Algae harvesting for biofuel production: Influences of UV irradiation and polyethylenimine (PEI) coating on bacterial biocoagulation

Bioresource Technology 166 (2014) 266–272

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Algae harvesting for biofuel production: Influences of UV irradiation and polyethylenimine (PEI) coating on bacterial biocoagulation Michael Agbakpe a,1, Shijian Ge a,1, Wen Zhang a,⇑, Xuezhi Zhang b, Patricia Kobylarz a a b

John A. Reif, Jr. Department of Civil and Environmental Engineering, New Jersey Institute of Technology, Newark, NJ 07102, United States Laboratory for Algae Research and Biotechnology, Department of Applied Sciences and Mathematics, Arizona State University, Mesa, AZ 85212, United States

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 PEI coating on bacteria dramatically

PEI-coated E. coli cells

Chlorella

4

UV365

3

OD 680

enhanced the algal harvesting efficiency.  UV365 irradiation could also improve the biocoagulation process.  Potential energy barrier exists between the interacting bacterial and algal cells.

2 1 0 0

10

20

30

40

50

Time elapsed (min) .

Uncoated E. coli under UV365 illumination at 1.6 mW cm

-2

Uncoated E. coli without UV365 illumination No addition of E. coli cells and no UV365 illumination .

PEI-coated E. coli under UV365 illumination at 1.6 mW cm

-2

PEI-coated E. coli without UV365 illumination

a r t i c l e

i n f o

Article history: Received 26 March 2014 Received in revised form 15 May 2014 Accepted 17 May 2014 Available online 23 May 2014 Keywords: Biocoagulation Algal harvesting Coagulation efficiency Biofuel DLVO

a b s t r a c t There is a pressing need to develop efficient and sustainable separation technologies to harvest algae for biofuel production. In this work, two bacterial species (Escherichia coli and Rhodococus sp.) were used as biocoagulants to harvest Chlorella zofingiensis and Scenedesmus dimorphus. The influences of UV irradiation and polyethylenimine (PEI)-coating on the algal harvesting efficiency were investigated. Results showed that the UV irradiation could slightly enhance bacteria–algae biocoagulation and algal harvesting efficiency. In contrast, the PEI-coated E. coli cells noticeably increased the harvesting efficiencies from 23% to 83% for S. dimorphus when compared to uncoated E. coli cells. Based on the soft-particle Derjaguin– Landau–Verwey–Overbeek (DLVO) theory, an energy barrier existed between uncoated E. coli cells and algal cells, whereas the PEI coating on E. coli cells eliminated the energy barrier, thereby the biocoagulation was significantly improved. Overall, this work presented groundwork toward the potential use of bacterial biomass for algal harvesting from water. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Biofuels have been proposed as a suitable sustainable replacement to fossil oil. Algal biomass is emerging as a renewable feedstock for biofuels and bioproducts due to their biotechnological advantages such as high productivity, use of nonarable land, growth ⇑ Corresponding author. Tel.: +1 (973) 596 5520; fax: +1 (973) 596 5790. E-mail address: [email protected] (W. Zhang). Michael Agbakpe and Shijian Ge contributed equally to this paper as co-first authors. 1

http://dx.doi.org/10.1016/j.biortech.2014.05.060 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

in wastewater, and ease of handling in liquid medium (Singh et al., 2014). Furthermore, efficient harvesting or removing algae from water is not only critical for biofuel production but also important for maintaining sanitary quality of drinking water treatment processes. Algal biomass harvesting cost, however, constitutes a significant part of the total biomass production cost owing to the small size of algal cells (typically 2–20 lm in diameter) and their low densities in growth media (e.g., 0.5–5 g-dry weightL1 growth medium) (Powell and Hill, 2013). Furthermore, algae removal from water is also critical for water treatment to cope with increasing deterioration water quality due to eutrophication. To make the

