 (▷), and 2.83 ( )

135 mL g−1 VSS and 0.3–0.6 g BOD5 g−1 VSS day−1) (Xavier et al. 2009). In phase II, maximum values of PHAaccumulating cells were obtained under operational conditions of an OLR of 2.83 kg BOD5 m−3 day−1 and a nutrient ratio of 100:1:0.3 with 89.41 %, obtaining similar values for different OLRs and nutrient ratios, specifically 88.11 % for 0.81 kg BOD5 m−3 day−1 and 85.09 % for 2.67 kg BOD5 m−3 day−1 for a nutrient ratio of 100:5:1. The percentage of PHA-accumulating cells in phase II at different OLRs was stable with respect to VSS concentrations (2.63–3.02 g VSS L−1). Bengtsson et al. (2008) found PHA production of 48 % of dry weight of sludge in three different stages (acidogenic fermentation, batch systems, and activated sludge) under a COD/N/P ratio of the 100:0.030:0.001, while Yan et al. (2008) found PHA biosynthesis between 17.0 and 43.1 % of dry weight of sludge by batch experiments, in both cases with paper mill wastewater. However, Pozo et al. (2011) found PHA biosynthesis levels of 25.7 and 30.4 % under BOD5/N/P ratios of 100:5:1 and 100:1:0.2, respectively, in kraft mill wastewater.

150 125 100 75 50 25 0 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

-1 -1 F M ( g BOD5 g VSS. d )

-1

0.8

0.9

1.0

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in the range of 35–135 mL g−1 VSS under F M−1 conditions between 0.3 and 0.6 g BOD5 g−1 VSS day−1 (Xavier et al. 2009). During phase I, the sludge sedimentation was low when the OLR was ranged between 0.13 and 0.56 kg BOD5 m−3 day−1, with average values of 167.54 and 71.72 mL g−1 VSS, respectively, for SVI; meanwhile, the F M−1 was 0.13 and 0.15 g BOD5 g−1 VSS day−1, respectively. During this operation, part of the biomass was washed out by MBBR. By increasing the OLR from 2.36 to 2.99 kg BOD5 m−3 day−1, the flocculent sludge characteristics were obtained with average values between 94.59–113.83 mL g−1 VSS and 0.32–0.37 g BOD5 g−1 VSS day−1 for SVI and F M−1, respectively. In phase II, the behavior of the sludge was flocculent under OLR 0.81 and 2.67 kg BOD5 m−3 day−1. The average SVI values were 97.30–122.68 mL g−1 VSS and 0.57–0.59 g BOD5 g−1 VSS day−1. However, a proliferation of filamentous bacteria began to appear when the OLR was increased to 2.83 kg BOD5 m−3 day−1 with a SVI between 17.85 and 145.98 mL g−1 VSS and F M−1 of 0.69 g BOD5 g−1 VSS day−1, generating bulking in the MBBR. Jarpa et al. (2012) and Bengtsson et al. (2008) indicate that PHA accumulation is associated with flocculent sludge. On the other hand, Xavier et al. (2009) obtained flocculent biomass characteristics with an OLR of 0.14–1.09 kg BOD5 m−3 day−1 and nutrient ratios of 100:5:1 and 100:1:1; meanwhile, Pozo et al. (2011)

3.4 Organic Matter Removal Efficiency Organic matter removal efficiency was determined for MBBR systems operating at different OLRs and BOD5/N/P ratios. Figure 3 shows BOD5 removal efficiency during phases I and II. Phase I obtained a maximum BOD5 efficiency of 95.60 % under an OLR of 0.56 kg BOD5 m−3 day−1 and a nutrient ratio of 100:5:1. BOD 5 efficiency decreased to 87.2 % when OLR was increased to 2.99 kg BOD5 m−3 day−1 and the nutrient ratio decreased to 100:1:0.3. During phase II, maximum efficiency of BOD5 removal (97.10 %) was obtained at an OLR of 0.81 kg BOD5 m−3 day−1 and a BOD5/N/P ratio of 100:5:1. However, BOD5 removal dropped to 84.6 % when the MBBR was operated at an OLR of 2.83 kg BOD5 m−3 day−1 and a nutrient ratio of 100:1:0.3. Chamorro et al. (2010) obtained similar vBOD5 removal values in kraft mill wastewater treated in a MBBR (75–95 %) with 0.07–1.19 kg BOD5 m−3 day−1. COD performance and total phenolic compounds were also evaluated (Fig. 4). Maximum COD removal

