Feasibility of microbially induced carbonate precipitation through a Chlorella-Sporosaricina co-culture system

Algal Research 47 (2020) 101831

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Feasibility of microbially induced carbonate precipitation through a Chlorella-Sporosaricina co-culture system

T

⁎

Pinpin Xua,1, Hua Fana,1, Lijian Lengb, Liangliang Fana, Shuhua Liuc, Paul Chend, , ⁎ Wenguang Zhoua, a

School of Resources, Environmental & Chemical Engineering, Key Laboratory of Poyang Lake Environment and Resource Utilization, Ministry of Education, Nanchang University, Nanchang 330031, China b School of Energy Science and Engineering, Central South University, Changsha 410083, China c State Key Laboratory of Water Resources and Hydropower Engineering Science, Wuhan University, Wuhan 430072, China d Bioproducts and Biosystems Engineering Department, University of Minnesota, Saint Paul, MN 55108, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Microbially induced carbonate precipitation Chlorella-Sporosaricina co-culture Growth conditions optimization Calcification rate Shape and morphology

Microbially induced carbonate precipitation, an eco-friendly and energy-efficient technology, has attracted many researchers' attention because of its potential use for soil solidification, crack repair and concrete selfrepair, etc. In this study, the feasibility of using enhanced co-culture of Chlorella sp. and Sporosaricina pasteurii under mixotrophic conditions to induce carbonate precipitation was investigated. The effects of different growing conditions on microbial growth and their relationship with microbially induced carbonate precipitation were evaluated. The results showed that under optimal growth conditions of algae-bacteria inoculation volume ratio of 3:2 (v/v), initial pH value of 9.0 and glucose concentration of 1 g.L−1, the algae-bacteria co-culture system was able to effectively induce carbonate precipitation and the Chlorella sp. biomass in the co-culture system was significantly improved by 37.74%. The Ca2+ rate was reduced by 60.4% in the co-culture system, which was higher than that in the mono-culture system. The calcification rate constant in the co-culture was 0.3514. The crystalline mineral particles were calcite crystals as determined by X-ray diffraction. It was observed through a scanning electron microscope that mineral particles piled up together and some microbial cells were deposited on the crystal surface. These results suggest that microbially induced carbonate precipitation through Chlorella-Sporosaricina co-culture is feasible, but more studies are necessary to understand mechanisms and optimize the process.

1. Introduction Microbially induced carbonate precipitation is a biological process in which bicarbonates produced by microorganisms react with calcium ions to form calcium carbonates that precipitate out. For decades, microbially induced carbonate precipitation has been used for soil property improvement, metal remediation, carbon sequestration, and construction restoration [1,2]. A variety of microorganisms including bacteria, fungi, and algae are capable of inducing carbonate precipitation through a number of biological pathways. Bacteria, such as Sporosaricina pasteurii (S. pasteurii), are common calcium carbonate precipitation inducers [3–6]. There were only a few studies that reported microbially induced carbonate precipitation through fungi. For example, Fang et al. screened a urea-producing Penicillium CS1 from

sewage activated sludge and used it to induce carbonate precipitation for preparation of cement column with enhanced compressive strength [7]. Fungi induced deposition was also used to immobilize heavy metals in tailings [8], which effectively decreased exchangeable heavy metals. In addition to bacteria and fungi, most of the photosynthetic autotrophic microorganisms, such as green algae [9] and cyanobacteria [10–12], can also induce calcium carbonate precipitation for carbonate deposition and carbon sequestration. CO2 is used by photosynthetic microorganisms in their metabolic process (Eq. 1) where there is a dynamic equilibrium between HCO3– and CO32– as shown in Eq. 2. Microalgae utilize CO2 or HCO3– and release OH−, resulting in an increase in pH (Eq. 3) [13]. Calcium carbonate is produced when this reaction occurs in the presence of calcium ion in the system (Eq. 4) [14].

⁎

Corresponding authors. E-mail addresses: [email protected] (P. Chen), [email protected] (W. Zhou). 1 These authors contributed equally to this work and should be considered co-first authors. https://doi.org/10.1016/j.algal.2020.101831 Received 15 November 2019; Received in revised form 4 February 2020; Accepted 4 February 2020 2211-9264/ © 2020 Published by Elsevier B.V.

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CO2 + H2 O → CH2 O + O2

(1)

2HCO3− ↔ CO2 + CO3− + H2 O

(2)

CO3−

+ H2 O ↔

HCO3−

(3)

Ca2 +

HCO3−

OH−

+

+

+

OH−

microbial growth was investigated and its best culture conditions were also optimized. Moreover, the carbonate precipitation capacity of three cultivating models, namely mono-culture Chlorella sp., co-culture of Chlorella and S. pasteurii, mono-culture S. pasteurii, under the same calcium concentrations were investigated and their corresponding precipitates were characterized using X-ray diffraction (XRD) and scanning electron microscope (SEM).

(4)