M. Agbakpe et al. / Bioresource Technology 166 (2014) 266–272

large-scale production economically viable, there is therefore a pressing need to develop low cost harvesting technology (Prochazkova et al., 2013). Most conventional methods for algal harvesting such as sedimentation by gravity, centrifugation, microstraining, coagulation, chemical precipitation, filtration and flotation are often energy- or time-consuming (Vandamme et al., 2013). For instance, centrifugation might be feasible for small scales but too expensive and energy-intensive for large-scale algal separation (Wijffels and Barbosa, 2010). Membrane filtration has also been considered for microalgae harvesting but needs to overcome the challenges in energy effectiveness and capital cost (Zhang et al., 2010). Chemical coagulation is a well-established and widely used method for algal removal. However, applications of high-dose synthetic chemical coagulants (e.g., aluminum and iron) in the harvesting processes may lead to negative impacts on downstream biofuel extraction and refinery processes. Sedimentation appears to be a relatively simple process where algal cells are concentrated by gravity. However, since sedimentation relies on precipitation of algal cells, large-sized algae would be collected more efficiently than small cells. Moreover, microalgae have densities comparable to water and commonly form stable suspensions due to their negative surface charge, which makes coagulation and precipitation of algae difficult. The US DOE has identified the cost-effective harvesting of algae as one of the ‘‘roadblocks’’ to economical biofuel production from algal biomass culture. Biocoagulation or bioflocculation/bioaggregation, in recent studies, have been successfully applied in removing algae in wastewater treatment and these bio-processes also present high potential as a green and chemical-free harvesting method for algae biofuel production (Manheim and Nelson, 2013). However, the underlying mechanisms of biocoagulation are not yet well understood and deserve further research. Some algae species coagulate more readily than others and such naturally bioflocculating algae can be mixed with other species to induce coagulation and flocculation (Taylor et al., 2012). Furthermore, bacteria or fungi can also induce bioflocculation of algae. Some fungi, for instance, have positively charged hyphae that can interact with the negatively charged algal cell surface and cause flocculation (Zhang and Hu, 2012). Specific consortia of bacteria can also induce flocculation of algae (Lee et al., 2009), and several bacteria have been identified as biocoagulatoin agents that can be used to aggregate algae (Nontembiso et al., 2011). Recent studies suggest that microbes are a necessary factor in the process of algal aggregation (Grossart et al., 2006). These coagulating or flocculating fungi or bacteria can be cultivated separately or in combination with the algae. Due to the rapid and inexpensive cultivation, bacterial biomass may be one of the potential biocoagulation and flocculation agents (Wang et al., 2012), which avoid the use of synthetic chemical coagulants and may enable the reuse of algae growth media. Nevertheless, bacteria and algae both have negative surface charge, which results in repulsive interactions and makes their biocoagulation less efficient. A few recent studies have investigated the use of positively charged (cationic) polymeric coagulants for algal harvesting (Milledge and Heaven, 2013; Vandamme et al., 2013). Polymer coagulants or flocculants reduce or neutralize the negative surface charge on cells and they can also bring particles together by physically linking one or more particles through a process called bridging. Cationic polymers doses of between 1 and 10 mg ml1 can induce flocculation of freshwater algae (Molina Grima et al., 2003). Thus, bacterial surface modification is important for improving the bioflocculation processes. The common modification is to coat bacteria with strong cationic electrolytes or polymers such as polyamine, poly(dimethyldiallylammonium chloride) (PDMAC) or poly(ethyleneimine) (PEI) . Thus far the pertinent research on surface modified bacteria as coagulant for harvesting algae is quite limited. Also, the biocoagulation efficiency using positively charged