100:5:1

BOD5:N:P

)

100:1:0.3

100

95

BOD5 removal (%)

Fig. 3 MBBR performance. BOD5 removal. Phase I ( ) and phase II (

obtained these optimal sludge characteristics at an OLR of 1.24 kg BOD5 m−3 day−1 and nutrient ratios of 100:5:1 and 100:1:0.2.

90

85

80

75

70 0.5

1.0

1.5

2.0

2.5 -

OLR (kg BOD 5 m 3

-1

)

3.0

3.0

Water Air Soil Pollut (2016) 227:299 BOD 5 : N: P -3

Page 7 of 8 299 100:1:0.3

100:5:1

-1

OLR (kg BOD5 5m/m3dd))

0.56

0.13

2.36

2.99

2.99

4 Conclusions

100

(a)

90 80 70

COD, Total phenolic compounds removal (%

60 50 40 30 20

10 0 0

50

100

150

200

250

300

100:1:0.3

100:5:1 2.67

0.81

2.83

100

(b)

90 80

The maximum percentage of PHA-accumulating cells was 85.10 % under OLR 2.99 kg BOD5 m−3 day−1 and BOD5/N/P ratio of 100:5:1 from the PGW influent. On the other hand, the maximum BOD5 removal by MBBR was of 95.60 % at OLR 0.56 kg BOD5 m−3 day−1 and a nutrient ratio of 100:5:1. However, in the case of the TMP influent, the maximum percentage of PHAaccumulating cells was 89.41 % for 2.83 kg BOD5 m−3 day−1 and a BOD5/N/P ratio of 100:1:0.3, decreasing BOD5 efficiency to 84.6 % and affecting sludge characteristics for these operating conditions. These results show that PHA biosynthesis is possible for PGW and TMP influents used as substrates.

70

Acknowledgments This work was supported by FONDECYT 1120664 and CONICYT/FONDAP 15130015 grants.

60 50 40 30 20

References

10 0

0

20

40

60

80

100

120

140

160

Time (d)

Fig. 4 MBBR performance. a Phase I. b Phase II. COD (■) and total phenolic compound (UV215 nm) (□) removal

during phase I was 64.92 % at an OLR of 2.99 kg BOD5 m−3 day−1 and a BOD5/N/P ratio of 100:1:0.3, while for total phenolic compounds, it was 41.01 (Fig. 4a). In phase II, maximum COD removal was 76.5 % at an OLR of 0.81 kg BOD5 m−3 day−1 and a nutrient ratio of 100:5:1. Total phenolic compound removal efficiency was 52.77 % at an OLR of 0.81 kg BOD5 m−3 day−1 and a nutrient ratio of 100:5:1. Pozo et al. (2012) indicated that MBBRs are highly efficient at removing COD (24–82 %). Xavier et al. (2009) achieved over 45 % removal of total phenolic compounds. These results are in accord with Odegaard (2006), who found that COD removal efficiency decreases when HRT decreases. Furthermore, reduced organic removal efficiency is also due to the decrease in the BOD5/N/P ratio from 100:5:1 to 100:1:0.3. Diez et al. (2002) found that a low nutrient ratio (100:2.5:0.5) affects microorganism growth. Moreover, limited nutrient conditions cause bulking in the reactor. Several authors have shown that the origin of recalcitrant COD is the molecular weight fraction of lignin above 1000 Da (Jarpa et al. 2012; Vidal et al. 2001).

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