↔ CaCO3 + 2H2 O

Zhu et al. [15] demonstrated the microbial calcification potential of three cyanobacterial species, namely Synechocystis sp. PCC6803, Synechococcus sp. LS0519, and Synechococcus sp. PCC8806 by monitoring the changes in chemical composition and morphological and spectroscopic properties of mortar surfaces. Moreover, as reported by Yan et al. [16], Synechocystis sp. PCC6803 cultured with the BG-11 medium could induce calcium carbonate deposition. Compared with bacteria-based microbially induced carbonate precipitation, algae-based offers following additional advantages: 1) significantly higher carbon sequestration efficiency: the fixation efficiency of Chlorella sp. and Spirulina platensis were 46% and 39%, respectively, and calcite formation was induced by the two species [17]; 2) the ease of cultivation in a mild environment: algae can be easily cultivated under sunlight, and does not require precisely controlled environment like for other microorganisms such as bacteria [18]; Besides, the variety of the precipitation varies with surrounding environment. For example, many freshwater algae have been reported to induce calcium carbonate precipitation when they lived in a calcium supersaturated environment [19,20]. However, the long growth period to accumulate sufficient algae biomass is a main problem for algae induced calcium carbonate precipitation. It has been reported that lake calcium carbonate deposits were caused by the synergetic actions of microalgae and bacteria [21], suggesting that co-culture of microalgae and bacteria may improve biomineralization. It is well known that algae and bacteria have been coexisting for a long time in the ecosystem with a complex relationship, and there are many complex relationships between them from symbiosis to parasitism [22]. Many microalgae culture systems were maintained by co-culture with bacteria, which plays an important role in wastewater treatment using microalgae. Some researchers believed that pollutants (such as nitrogen and phosphorus) were absorbed and degraded in the microalgae and bacteria co-culture system significantly [23,24]. Higgins et al. demonstrated that Chlorella minutissima grew more rapidly and showed higher densities when it was co-cultured with Escherichia coli [25]. Bell [26] and Guo [27] indicated that the production of triglyceride was enhanced under the mixed culture of Chlorella vulgaris and Pseudomonas sp. Though pioneer work on co-cultural of algae and bacteria has been conducted, the feasibility of microbially induced carbonate precipitation through either mono-culture of Chlorella sp., an easy-to-grow the algae with relatively low-cost, or co-culture of Chlorella sp. with S. pasteurii, an ideal bacteria for biologically induced carbonate precipitation without causing harmful disease, has not been explored. Therefore, the aim of this study was to determine the feasibility of microbially induced carbonate precipitation with both the mono-culture and co-culture system, in hope to provide a new and distinct method and useful guidance for the research on microbial mineralization. In this study, the effect of different parameters, including inoculation ratio, initial pH value, and glucose concentration, on the

2. Materials and methods 2.1. Microbial strains and culture conditions The Chlorella sp. used in this study was isolated from a local wastewater treatment plant. The bacterium Sporosaricina pasteurii (S. pasteurii ATCC 6453) was purchased from ATCC (American Type Culture Collection, Maryland, USA). The culture mediums for Chlorella sp. and S. pasteurii were BG-11 and NH4-YE (10 g.L−1 of (NH4)2SO4, 20 g.L−1 of yeast extract), respectively. The Chlorella sp. and S. pasteurii were cultured in standard 250 mL Erlenmeyer flasks filled with 120 mL sterile BG-11 and NH4-YE medium, respectively. The algae and bacteria were cultured at 30 °C in an orbital shaker at 120 rpm under continuous illumination at a light intensity of 100 μmol photons m−2 s−1. After it reached the stable growth phase, the algae and bacteria cells were used as inoculum for the next experiment. 2.2. Effect of culture conditions on microbial growth The protocol used to optimize the culture conditions would first evaluate the effect of individual conditions using a one-factor-at-a-time scheme and then to study interactions among the factors. Key culture parameters affecting the growth of algae and bacteria, such as incubation volume ratios of algae-bacteria (IVR = 4:1, 3:2, 2:3, and 1:4), initial medium pH values (pH = 5.0, 7.0, 9.0 and 11.0), and glucose concentration (GC = 0, 1, 2, and 4 g.L−1) were studied. An orthogonal experiment was conducted to further optimize culture conditions. The optimal conditions were then used to determine the effect of culture condition as shown in Section 2.2. The microbes were cultured in the improved Bold Basal Media (BBM) [28] supplemented with glucose as a carbon source. The microbial cells of all samples (mono-culture Chlorella sp., co-culture of Chlorella and S. pasteurii, mono-culture S. pasteurii) were inoculated (10% v/v) into separate sterile Erlenmeyer flask (containing 100 mL medium) at 30 ± 0.2 °C in orbital shaker at 120 rpm under continuous illumination at an intensity of 100 μmol photons m−2 s−1.The optimal conditions were used for the following microbially induced carbonate precipitation tests as shown in Section 2.3. The contents of the co-culture groups were shown in Table 1 and the mono-culture groups (IVR = 1:0 and 0:1) and the control groups (without any microbial cells in improved BBM culture medium) as same as the co-culture groups. 2.3. Microbially induced carbonate precipitation tests The microbially induced carbonate precipitation through the different culture microbial system was carried out in the modified BBM medium supplemented with high concentration of calcification solution

Table 1 The contents of the co-culture groups. Inoculation volume ratio IVR (v/v) 4:1 3:2 2:3 1:4

pH 9.0

Initial pH −1

GC (g.L 1

)

pH 5.0 7.0 9.0 11.0

Glucose concentration IVR (v/v) 3:2

GC (g.L 1

−1

)

GC (g.L−1) 0 1 2 4

Note: IVR is Inoculation volume ratio of algae-bacteria; pH is the initial pH of the medium; GC is the Glucose concentration. 2

IVR (v/v) 3:2

pH 9.0

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model (Eq. 7) according to Stocks-Fischer et al. [32,33] was used to fit the experimental data:

(0.1 M Ca2+and urea (0.1 M, which as an induction factor for urease)) under the optimal culture conditions experimentally determined in Section 2.2 (IVR = 3:2, GC = 1 g.L−1, pH = 9.0). During the test, 50 mL culture broth with microbial cells that grew in stationary phases mixed with 50 mL calcification solution was placed in sterile Erlenmeyer flasks at 30 ± 0.2 °C in an illuminated incubator under continuous illumination at a light intensity of 100 μmol photons m−2 s−1. The experimental period was 20 days and the samples were shaken well twice a day.

y = A2 +

(7)

where y is the amount of calcium carbonate precipitate; A1 is the minimum amount of precipitate, A2 is the maximum amount of precipitate; x is the reaction time (d); t is the time at the maximum (dy/dx), and k is the rate constant. 2.4.5. Morphology and composition of the precipitants At the end of the culture experiment, the solution was poured out from the flask and the crystals remaining in the bottom were collected, which then washed with distilled water twice and freeze-dried. The dried material was then analyzed using Scanning Electron Microscope (JSM-6701F, JEOL Corporation, Japan) and X-ray diffraction (Bruker Corporation, Germany)) to confirm its composition and crystal morphology. The samples were coated with graphite to provide electrical conductivity before analyzed by SEM.