267

bacteria toward algal harvesting is likely influenced by many operational parameters such as the dose of bacterial agents, surface coating, and light conditions (Kolappan and Satheesh, 2011). The specific objectives of this work are (i) to investigate the effects of bacteria cells coated with cationic polymer PEI on bacteria–algae aggregation and algal recovery efficiency, (ii) to study roles of UV irradiation in the process of bacteria–algae biocoagulation; and (iii) to explore the thermodynamic interactions between algal and bacteria cells using the soft-particle Derjaguin–Landau–Verwey–Overbeek (DLVO) theories. 2. Methods 2.1. Algae and bacteria cultures Two genera of fresh water algae and two species of bacteria were cultivated and used in this study. The two genera of freshwater algae inoculum of Chlorella zofingiensis (C. zofingiensis) and Scenedesmus dimorphus (S. dimorphus), were obtained from Laboratory for Algae Research and Biotechnology at Arizona State University. Both algae species were cultivated in the modified Bold’s Basal Medium (BBM) in 1000 ml flasks sparged with 5% CO2 at a loading rate of 8.5  104 L-CO2 min1(L-medium)1. The cultivation included a light–dark cycle (12 h/12 h) with a light intensity of about 1200 lux (approximately 27.4 lmoles m2 s1 or 4200 mWatt m2). The algae were cultivated at room temperature (26 ± 3 °C). During the growth period of 14 days, both culture were kept at the same pH of 7 ± 1 and dissolved oxygen (DO) of 18 ± 2 mg L1. The oxidation reduction potential (ORP) of 206 ± 37 mV was recorded for C. zofingiensis, and 170 ± 31 mV was recorded for S. dimorphus. The maximum algal concentrations were approximately 1.2 and 1.8 g L1 for C. zofingiensis and S. dimorphus, respectively in the medium suspension, which was directly used in biocoagulation experiments. Two bacteria species Escherichia coli (E. coli) K12 cells (strain 21) and Rhodococus sp. were purchased from E. coli Genetic Stock Center (Department of Biology, Yale University, New Haven, CT). The bacteria were cultured overnight in a Luria–Bertani (LB) broth medium in an incubator (Thermo Scientific, MAXQ 4450) at 37 °C. The mean cell concentrations of E. coli and Rhodococus sp. after overnight incubation were determined to be 968 ± 42 and 824 ± 23 mg L1, respectively. Bacteria were freshly prepared for subsequent surface coating or coagulation experiment. Bacterial biomass was rinsed by phosphate buffer saline (PBS) to remove the residual medium constituents and concentrated to approximately 23 g L1 with centrifugation (3000g for 5 min) before use. More details about algal and bacterial cultivation are provided in Section S1 of the Supporting Information (SI). 2.2. Characterization Algal and bacteria size distribution were determined with Multisizer 3 Coulter Counter instrument (Beckman Coulter). Hydrodynamic diameters and the bacteria size distribution was characterized by the dynamic light scattering (DLS) technique performed with a Malvern Instruments Zetasizer Nano ZS. The zeta potentials of algae and bacteria were also measured with the same DLS instrument using the folded capillary cell (DTS1060, Malvern Instruments). The morphology and aggregation of bacteria and algae were imaged and examined using a light microscope (Eclipse Ti, Nikon Instruments Inc.). 2.3. Algae–bacteria biocoagulation experiments 2.3.1. Concentration effects of uncoated bacteria The algae–bacteria biocoagulation was performed in 300-ml glass beakers as detailed in Section S2. Algal suspension was

M. Agbakpe et al. / Bioresource Technology 166 (2014) 266–272

thoroughly mixed with bacterial cells with a magnetic stirrer at a speed 250 rpm for 5 min and natural settling afterwards. The bacterial dose effect on the harvesting efficiency of algae cells was investigated without UV irradiation with E. coli and Rhodococus sp. used as biocoagulants. The bacterial dose was expressed as the bacteria–algae mass ratios. The ratios ranged from 0, 22 ± 1, 42 ± 1 and 212 ± 4 mg g1 for C. zofingiensis and 0, 14 ± 1, 28 ± 1, 132 ± 3 mg g1 for S. dimorphus, respectively. Time resolved OD680 of the supernatant were measured by triplicate sampling of 1.5 ml at an approximate depth of 1 cm below surface of the liquid. 2.3.2. UV effects with uncoated bacteria As shown in Fig. S2a, the UV365 irradiation was provided by placing a UV lamp on top of the suspension. Different UV intensities were obtained by varying the distance between the UV lamp and the beaker. Control experiments were performed (1) with E. coli addition but no UV irradiation and (2) without E. coli addition and UV irradiation. The OD680, ORP, DO, and pH were all simultaneously monitored and recorded every 5 min throughout the experimental period. 2.3.3. Biocoagulation with PEI-coated bacteria with and without UV 50% PEI (wt.%, equivalent to approximately 500 g L1) purchased from Sigma–Aldrich was diluted to 0.1–10% (wt.%) in DI water with pH adjusted to 7.0. The PEI coating on bacteria was obtained by mixing different amounts of PEI into the E. coli suspension at different mass ratios (g-E. coli g1-PEI) ranging from 1:10 to 1:1000. The mixing was stabilized for 10–25 min. The PEI coating on E. coli cells was instantly indicated by the zeta potential changes of the coated bacteria, measured by DLS. After the optimum PEI coating concentration was determined, the mixture was centrifuged and washed with DI water three times to remove any remaining PEI. The experiments about PEI coating effects on the biocoagulation efficiencies were conducted under the same above UV irradiation and other experimental conditions. As a control, only PEI and algae mixture without E. coli cells and UV irradiation was also prepared and studied. 2.4. Harvesting efficiency evaluation Three indicators were used to evaluate the biocoagulation performance. These indicators are the algal harvesting efficiency (HE), recovery efficiency (RE) and recovery capacity (RC, g-DCW.g-bacteria1), which were calculated as follows (Hu et al., 2013):

ðC 0  C t ÞV RCðg DCW  g bacteria Þ ¼ m   C t =C 0 REð%Þ ¼ 1   100% C 0t =C 00 1

ð1Þ ð2Þ

U vdw ¼

  A132 a1 a2 1 6hða1 þ a2 Þ 1 þ 11:12h=kc

ð4Þ

The electrostatic interaction energy is calculated using the linearized version of the Poisson–Boltzmann expression:

" #   2pa1 a2 nkB T 2 2U1 U2 1 þ ejh 2 2jh þ lnð1  e U EL ¼ ðU1 þ U2 Þ 2 ln Þ ða1 þ a2 Þj2 1  ejh U1 þ U22 ð5Þ where all the equation parameters are summarized in Table S1. 2.6. Statistical analysis All experiments were carried out at room temperature of 25 ± 3 °C with triplicate sampling and testing. The presented results are mean values ± standard deviation from three independent experiments. The differences in harvesting efficiencies (HE) between test groups and the control group were tested for significance using one way analysis of variance (ANOVA) at a significant level of 0.05. 3. Results and discussion 3.1. Characterization of bacteria and algae cells 3.1.1. Growth kinetics of algae Algal growth kinetics indicated by OD680 together with nutrient changes is presented in Fig. 1. The algal concentrations increased approximately from an initial 0.2–0.8 g L1 and from 1 to 1.5 g L1 at steady state for C. zofingiensis and S. dimorphus, respectively. 1 The NO to 0.26 ± 3 -N concentration dropped from 14 ± 2 mg L 0.05 mg L1 for both cases. The cell density increased from 2.50  106 to 2.21  108 cell mL1 for C. zofingiensis, and 1.15  106 to 3.81  107 for S. dimorphus. The growth kinetics and nutrient assimilation are comparable to other studies (Zhang et al., 2012). In addition, the hydrodynamic diameters were 3.1 ± 0.1 lm and 2.0 ± 0.5 lm for E. coli and Rhodococus sp., respectively. The average algal length and width, as measured by the optical microscope (n = 50), were 5.3 ± 1.0 lm and 4.1 ± 0.6 lm respectively for C. zofingiensis and 7.6 ± 1.7 lm and 3.7 ± 0.9 lm for S. dimorphus. The algal cell size distributions for bacteria and algae cells are presented in the Section S5 of SI (Fig. S3).

ð3Þ

where C0 and Ct are the concentration of algal cells before and after 0 0 harvesting (g L1), C0 and Ct are the concentration of algal cells without bacteria in control group (g L1), V is the volume of cell suspension and m is the mass of bacteria cells used for harvesting (g). 2.5. Colloidal interaction analysis Quantitative information on the nonspecific interaction forces between bacteria and algae cells can be directly obtained with the Ohshima’s soft-particle DLVO theory assuming that Lifshitz-van der Waals and electrostatic forces are the dominant forces (Ohshima, 1995). The computation methods for the van der Waals and electrostatic forces vary with the geometry of the interacting entities. Because bacterial cells usually are 1.5–2 lm

3

14 10

2

6 1

2

0

NO3- as N (mg.L-1)

HEð%Þ ¼ 1  ðC t =C 0 Þ  100%

in length and 0.5 lm in width, while algal cells are approximately 2–10 lm in length and 2–5 lm in width , their interactions can be modeled as particle–particle geometry. The retarded Lifshitz-van der Waals interaction energy for sphere–sphere geometry is calculated as when h < kc/4p and h < ai (Schenkel and Kitchener, 1960):

OD680

268

-2 0

4

8

12

16

Time (d) OD680 -C. zofingiensis NO3- - C. zofingiensis

OD680 – S. dimorphus NO3- - S. dimorphus

Fig. 1. The evolution of OD680 and NO 3 -N of the cultivated algae with time.

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Zeta Potential (mV)

10 0 -10 -20 -30 -40 -50 -60 1

3

5

7

9

pH E. coli Chlorella zofingiensis

Rhodococus sp. Scenedesmus dimorphus

Fig. 2. Zeta potentials of bacteria and algae in DI water at different pHs.