2.4. Analytical methods 2.4.1. Chlorophyll A for measuring algal biomass The traditional measurement of optical density (OD) of the culture suspension at 680 nm [29] could not accurately reflect the biomass of algae in the algae-bacteria symbiotic system due to the presence of bacteria. However, since the bacteria do not contain chlorophyll A, a measurement of chlorophyll A could indicate the algal biomass. The measurement of chlorophyll A was conducted as follows [30]: the microbial pellet in 5 mL culture solution was collected and then centrifuged at 4000 ×g for 20 min; after centrifuge, 3–5 mL methanol (99.5%) was added into the precipitation for extract chlorophyll by oscillating under weak light at 5 min; the samples with methanol at 4000 ×g for 20 min and pigment extracted was used to measure the OD at 666, 653 nm to calculate the content of Chl. A (chlorophyll A) according to Eq. (5):

Chl. A (mg/L) = 15.65 × A666 –7.34 × A534

A1 − A2 1 + e k (x − t )

2.5. Statistical and analysis All the results were calculated as the average value of three replicates and the standard deviation (SD) was calculated as well. Statistical treatment of data was performed with the software Origin 8.0. An analysis of variance (One-way ANOVA) with a Tukey correction was carried out using IBM SPSS Statistics to indicate any significant differences among groups. Differences were considered statistically significant at P values < 0.05.

(5)

where, A666 and A653 are the absorbance of the pigment extract at wavelength 666, 653 nm, respectively.

3. Results and discussion 2.4.2. Optical density for measuring bacterial biomass The general process to determine the biomass of bacteria involves the separation of bacteria cells from algae cells and determination by the optical density. To separate bacteria from algae, the culture broth was subjected to low-speed centrifugation at 1000 ×g for 5 min. Then, the larger Chlorella sp. (about 5 μm) cells were precipitated to the bottom while the smaller S. pasteurii cells (about 1 μm) were in the upper suspension. And it was confirmed that the two microorganisms as observed by light microscopes. Before measuring the OD, a blank (uninoculated medium) was used to calibrate the spectrophotometer (DR Corporation, America). Then the supernatant of the centrifuged solution was collected and measured at a wavelength of 600 nm to calculate the biomass of the bacteria.

3.1. Effect of different inoculation volume ratios of algae-bacteria on microbial growth for co-culture 3.1.1. Effect of different inoculation volume ratios on the growth of Chlorella sp. The effect of IVR (inoculation volume ratios of algae-bacteria) on the chlorophyll A content in Chlorella sp. is shown in Fig. 1. The chlorophyll A content in the mono-culture of S. pasteurii group (no algae) and the control group (no algae and no bacteria) was close to zero, while Chlorella sp. and Chlorella-S. pasteurii system showed a higher value. Among different levels of IVR, co-culture system with decreasing IVR, i.e., 4:1, 3:2, 2:3, and 1:4, were 12.44 ± 0.35 mg.L−1, 16.35 ± 0.88 mg.L−1, 11.38 ± 1.18 mg.L−1, and 10.39 ± 0.23 mg.L−1, respectively, while the chlorophyll A content of mono-culture of Chlorella sp. was 12.88 ± 0.79 mg.L−1, 11.59 ± 0.79 mg.L−1, 11.87 ± 0.58 mg·L−1, 11.18 ± 0.54 mg.L−1. Regardless of the cultivation time, in general, higher content of chlorophyll A was observed in the co-culture system than in the Chlorella sp. mono-culture group when IVR was higher than 1 (Fig. 1(a) and (b)). However, the opposite trend was observed when the IVR to < 1 (Fig. 1(c) and (d)). As shown in Table 2, the mean specific growth rate μc of Chlorella sp. with different IVR in the co-culture system was higher than the μm in the mono-culture system, and Chlorella sp. growth rate increased with increasing bacteria. Therefore, it can be concluded from the results that the co-culturing with a certain amount of bacteria benefited the biomass development of Chlorella sp., which is in agreement with the previous study [34]. However, when the initial bacteria were more than the algae (Fig. 1(c) and (d)), a slower growth was observed, indicating the growth of Chlorella sp. was inhibited by coculture of high concentration of bacteria, which could be caused by competition for the nutrients between the S. pasteurii and Chlorella sp. Based on the experiment results and statistical analysis of Fig. 1 and Table 2, the optimal IRV was 3:2 (v/v). Under this condition, the growth of the Chlorella sp. was significantly increased up to 37.74%. In

2.4.3. Specific growth rate The specific growth rate was determined by biomass accumulated and time of unicellular microbe Eq. (6) on data from exponential growth [31].

μn =

Ln (Nn/ Nn − 1 ) (n = 1, 2, 3…) tn − tn − 1

(6)

where, μn is the specific growth rate in the n day, N is the concentrations of unicellular, t is the time (days); The mean specific growth rate μx is the average specific growth rate of unicellular microorganisms from the beginning of the experiment to the maximum concentration. 2.4.4. Ca2+ concentration, pH and deposition dynamical models To determine precipitated carbonates, the concentration of free Ca2+ in the culture broth was measured using Inductively Coupled Plasma Emission Spectrometer (ICP-OES, Varian Corporate, America) every 4 days for a total of 5 times after pretreated by the 0.45 μm Glass Fiber Filter (Jinteng, Tianjin, China) to minimize the influence of large particles. All pH values of the sample solutions were monitored using a pH probe (PHSJ-4A, Rex Electric Chemical, China). Based on our preliminary evaluation of the experimental data, a modified exponential 3

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Chlorella sp. Chlorella-S.pasteurii S. pasteurii Control IVR= 4:1

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(a)

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The content of chlorophyll A /mg.L-1

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time/d Chlorella sp. Chlorella-S.pasteurii S. pasteurii Control IVR= 2:3

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time/d

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The content of chlorophyll A/mg.L-1

(b)

12 10 8 6 4 2 0

Chlorella sp. Chlorella-S.pasteurii S. pasteurii Control IVR= 1:4

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0

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Fig. 1. The chlorophyll A content (mean ± standard deviation of n = 3 replicates) co-culture of algae-bacteria with different IVR (Inoculation volume ratio of algaebacteria): (a) 4:1; (b) 3:2; (c) 1:1; (d) 2:3.

growth and biomass accumulation of algae. The reason could be that bacteria can secrete growth-promoting factors that can benefit the growth of algae. For example, Croft et al. [36] found that the bacteria living in the algal culture would support the growth algae by synthesizing vitamin B12. Similarly, the growth of green alga, Lobomonas rostrate was supported by vitamin B12 from the heterotrophic bacterium Mesorhizobium sp. [37]. In our study, S. pasteurii could generate nutrients to promote the growth of Chlorella sp., which will be analyzed later.

Table 2 The mean specific growth rate (mean ± standard deviation) of Chlorella sp. and S. pasteurii with different inoculation volume rations of algae-bacteria under different culture systems. Microbiological species

Chlorella sp.

S. pasteurii

IVR (v/v)

4: 3: 2: 1: 4: 3: 2: 1:

1 2 3 4 1 2 3 4

μx Mean specific growth rate μm mono-culture

μc co-culture

0.4119 0.5605 0.3970 0.4571 0.5806 0.4759 0.4100 0.4759

0.5620 ± 0.0852 0.6055 ± 0.0570 0.6127 ± 0.0357 0.7131 ± 0.0123 1.341 ± 0.064 2.243 ± 0.127 0.4160 ± 0.0364 0.8443 ± 0.0567

± ± ± ± ± ± ± ±

0.1135 0.0271 0.1273 0.0023 0.0123 0.0346 0.0234 0.0987

3.1.2. Effect of different inoculation volume ratios on the growth of S. pasteurii The growth of S. pasteurii in the co-culture system with different inoculation ratios is shown in Fig. 2. It could be observed that the maximum OD600 concentration of co-culture system with four IVRs reached to 0.1840 ± 0.0107, 0.2375 ± 0.0757, 0.2670 ± 0.0062, and 0.2725 ± 0.0304, respectively, while the OD600 concentration of only S. pasteurii group was 0.6432 ± 0.0063, 0.6814 ± 0.0007, 0.7373 ± 0.0063, and 0.6875 ± 0.0042, respectively. This suggests that the growth of bacteria in the co-culture system was inhibited, which could be because the composition of the co-culturing medium was mainly designed for the growth of Chlorella sp. However, the changes in the mean specific growth rate μc of S. pasteurii in the coculture system under different IVR experiments did not show the same trend as Chlorella sp. (Table 2). The μc of S. pasteurii in IVR of 4:1 (v/v) and 3:2 (v/v) were higher than that μm of S. pasteurii, which showed opposite results between IVR of 2:3 (v/v) and 1:4 (v/v). The results indicated that the IVR also had some effects on the bacteria growth in

Note: μc is the mean specific growth rate of algae-bacteria under the co-culture system; μm is the mean specific growth rate of algae-bacteria under the monoculture system.

addition, analysis of variance with Tukey was conducted on the concentration of chlorophyll A for algae-bacteria. It was clear that there were significant differences between the volume ratio of 3:2 (v/v) and algae mono-culture group (p < 0.05), but no significant differences between other experimental groups and Chlorella sp. mono-culture group (p > 0.05). Therefore, it can be safely concluded that the optimal IVR for Chlorella sp. grew in the co-culture system was 3:2 (v/v). Similar results have been reported by Le Chevanton et al. [35]. In their study, Dunaliella sp. in mixed culture with bacteria promoted the

4

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0.8 0.7

Chlorella sp. Chlorella-S.pasteurii S. pasteurii Control IVR= 3:2

The concentration of S.pasteurii/OD600

The concentration of S.pasteurii/OD600

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(a)

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(c)

The concentration of S.pasteurii/OD600

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time/d

(d)

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time/d

Fig. 2. The OD600 concentration (mean ± standard deviation of n = 3 replicates) of S. pasteurii in the co-culture system of algae-bacteria with different IVR (Inoculation volume ratio of algae-bacteria): (a) 4:1; (b) 3:2; (c) 1:1; (d) 2:3.

3.2. Effect of different initial pH values on microbial growth for co-culture

the co-culture system. In addition, compared with the curve of S. pasteurii, the bacillus grew in a small amount in the early stage no matter how much of Chlorella sp. was inoculated initially, whereas the concentration of S. pasteurii began to decline on the third day. It indicated that bacteria were inhibited in the co-culture system, which might be attributed to the rapid growth of Chlorella sp. (as shown in Fig. 1). The autotrophic algae can fix CO2 through the Calvin cycle to form organic carbon molecules which is eventually leaked into the surrounding water environment as dissolved organic matters (DOM), including dissolved organic carbon (DOC), dissolved organic nitrogen (DON), and dissolved organic phosphorus (DOP) [38], which could be used by bacteria. Besides, there was no significant difference (p > 0.05) in the biomass of S. pasteurii among the experimental groups according to the analysis of variance. Based on the results shown in Figs. 1 and 2, it could be clearly concluded that both Chlorella sp. and S. pasteurii could grow rapidly and grow together in the early stage in the co-culture system because there were adequate nutrients to support bacteria and algae growth in the initial stage. In summary, it can be concluded that the IVR significantly affected the growth of Chlorella sp. but had little effect on the growth of S. pasteurii, which could be attributed to the fact that the composition of the medium was mainly designed for the growth of Chlorella sp. Furthermore, the algae biomass accumulation was significantly promoted (p < 0.05) in the co-culture system at volume inoculation ratio of 3:2 (v/v), which may be attributed to the growth-promoting substances such as vitamin B12 secreted by bacteria [39].