3.1.2. Zeta potentials of bacteria and algae As shown in Fig. 2, both algae and bacteria possessed negative surface charges at a wide pH range, which could be attributed to the presence of carboxylic, phosphoric, phosphodiester, hydroxyl and amine functional groups (Burnett et al., 2006; Hadjoudja et al., 2010). The zeta potentials at a pH of 7 ± 1 of the two algae, C. zofingiensis, S. dimorphus, were 15.6 ± 1.4 mV and 14.4 ± 1.2 mV respectively while those of the bacteria, E. coli and Rhodococus sp. in the algae medium were 4.2 ± 0.9 mV and 4.8 ± 0.4 mV respectively. In addition, the zeta potentials of bacteria and algae (in NaCl and CaCl2 electrolytes at a pH of approximately 7.6) became less negative with increasing ionic strength due to the compression of electrical double layer (Fig. S4). 3.2. UV irradiation effects and mechanisms Concentration effects of uncoated bacteria on biocoagulation and sedimentation of algae suspension without UV irradiation were investigated. As apparent in Fig. S5, the bacteria–algae biocoagulation at different bacterial additions did not significantly increase the algal sedimentation. Thus, the bacteria/algae mass ratio of 21 mg g1 was chosen throughout the following biocoagulation experiments. Fig. 3 indicates the changes of OD680 in two algae suspensions mixed with uncoated E. coli with and without UV irradiation. Compared with the control groups, OD680 of samples with UV irradiation at 1.6 mW cm2 decreased relatively faster for both algal species. Particularly, the sedimentation of algae cells appeared to be faster than that of samples without the UV irradiation. In addition, the OD680 of the bacterial or algal suspensions alone were observed to be stable under the same UV irradiation (results not shown), indicating that UV irradiation did not induce significant biocoagulation of bacterial or algal cells.

Table 1 summarize the calculated HE, RE and RC for different scenarios. For example, for S. dimorphus, the HE increased from 19 ± 2% without UV irradiation to 23 ± 2% with UV irradiation. Similar patterns were observed for RE and RC indicators, which both demonstrated that UV irradiation could result in pronounced biocoagulation and thus increase the algal harvesting efficiency. Similarly, both the HE and RE of C. zofingiensis were found to increase in the presence of UV irradiation by approximately 15–20%. However, the increase for S. dimorphus was not significant. Furthermore, the values of ORP, DO and pH in suspensions did not change notably throughout the experimental period (Section 5 in SI). The potential influence of UV irradiation on the bacteria–algae biocoagulation efficiencies could arise from the fact that UV irradiation can alter the surface characteristics of microorganisms. For instance, the UV irradiation could alter the hydrophobicity of the bacterial surface, which became more hydrophilic with UV treatment (Li and Logan, 2005). Thus, the interaction between bacteria and algae might be more facilitated by the possible reduction of the hydrophobic repulsion. Moreover, UV irradiation oxidizes the surface polymers of the bacteria and probably leads to the loss of outer layer structures (e.g., flagella and pili) of the cell surface and thus affects the steric interaction between bacteria and algae (Li and Logan, 2005). Finally, bacteria could be inactivated by UV irradiation as a result of photochemical damage to their nucleic acids and consequently the normal cell functions or reproduction would be negatively affected. For example, the amount of carbohydrate and protein in the EPS synthesized by the bacteria under UV irradiation was found much less than that of the control (Kolappan and Satheesh, 2011). The impacts on surface components such as EPS may influence the bacteria–algae interaction and biocoagulation. Similarly, UV irradiation may have also affected algal surface characteristics (Tao et al., 2010). Ou et al. (2011) demonstrated that UV-C could effectively damage the Microcystis aeruginosa (FACHB-912) cells, most likely via a 3-step procedure, including impairment of photosynthesis system, decomposition of cytoplasmic inclusions, and cell cytoclasis. Thus, considerable decrease of algal cell integrity (e.g., membrane disintegration) would be highly possible in the presence of UV irradiation, although it was not examined in current study. There were no significant differences in the OD680 between the UV irradiated culture without biocoagulation and the control, indicating that the changing in algae surface by UV irradiation was not obvious under the experiment conditions. 3.3. PEI coating effects on algal harvesting efficiency 3.3.1. Biocoagulation with PEI-coated bacteria with and without UV irradiation Biocoagulation depends largely on the surface properties of bacteria or algal cell surfaces (Vandamme et al., 2013). From the 4.0

(a)

4

(b)

3.5

OD680

OD680

3 2

3.0 2.5

1

2.0

0 0

10

20

30

40

50

0

No UV with E. coli

10

20

30

40

50

Time elapsed (min)

Time elapsed (min) 1.6 mW.cm-2 UV365 with E. coli

No UV and no E. coli

Fig. 3. Change in optical density of (a) C. zofingiensis and (b) S. dimorphus suspensions (the initial algal concentrations were 1.2 and 1.8 g L1 respectively) after mixing with uncoated E. coli with and without exposure to UV irradiation compared to that of algae suspension without any treatment.

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Table 1 Comparisons of computed HE, RE and RC of algae under different conditions. Algae

Condition

HE (%)

RE (%)

RC (g-algae g-bacteria1)

p-value

S. dimorphus

Control group (only algae) Algae with only PEI and without UV Uncoated E. coli mixed with algae without UV Uncoated E. coli mixed with algae with UV PEI-coated E. coli mixed with algae without UV PEI-coated E. coli mixed with algae with UV irradiation

12 ± 2 42 ± 2 19 ± 2 23 ± 2 81 ± 1 83 ± 1

– 35 ± 3 8±1 12 ± 1 78 ± 1 80 ± 1

– 34 ± 2 15 ± 2 18 ± 2 68 ± 4 70 ± 4

– 0.017 0.011 0.007 3  106 5  106

C.zofingiensis

Control group (only algae) Algae with only PEI and without UV Uncoated E. coli mixed with algae without UV Uncoated E. coli mixed with algae with UV PEI-coated E. coli mixed with algae without UV PEI-coated E. coli mixed with algae with UV irradiation

36 ± 3 38 ± 1 55 ± 4 56 ± 5 64 ± 1 70 ± 1

– 7±1 30 ± 8 31 ± 9 44 ± 3 53 ± 3

– 21 ± 1 27 ± 5 28 ± 5 35 ± 1 38 ± 1

– 0.261 0.075 0.083 0.003 0.001



HE, RE and RC denoted the algal harvesting efficiency, recovery efficiency and recovery capacity, respectively. When P values are less than 0.05, the results of the test groups are statistically different from those of the control groups. Apparently, the improved harvesting efficiencies for algae mixed with only PEI and uncoated E. coli cells with or without UV were not statistically different from the control groups, whereas other experimental groups led to significant improvement as evidenced by the P values of less than 0.05. 

(a)

4

3

OD680

OD680

(b)

4

3 2 1

2 1

0

0 0

10

20

30

40

50

Time elapsed (min) No UV with PEI-coated E. coli 1.6 mW.cm-2 UV365 with PEI-coated E. coli

0

10

20

30

40

50

Time elapsed (min)

No UV, no PEI and no E. coli With PEI only (PEI 0.1% w/w)

Fig. 4. Change in optical density of (a) C. zofingiensis at the initial concentration of 1.2 g L1 and (b) S. dimorphus at the initial concentration of 1.8 g L1 after mixing with PEIcoated E. coli at the mass ratio of 21 mg-E. colig-algae1 with/without exposure to UV irradiation compared to that of algae suspension without any treatment.

experimental results above, it became clear that the uncoated bacterial or algal cells, due to their negative surface charges, have strong electrostatic repulsion that greatly hampered the bacterial attachment to algae. Therefore, in studying the coating effect, E. coli cells were used as the model bacteria for the cationic polymer coating with PEI to modify the surface charge and improve the biocoagulation efficiencies. E. coli cells were only used here, because once coated with PEI, bacteria will primarily act as positively charged particles. The different bacterial species would behave similarly with respect to bacteria–algae interactions. With PEI coating, zeta potential of E. coli cells increased sharply from 16.9 ± 0.3 mV to 7.9–22.6 mV in the first 5 min at all PEI concentrations and afterwards leveled off in the following 25 min (Fig. S6a). The positive shift in zeta potential was almost linear with the exposed PEI concentration (Fig. S6b). The highest zeta potential (27.0 ± 1.2 mV) was observed at 10% (w/w) PEI with 25min exposure, which was adopted in the subsequent experiments. Fig. 4 indicates the changes of OD680 in two algae suspensions mixed with 10% PEI-coated E. coli with or without UV irradiation. In the first 10 min, the sedimentation of the coagulated algae– bacteria mixture was faster than that in the subsequent time. It was worth noting that, after PEI coating, E. coli cells demonstrated enhanced biocoagulation performance as the HE, RE and RC all increased by almost 46 times (Table 1), especially for S. dimorphus. Particularly, the HE increased significantly to approximately 81 ± 1% even without UV irradiation; the RC was also greatly improved by more than three folds. By contrast, the coagulation was not dramatically improved with addition of PEI only into the algal suspension at a PEI dose of up to 0.1% (w/w) (Fig. 4), indicative of the critical roles (e.g., increasing available surface areas for interactions) of bacteria biomass in this PEI-facilitated