3.2.1. Effect of different initial pH values on the growth of Chlorella sp. The pH value of the growth media not only affects the cell wall structure and alter the conformation of proteins protruding from the plasma membrane but also has an impact on the lipid structure and function of cellular membranes and the perturbation of the function of membrane-embedded proteins [40]. The effect of initial pH value on the chlorophyll A concentration is shown in Fig. 3. The maximum chlorophyll A concentration in the co-culture system with different pH values ranged from 5.0 to 11.0 were 15.73 ± 0.36 mg.L−1, 11.26 ± 0.53 mg.L−1, 19.48 ± 2.00 mg.L−1, and −1 8.892 ± 0.130 mg.L , respectively, while mono-culture Chlorella sp. showed their corresponding maximum chlorophyll A concentration as 10.58 ± 0.40 mg.L−1, 15.49 ± 0.70 mg.L−1, 14.88 ± 0.83 mg.L−1, and 15.46 ± 0.59 mg.L−1, at the 4 different pH values, respectively. Obviously, the chlorophyll A concentration at pH value of 9.0 (Fig. 3(c)) was the highest, which suggested that 9.0 was the best initial pH value for the growth of Chlorella sp. As shown in Table 3, the μc at different pH values were higher than the μm, the μc at pH value of 9.0 also was the highest, and the maximum chlorophyll A concentration occurred on day 6. It should be noted that the growth of Chlorella sp. was significantly inhibited in the algae-bacteria co-culture system at pH 7.0 and 11.0 (Fig. 3(b) and (d)) when compared with the Chlorella sp. groups. Analysis of variance with Tukey procedure showed there was a significant difference (p < 0.05) in growth for the pH value of 9.0 and 11.0 for the control and no significant difference (p > 0.05) 5

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Fig. 3. The chlorophyll A concentration (mean ± standard deviation of n = 3 replicates) in the co-culture of algae-bacteria with different initial pH value: (a) 5.0; (b) 7.0; (c) 9.0; (d) 11.0.

inorganic carbon at high alkaline pH value and almost no carbon is accessible for the algae, because algae use only bicarbonate ion (HCO3−) [43]. It was reported that the growth of three dinoflagellate algae whose growth was inhibited at high pH value [44].

Table 3 The mean specific growth rate (mean ± standard deviation) of Chlorella sp. and S. pasteurii with different initial pH under different culture systems. Microbiological species

Chlorella sp.

S. pasteurii

Initial pH

5.0 7.0 9.0 11.0 5.0 7.0 9.0 11.0

μx Mean Specific growth rate (d−1) μm mono-culture

μc co-culture

0.4479 0.5047 0.5111 0.3136 0.7947 0.7725 0.4953 0.3780

0.7189 0.6347 0.7560 0.4657 0.5243 0.4234 0.4893 0.3457

± ± ± ± ± ± ± ±

0.0345 0.0203 0.0676 0.0235 0.1275 0.0917 0.1975 0.0657

± ± ± ± ± ± ± ±

3.2.2. Effect of different initial pH values on the growth of S. pasteurii The growth of S. pasteurii in the algae-bacteria co-culture at different initial pH values is shown in Fig. 4. Compared with the maximum OD600 bacterial concentrations of mono-culture of S. pasteurii group (0.5988 ± 0.0007, 0.6395 ± 0.0025, 0.6890 ± 0.0127 and 0.5265 ± 0.0052 at four different pH values), the maximum OD600 bacterial concentration of co-culture group at different pH were only 0.1210 ± 0.0248, 0.1362 ± 0.0339, 0.1774 ± 0.0042, and 0.1994 ± 0.0010, respectively, suggesting that the growth of bacteria in the experimental group of mixed algal bacteria system was significantly inhibited and increased with increasing pH value. Interestingly, the μc of S. pasteurii at different pH values were all lower than theμm, which also proved that the pH of medium not only affect the biomass of bacteria but also play an important on the growth rate of S. pasteurii in the co-culture system. Compared with mono-culture S. pasteurii, the growth of bacteria in the co-culture was limited, which might be attributed to the rapid growth of Chlorella sp. (Fig. 3), which consumes the most of the nutrients. Since alkalis such as hydroxide ion, carbonates and bicarbonates may influence microbial growth by affecting the inorganic carbon source [45]. It also found out that pH can affect bacteria growth when bacterial concentration with increasing the

0.1228 0.0089 0.0567 0.0543 0.0507 0.1489 0.0754 0.0129

Note: μc is the mean specific growth rate of algae-bacteria under the co-culture system; μm is the mean specific growth rate of algae-bacteria under the monoculture system.

for other pH values. It suggests that Chlorella sp. grew better at the pH values of 9.0 than other values. It is known that pH value is an important parameter of the bicarbonate buffering system which determines the forms of inorganic carbon available to the algae [41], and affects the process associated with algal growth, e.g., metabolism, and uptake of ions [42]. The inhibition of the growth of Chlorella sp. at pH value of 11.0 can be explained by the fact that carbonate ion (CO32−) is the dominant form of 6

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Fig. 4. The OD600 concentration (mean ± standard deviation of n = 3 replicates) of S. pasteurii in the co-culture of algae-bacteria with different initial pH value: (a) 5.0; (b) 7.0; (c) 9.0; (d) 11.0.

16.90 ± 1.18 mg·L−1, 14.39 ± 1.07 mg·L-1, and 7.200 ± 0.312 mg·L−1, respectively, which suggested that the biomass of Chlorella sp. was highest under GC of 1 g.L−1. The μc of Chlorella sp. with different GC culture condition were all higher than the μm, and the μc increased with increasing of GC (Table 4), which indicated that the growth rate was promoted in early-stage (Fig. 5(c) and (d)) when the medium has higher organic carbon concentration. The growth rate of algae increased with increasing glucose concentrations, which can be explained by the fact that glucose is an important intermediate product for various metabolic precursors and plays an important role in the TCA cycle (Tricarboxylic Acid cycle) for ATPs (Adenosine Triphosphate) production by mitochondrial oxidative phosphorylation [51]. Compared with photoautotrophic and heterotrophic cultures, the biomass of Chlorella sp. in the co-culture system was enhanced [52,53]. Although Ye et al. [54] suggested that the growth of Chlorella sp. should increase when Chlorella sp. were cultured in medium with added extra organic carbon for photosynthesis and respiration, the biomass of Chlorella sp. in the co-culture system and mono-culture Chlorella sp. groups was inhibited when the concentration of glucose was 4 g.L−1, which was consistent with the previous theoretical and experimental studies [55]. Tan also indicated that the microalgal growth was strongly inhibited at glucose concentrations of 5 g.L−1 or higher [56]. High glucose concentration would render high osmotic pressure, and plasmolysis formed in the cell wall and produced harmful by-products, such as organic aid that inhibits microbial growth and metabolism. There was a significant difference (p < 0.05) for the glucose concentration at 4 g.L−1 when compared with the glucose concentration at 1 g.L−1 and 2 g.L−1.

pH value of the medium in the co-culture system in our study. It was observed that there was no significant difference in bacterial growth between the co-culture and the mono-culture systems, and no significant difference (p > 0.05) bacterial growth among all experimental groups within a culture system, be it mono-culture or co-culture. In this study, it was found that the pH value of the co-culture system of Chlorella sp. and S. pasteurii reached up to 7–9, but the optimal initial pH value was 9.0 according to Fig. 3 and Fig. 4. Generally, it is believed that the optimal pH value varies with different strains. It was reported that the optimal pH value for Chlorella sp. in mixed-culture fed-batch chemostat reactors was about 6.31 to 8.0 [46,47]. It is known that S. pasteurii grows in the medium with pH value ranging from 6.5 to 9.0 [48,49]. However, the optimum initial pH value was 9.0 for Chlorella sp. to grow well with S. pasteurii in this study, which may be closely related to the presence of bacteria and metabolic activity [50] in a mixed cultivation system. 3.3. Effect of different glucose concentration on microbial growth in algaebacteria co-culture system 3.3.1. Effect of different glucose concentrations on the growth of Chlorella sp. The effect of different GC (glucose concentrations) on the growth of algae is shown in Fig. 5. The maximum chlorophyll A in mono-culture Chlorella sp. system were 10.14 ± 0.81 mg.L−1, −1 −1 12.35 ± 0.02 mg.L , 12.81 ± 0.12 mg.L , and 8.260 ± 0.035 mg.L−1, while the maximum chlorophyll A contents in the co-culture system were 12.96 ± 0.55 mg·L−1, 7

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Fig. 5. The chlorophyll A contents (mean ± standard deviation of n = 3 replicates) in the co-culture system of algal-bacteria with initial GC (Glucose concentration): (a) 0 g.L−1; (b) 1 g.L−1; (c) 2 g.L−1; (d) 4 g.L−1.

0.1985 ± 0.0034 and 0.6946 ± 0.0443, respectively. However, as shown in Table 4, the μc of S. pasteurii in co-culture system in different GC (Glucose concentration) culture condition were lower than the μm of the bacteria and the μC increased with increasing GC, and but the μm of bacteria was the highest at GC of 4 g.L−1. Obviously, high concentrations of organic carbon would promote microorganism growth in the early stage, and the μm was higher than the μc, which may be ascribed to the possibility that most of the organic carbons were consumed by Chlorella sp. for growth in the co-culture system. The results showed that the growth of S. pasteurii in the co-culture system was limited but could grow at low concentration when compared with the S. pasteurii. When the glucose concentration was 0 g.L−1, few bacteria grew in Chlorella-S. pasteurii system, which might be related to the presence of Chlorella in the co-culture system. Watanabe [57] found that some photosynthetic products of algae would be discharged in the form of DOC and provided organic carbon for the growth of bacteria. This phenomenon explained why S. pasteurii could grow in a mixed system without the presence of glucose. Besides, the bacteria could grow well in glucose concentration 4 g.L−1 when the biomass of chlorella decreases in the co-culture system (Fig. 5 (d)), and it speculated that the cell concentration influences the growth of algae in the Chlorella-Sporosaricina co-culture system. However, when the bacteria reached a stable state, it is declined rapidly and began to die, as shown in Figs. 6 (c), (d). In addition, the bacterial growth in the glucose concentration 4 g.L−1 was significantly different from those at 0 and 1 g.L−1, and the biomass and growth of Chlorella sp. in the co-culture system were limited.

Table 4 The mean specific growth rate (mean ± standard deviation) of Chlorella sp. and S. pasteurii with different inoculation volume rations of algae-bacteria under different culture systems. Microbiological species

Chlorella sp.

S. pasteurii

GC/(g.L−1)

0 1 2 4 0 1 2 4

μx Mean Specific growth rate μm mono-culture

μc co-culture

0.4408 ± 0.0898 0.4737 ± 0.0178 0.4797 ± 0.0347 0.4066 ± 0.0789 0.2869 ± 0.0487 1.156 ± 0.130 1.254 ± 0.175 0.8628 ± 0.2071

0.5667 ± 0.1033 0.7333 ± 0.0875 0.5843 ± 0.0459 1.407 ± 0.298 0.0501 ± 0.0678 0.3376 ± 0.0267 0.4139 ± 0.0590 0.5539 ± 0.0542

Notes: μc is the mean specific growth rate of algae-bacteria under the co-culture system; μm is the mean specific growth rate of algae-bacteria under the monoculture system.