biocoagulation process. The PEI dose of 0.1% (w/w) was chosen because 0.1% was the relevant PEI concentration present in the mixture when applying PEI-coated E. coli cells. Similar positive effects on the algae sedimentation rates and harvesting efficiencies were observed with bacteria as biocoagulants (Grossart et al., 2006; Oh et al., 2001). For example, Paenibacillus sp. AM49 effectively coagulated with Chlorella vulgaris from large-scale cultures (Oh et al., 2001). In addition, the UV irradiation promoted the biocoagulation and sedimentation slightly (the HE increased from 81% to 83% in Table 1). These observations again confirmed that the algae harvesting rates and efficiency with bacterial coagulants improves upon UV irradiation. 3.3.2. Interaction energy between PEI-coated bacteria and algae using soft-particle DLVO theory Examination of the surface interaction characteristics via DLVO theories would allow us to obtain a better understanding of biocoagulation processes and how physicochemical properties of interacting particles could facilitate the biocoagulation processes. In this work, the enhanced biocoagulation of PEI-coated E. coli cells toward algal cells was clearly attributed to the electrostatic attraction due to the positively charged E. coli cells and negatively charged algal cells. This notion was verified by the comparison of the total interaction energies (Utotal = Uvdw + UEL) for the uncoated and coated E. coli cells using the soft-particle DLVO as shown Fig. 5. Clearly, without the PEI coating on bacterial surface, a great energy barrier existed preventing or lowering the biocoagulation process, whereas with the PEI coating the energy barrier diminished or was completely removed, meaning that the biocoagulation between E. coli cells and algal cells are thermodynamically favorable. Moreover, the interaction energies between the bare

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(a) 100000

(b)

Interaction energy (kBT)

50000 0 0

10

20

30

40

50

60

-50000 -100000 -150000 -200000

PEI-coated E.coli-Chlorella PEI-coated E.coli-Scenedesmus Uncoated E.coli-Chlorella Uncoated E.coli-Scenedesmus

Interaction distance (nm)

Fig. 5. (a) Comparison of interaction energies between PEI-coated or uncoated E. coli cells and algae in algal medium using soft-particle DLVO without considering the acid– base interaction energy; (b) Mixture images of C. zofingiensis with uncoated (up) and coated E. coli cells (down).

(uncoated) bacteria and algal cells alone was also assessed to better understand their colloidal stability (See Figs. S7 and S8). The soft-particle DLVO predictions correctly explains the experimental data presented previously, however, it stills suffers the inherent limitations from the classical DLVO theory. For example, the effects of surface roughness or surface structural on the interaction energy have not been considered by DLVO (Snoswell et al., 2005). In fact, the structural moieties on microbial cells such as bacterial pilus and fimbria do play significant roles in surface interactions and attachment. However, in the DLVO model, the interacting bacteria and algae are assumed to be molecularly smooth, which in reality is impossible. Another significant assumption is the shape, which is a perfect sphere for bacteria and algae in the model calculation. However, the actual shapes bacteria and algae vary significantly even for the same kind. The last but not the least, the DLVO theory is preferably used for monovalent salts at relatively low concentrations. In fact, there is a zoo of complex interfacial forces (also called non-DLVO forces) involved in many colloidal systems, to name a few, hydration force (Chang and Chang, 2002), hydrophobic force (Ong et al., 1999), and steric force (Rijnaarts et al., 1999). Extended DLVO or EDLVO theory that usually considers Lewis acid–base interactions in addition to the classical DLVO forces was found to lead to better predictions for many biocoagulation observations (Van Oss, 2006). Thus, the future work may incorporate the non-DLVO forces for the predictions of interaction energy. 3.4. Bacteria–algae biocoagulation mechanisms and implications for algal harvesting The successful biocoagulation between E. coli cells and two algae species could be explained by two plausible mechanisms: the direct attachment between bacteria and algae, and the bridging between the biopolymers such as carbohydrates and proteins excreted by bacteria and algae. Wang et al. (2012) demonstrated the remarkable ability of a bacteria strain (HW001) in aggregating and harvesting several algae such as Nannochloropsis oceanica IMET1, N. oceanica CT-1, Tetraselmis suecica, Tetraselmis chuii and the cyanobacterium Synechococcus WH8007. Scanning electron microscopy (SEM) further revealed the attachment between bacterial and algal cells, which was congruent with our microscopic observation. Another study also indicated that inside the aggregates of Nannochloropsis, algal cells were stuck together with bacteria and cell wall debris (Rodolfi et al., 2003). In this study, the direct interaction between E. coli and C. zofingiensis and S. dimorphus may have played