3.3.2. Effect of different glucose concentrations on the growth of S. pasteurii The effect of different glucose concentrations on the growth of bacteria under the co-culture model is shown in Fig. 6. The OD600 concentration of bacteria in the Chlorella sp. and the control group is very low, while higher OD600 concentration was observed in Chlorella-S. pasteurii co-culture and S. pasteurii mono-culture. With the increase in glucose concentration, the highest OD600 concentration of Chlorella-S. pasteurii system was 0.0356 ± 0.0079, 0.1898 ± 0.0007, 8

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Fig. 6. The OD600 concentration (mean ± standard deviation of n = 3 replicates) of S. pasteurii in the co-culture system of algae-bacteria with initial GC (Glucose concentration): (a) 0 g.L−1; (b) 1 g.L−1; (c) 2 g.L−1; (d) 4 g.L−1.

control group decreased slightly, which could be attributed to the chemical reactions of calcium phosphate precipitation in the presence of positive phosphate in the medium. Moreover, the quantitative analysis of the induced carbonate precipitation process using the modified exponential model shows that the data from all three microbial systems fitted the modified exponential model well, indicating that the microbially induced carbonate precipitation not only occurs in Chlorella sp. and S. pasteurii but also occur in the co-culture system. As shown in Table 5, the R2 (The correlation coefficient squared) values of three culture systems were 0.9993, 0.9988, and 0.9970 respectively. The high correlation coefficient squared (R2 > 0.9, α < 0.05) suggested that the exponential model can be employed to predict the process along with incubation time. However, the R2 value of the blank control group was −0.7175, suggesting that the modified exponential model could not be used to fit the reaction in the control group. The reason could be due to the chemical

In summary, algae and bacteria can grow together when the glucose concentration is 1 g.L−1 based on the comprehensive analysis from Fig. 5, Fig. 6 and Table 4.

3.4. Microbially induced carbonate precipitation through algae-bacteria coculture system 3.4.1. The change in calcium ion concentration in algae-bacteria co-culture based microbially induced carbonate precipitation It has been discussed previously that different parameters including IVR, initial pH value, and GC influenced the growth of microorganisms in the co-culture of algae-bacteria. The optimal culture conditions, i.e., the inoculation volume ratio of 3:2 (v/v), initial pH value of 9.0 and glucose concentration of 1 g.L−1 were used in the study of microbially induced carbonate precipitation through algae-bacteria co-culture. The change in Ca2+ concentration in the co-culture system was used as an indicator of how much calcium precipitated. The change in Ca2+ concentration was plotted against culture time (Fig. 7(a)). The data were fitted into Eq. 7 and the fitted curves showing the estimated precipitated calcium are shown in Fig. 7(b) and the kinetic parameters are presented in Table 5. As shown in Fig. 7(a), the maximum reduction in calcium ion in the algae-bacteria co-culture system was 60.40%, which was higher than that of mono-culture algal group (47.78%) and bacterial group (41.26%) after 20 days, indicating that the co-culture system was better than the mono-culture system of Chlorella sp. and S. pasteurii induced the deposition as well. Interestingly, the Ca2+ concentration in the

OH−

reaction (Ca2 + + H2 PO4−/HPO4− → CaPO4 ↓ + H2 O) take place to form calcium carbonate precipitates in the control group. In addition, the rate constant of precipitation in the co-culture system was 0.3514, which was higher than that of mono-culture Chlorella sp. system (0.0941), indicating that the process of microbially induced carbonate precipitation was promoted in the co-culture system due to that the presence of S. pasteurii. However, the higher rate constant of precipitate in S. pasteurii was due to the higher activity bacteria in the early stage. Besides, it is known that the pH value also is very important during the process of microbially induced carbonate precipitation, which can not only affect the concentration of carbonate ions and induce a shift in 9

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Ca2+ precipitation capacity/g.L-1

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time/d Fig. 7. Microbially induced carbonate precipitation in the algae-bacteria system: (a) The Ca2+ concentration (mean ± standard deviation of n = 3 replicates) as a function of cultivation time; (b) Exponential model fitting of Ca2+ precipitation; (c) The change in pH value (mean ± standard deviation of n = 3 replicates) in the free solution.

3.4.2. Morphology analysis of mineral particles The crystal structure and morphology of mineral particles introduced by different culture systems, in terms of mono-culture of Chlorella sp., co-culture system of Chlorella sp.- S. pasteurii, and monoculture system of S. pasteurii, are shown in Figs. 8(a) to (c). Compared with Fig. 8(d) where the minerals were generated by chemical reaction, the particles were amorphous and agglomerate and most of the minerals in three culture systems were prismatic. However, the morphology of the minerals was different within the three samples, for example, the mineral particle sizes induced by Chlorella sp. (Fig. 8(a)) and co-cultured Chlorella sp. and S. pasteurii (Fig. 8 (b)) was larger than that induced by S. pasteurii (Fig. 8 (c)) suggesting that the difference was related to the presence of microalgae. Santomauro et al. [9] also indicated the presence of living microalgae had a great influence on the precipitation of calcium carbonate crystals. There were some visible elliptical-shaped cells whose diameter ranged from 1 to 5 μm and embedded in the crystals or attached to the surface of the crystals when in mono-culture Chlorella sp. and the co-culture system, which appeared to be the features of algal cells or bacterial cells [15,60]. Moreover, the presence of crystalline calcite associated with microbe indicates that microbial cells served as a nucleation site during the mineralization process [61]. However, the mineral particles in Fig. 8 (b) were stacked together, the single-crystal structure with faintly visible prismatic

Table 5 The correlation analysis of the Exponential model (mean ± standard deviation). Experiments

k

R2

Chlorella sp. Chlorella-S. pasteurii S. pasteurii Control

0.0941 ± 0.0214 0.3514 ± 0.0244 0.5117 ± 0.0861 *

0.9993 0.9988 0.9970 −0.7175

Note: k is the rate constant of precipitation process; R2 is the correlation coefficient squared; * represent the value is meaningless.

the bicarbonate equilibrium when the pH rises in the aqueous environments (HCO3− + OH− → CO3− + H2 O) [58], but it also ensures that calcium carbonate does not dissolve. The change in pH in the free solution is shown in Fig. 7 (c). The initial pH value in different culture systems was significantly different before induced precipitation and the consumption of Ca2+ gradually rose with the pH value increasing in the free solution. And the pH value of the solution with different culture systems was alkaline (pH ≈ 8.0), and it is also proved that the alkaline environment beneficial to the process of calcification [59].

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(caption on next page) 11

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Fig. 8. SEM images of mineral particles induced by the different systems: (a) mono-culture system of Chlorella sp.; (b) co-culture system of algae-bacteria; (c) monoculture system of S. pasteurii; (d) from the chemical reaction.

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2θ/(°) Fig. 9. XRD pattern of mineral particles induced by the different systems: (a) mono-culture system of Chlorella sp.; (b) co-culture system of Chlorella-S. pasteurii; (c) mono-culture system of S. pasteurii.

when the concentration of Ca2+ was in the aqueous environments. The increase in pH also was measurable in the bulk solution, similar to calcium carbonate deposition mediated by other freshwater algae [13]. Moreover, since the cell surfaces of S. pasteurii and Chlorella sp. (whose cell walls are mainly composed of polysaccharides and proteins [65]) are both negatively charged, the positively charged calcium ions would be easily adsorbed on the surfaces of cells in the aqueous environments. In addition, many extracellular polymers may be secreted by the co-culture system during the growth of microorganisms. Extracellular polymers [66] contain organic molecules with a large number of negatively charged functional groups, such as carboxyl, hydroxyl, carbonyl, etc., which can provide active sites for cationic Ca2+, and promote the process of microbially induced carbonate precipitation. Therefore, it was observed that the co-culture system was more effective than the mono-culture systems. It has been reported that the cations adsorbed by excessive extracellular polymers may be wrapped by polymeric complexation and coacervates [67], which may reduce the free calcium ion concentration and weaken calcification. On the other hand, the extracellular polymers may be degraded by aerobic microorganisms in an alkaline environment, which reduces the absorption of a calcium ion by extracellular polymer, allowing greater calcium carbonate precipitation [68]. Therefore, the calcification rate of the coculture system was higher than that of the mono-culture Chlorella sp. system. However, the specific mechanism of calcium carbonate induced by the co-culture of Chlorella sp. and S. pasteurii has not been clearly understood and needs to be investigated further.

structures was difficult to identify. This could be attributed to some viscous substances (such as extracellular polymeric substances) secreted by the algae-bacteria symbiotic system, causing the crystals to stack together. The XRD pattern of the mineral particles in the algae -bacteria system is plotted in Fig. 9 (a) to (c). It was found that the main mineral particles induced by microorganisms were in the form of calcite crystal. Compared with standard calcite, the strongest diffraction peak is (104) crystal plane, the strongest diffraction peak of S. pasteurii group is (024) crystal plane (Fig. 9 (c)) while that of Chlorella sp. group and ChlorellaS. pasteurii are still (104) crystal plane (Fig. 9 (a) and (b)), suggesting that the preferential orientation occurred during induced carbonate precipitation process induced by Chlorella sp. Since the phenomenon of preferential orientation could be due to the fast growth rate of the crystal plane [32], the shift of strongest peak could be attributed to the fast growth of calcite by S. pasteurii. Besides, the occurrence of preferential orientation was closely related to biological macromolecules produced by microbes through the metabolism [62], which proved that crystal morphology is associated with microbial activity. Generally, the process of microbially induced carbonate precipitation mainly depends on four key factors [59]: (1) Ca2+concentration; (2) the dissolved inorganic carbon (DIC) concentration; (3) the pH; and (4) the effective nucleation sites. Our preliminary experiments have shown that the Chlorella sp. could grow well with bacteria S. pasteurii which provided a co-culture system to induced precipitation and promote the growth of microalgae. S. pasteurii is known to secrete the urease, which helps decompose urea to produce HCO3– and ammonia [63]. In addition, Chlorella sp. could use HCO3– in the culture media and resulting in an increased extracellular CO32– concentration to provide DIC [64] as indicated in Eq. 2. Compared with the mono-culture system, S. pasteurii in the co-culture system not only hydrolyzes urea to produce HCO3– but also provides CO2 through respiration to participate in the photosynthesis of Chlorella sp. The microbial cells in the co-culture system also could provide effective nucleation sites (shown in Fig. 8)

4. Conclusions In this paper, the feasibility of carbonate precipitation induced by co-culture system consisting of a freshwater microalga Chlorella sp. and a urease producing bacterium S. pasteurii was investigated. The following conclusions can be drawn: 1) The biomass accumulation of Chlorella sp. was enhanced by S. pasteurii, and the optimal conditions 12

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for the co-culture of the two microorganisms were inoculation volume ratio of 3:2 (v/v), initial pH value of 9.0 and glucose concentration of 1 g.L−1; 2) the co-culture system was able to induce calcium carbonate precipitation when sufficient calcium ions were present in the culture media. The Ca2+ reduction rate of Chlorella sp. and S. pasteurii in the coculture was 60.4%, which was higher than that in the mono-culture system, and the calcification rate was 0.3514; 3) the composition of the carbonate crystals was calcites, and the morphology and size of mineral particles in the experimental groups were different. Although it has been proved that the co-cultivation of Chlorella sp. and S. pasteurii benefited the microbially induced carbonate precipitation process, the mechanism involved in the functioning of this algae-bacteria co-culture system still need to be further studied.

[8]

[9]

[10]

[11]

CRediT authorship contribution statement

[12]

Pinpin Xu: Writing - original draft, Data curation, Methodology. Hua Fan: Writing - original draft, Data curation, Methodology. Lijian Leng: Supervision, Methodology. Liangliang Fan: Supervision, Methodology. Shuhua Liu: Supervision, Methodology. Paul Chen: Writing - review and editing. Wenguang Zhou: Writing - review and editing.

[13]

[14]

[15]

Declaration of competing interest

[16]

The authors declare that they have no competing interests. [17]

Acknowledgments This study was supported by the National Natural Science Foundation of China (31960734, 51668044), the Key Research Development Program of the Jiangxi Province of China (20171BBG70036 and 20181BBH80004), and the Talent Program for Distinguished Young Scholars of Jiangxi Province of China (20171BCB23015).

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[19]

[20]

Author's contributions

[21]

PX and HF participated equally in designing, performing the experiments, analyzing the data and writing the initial draft; LJL, LLF, and SHL assisted in conducting the experiment and critical revision of the manuscript. PC and WGZ critically reviewed the manuscript. All authors read and approved the final version of the manuscript. All authors read and approved the final version of the manuscript.

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