Bacteria

Polymer

Algae

Bridging

Patching

Fig. 6. Schematic view of the bridging and patching mechanisms involved in polymer induced biocoagulation.

the dominant role in the biocoagulation and the improved algal harvesting. Biopolymers or EPS bridge the microbial cells with the substratum and even permit negatively charged bacteria to adhere both negatively and positively charged surfaces. Particularly, adsorption of cationic polymers (carbohydrates and proteins) excreted by the microorganisms could also contribute to biocoagulation via two sub-mechanisms called bridging and patching (Salim et al., 2011) as illustrated in Fig. 6. As mentioned above, the PEI polymers alone did not significantly improve algal biocoagulation unlike PEIcoated E. coli cells. Therefore, bacterial cells could act as a shuttle for distributing positive charges and increasing the positive charged surface areas. The positively charged polymers bind partly or completely to algal cells. If the polymers bind partly, the unoccupied part of the polymers can bind to other algal cells, thereby bridging them and resulting in a network of polymers and algal cells. If the polymers bind the algal cells completely because they are too short to bind others as well, they adsorb (patch) to the surface and can create positive charges locally. These charges attract other algal cells and also result in biocoagulation of the cells. Although this study did not examine the production of EPS on bacterial or algal surface, it is evident that no or at least no significant amount of cationic polymers were produced by the tested bacteria

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or algae due to their overall negative surface charge as indicated by Fig. 2. Coagulation/flocculation/sedimentation has been considered an inexpensive harvesting method than flotation, membrane and centrifugation (Milledge and Heaven, 2013; Wiley et al., 2011). Algae–bacteria biocoagulation represents a potentially sustainable and low-cost engineering process to harvest algae (Wang et al., 2012). The cost may be contributed by the preparation of bacterial agents and the operation of coagulation systems (Powell and Hill, 2013). In large scale algal harvesting, the bacterial biocoagulant can be produced using fermentation, which will make it more cost effective than biopolymer such as chitosan. The use of bacteria biocoagulant is the same as using inorganic or biopolymers. There have been many large water or wastewater facilities with coagulation processes in operation at different scales. However, the potential barriers to developing economically viable biocoagulation processes could be seeking cheap sources or cultivation approaches for large quantities of bacterial biomass as the coagulating agents and optimization of the process parameters such as UV irradiation to increase the coagulation and settling efficiencies. 4. Conclusion The biocoagulation efficiencies of the bacteria–algae suspension were significantly increased with the PEI-coated E. coli cells and UV illumination. UV365 irradiation likely altered bacterial surface characteristics, which influenced bacteria–algae biocoagulation. In contrast, the PEI coating shifted the surface charge of E. coli cells from negative to positive charges, which resulted in strong electrostatic attraction between bacteria and algae cells and thus enhanced the biocoagulation. The soft-particle DLVO theory indicated the PEI-coating eliminated the energy barrier that could exist between bacterial and algal surfaces and correctly explained the enhanced biocoagulation mechanisms with PEI-coated E. coli cells. Acknowledgements This study was supported by the Research Startup Fund at NJIT and National Science Foundation Grant CBET-1235166. The authors would like to thank Drs. Liping Wei, Cheul Cho and Megha Thakkar for their help with microscopic imaging and algal size distribution. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.05. 060. References Burnett, P.-G., Heinrich, H., Peak, D., Bremer, P.J., McQuillan, A.J., Daughney, C.J., 2006. The effect of pH and ionic strength on proton adsorption by the thermophilic bacterium Anoxybacillus flavithermus. Geochim. Cosmochim. Acta 70, 1914–1927. Chang, Y.-I., Chang, P.-K., 2002. The role of hydration force on the stability of the suspension of Saccharomyces cerevisiae – application of the extended DLVO theory. Colloids Surf., A 211, 67–77. Grossart, H.P., Czub, G., Simon, M., 2006. Algae–bacteria interactions and their effects on aggregation and organic matter flux in the sea. Environ. Microbiol. 8, 1074–1084. Hadjoudja, S., Deluchat, V., Baudu, M., 2010. Cell surface characterisation of Microcystis aeruginosa and Chlorella vulgaris. J. Colloid Interface Sci. 342, 293– 299.

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