Volume 5 Issue 4
Oct.  2020
Article Contents
Turn off MathJax

Citation:

Extraction of Allelochemicals from Poplar Alkaline Peroxide Mechanical Pulping Effluents and Their Allelopathic Effects on Microcystis aeruginosa

  • Corresponding author: Tingzhi Liu, liutz@tust.edu.cn
  • Received Date: 2020-04-10
    Accepted Date: 2020-06-04
    Fund Project:

    National Undergraduate Innovation Program 2016100570039

    Tianjin Research Program of Application Foundation and Advanced Technology 14JCZDJC40500

  • In this study, allelochemicals were extracted from pulping effluents rather than from the raw material of plants. Herein, five organic solvents (ethyl acetate (EAC), methyl tert-butyl ether (MTBE), dichloromethane (DCM), carbon tetrachloride (CTC), and petroleum (PE)) were applied to separately extracting the allelochemicals from alkaline peroxide mechanical pulp (APMP) effluents. The results from the algal density, inhibition ratio, and optical density of 446 nm (OD446nm) concluded that the extractives from the APMP effluents can act as effective allelochemicals and showed noticeable allelopathic inhibition effects on Microcystis aeruginosa growth. The results indicated that organic solvent extraction could be a practical approach to isolate the allelochemicals from the APMP effluents, which would broaden the potential application of the APMP effluents in the production of antimicrobial agents and other value-added materials.
  • 加载中
  • [1]

    Barnes, J.P., Putnam, A.R., Burke, B.A., Aasen, A.J., 1987. Isolation and characterization of allelochemicals in rye herbage. Phytochemistry 26, 1385-1390. doi: 10.1016/S0031-9422(00)81818-X
    [2]

    Chen, Y., Zhou, S.M., Wang, Y.C., Li, L.B., 2017. Screening solvents to extract phenol from aqueous solutions by the COSMO-SAC model and extraction process simulation. Fluid Phase Equilibria 451, 12-24. doi: 10.1016/j.fluid.2017.08.007
    [3]

    Coll, J.C., Bowden, B.F., Tapiolas, D.M., Dunlap, W.C., 1982. In situ isolation of allelochemicals released from soft corals (Coelenterata:Octocorallia):a totally submersible sampling apparatus. J. Exp. Mar. Biol. Ecol. 60, 293-299. doi: 10.1016/0022-0981(82)90166-6
    [4]

    Covington, A.K., Brown, O.R., Criss, C.M., Dickinson, T., Fernández-Prini, R., Garnsey, R., Garnsey, R., Gough, T.E., Irish, D.E., King, E.J., 2012. Physical chemistry of organic solvent systems. In:Springer Science & Business Media. London, England:Harlesden, 1-21.
    [5]

    Dachuri, V., Boyineni, J., Choi, S., Chung, H.S., Jang, S.H., Lee, C., 2016. Organic solvent-tolerant, cold-adapted lipases PML and LipS exhibit increased conformational flexibility in polar organic solvents. J. Mol. Catal. B:Enzym. 131, 73-78. doi: 10.1016/j.molcatb.2016.06.003
    [6]

    DellaGreca M., Zarrelli A., Fergola P., Cerasuolo M., Pollio A., Pinto G., 2010. Fatty acids released by Chlorella vulgaris and their role in interference with Pseudokirchneriella subcapitata:experiments and modelling. J. Chem. Ecol. 36, 339-349. doi: 10.1007/s10886-010-9753-y
    [7]

    Freitas, A.C., Ferreira, F., Costa, A.M., Pereira, R., Antunes, S.C., Gon alves, F., Rocha-Santos, T.A., Diniz, M.S., Castro, L., Peres, I., Duarte, A.C., 2009. Biological treatment of the effluent from a bleached kraft pulp mill using basidiomycete and zygomycete fungi. Sci Total Environ. 407, 3282-3289. doi: 10.1016/j.scitotenv.2009.01.054
    [8]

    Gahukar, R.T., 2012. Evaluation of plant-derived products against pests and diseases of medicinal plants:a review. Crop. Prot. 42, 202-209. doi: 10.1016/j.cropro.2012.07.026
    [9]

    Geller, D.P., Das, K.C., Bagby-Moon, T., Singh, M., Hawkins, G., Kiepper, B.H., 2018. Biomass productivity of snow algae and model production algae under low temperature and low light conditions. Algal Res. 33, 133-141. doi: 10.1016/j.algal.2018.05.005
    [10]

    Hideno, A., 2017. Short-time alkaline peroxide pretreatment for rapid pulping and efficient enzymatic hydrolysis of rice straw. Bioresour. Technol. 230, 140-142. doi: 10.1016/j.biortech.2017.01.058
    [11]

    Hong, Y., Hu, H.Y., Sakoda, A., Sagehashi, M., 2010. Isolation and characterization of antialgal allelochemicals from Arundo donax L. Allelopathy Journal 25, 357-368.
    [12]

    Jiang, Z.Y., Guo, P.Y., Chang, C., Gao, L.L., Li, S.X., Wan, J.J., 2014. Effects of allelochemicals from Ficus microcarpa on Chlorella pyrenoidosa. Braz. Arch. Biol. Technol. 57, 595-605.
    [13]

    Li, F.M., Hu, H.Y., 2005. Isolation and characterization of a novel antialgal allelochemical from Phragmites communis. Appl. Environ. Microbiol. 71, 6545-6553. doi: 10.1128/AEM.71.11.6545-6553.2005
    [14]

    Li, L., 2016. Etraction of allelochmicals from poplar APMP effluents and its inhibition on Chlorella pyrenoidosa. Tianjin:Tianjin University of Science and Technology.
    [15]

    Li, L., Hou, W.H., 2007. Inhibitory effects of liquor cultured with Nelumbo nucifera and Nymphaea tetragona on the growth of Microcystis aeruginosa. Huan Jing Ke Xue 28, 2180-2186.
    [16]

    Li, W.X., Qi, S., Wang, N., Fei, Z.H., Farajtabar, A., Zhao, H.K., 2018. Solute-solvent and solvent-solvent interactions and preferential solvation of limonin in aqueous co-solvent mixtures of methanol and acetone. J. Mol. Liq. 263, 357-365.
    [17]

    Liu, G., Ke, M., Fan, X., Zhang, M., Zhu, Y., Lu, T., Sun, L., Qian, H., 2018a. Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish. Chemosphere 192, 289-296. doi: 10.1016/j.chemosphere.2017.10.167
    [18]

    Liu, G.T., Zhou, C.F., Sun, L.F., Zhu, W.W., Jiang, H., Wang, H.X., An, S.Q., 2011a. Effects of Eichhornia crassipes allelochemicals on the growth of two mono-and co-cultured algae Microcystis aeruginosa and Scenedesmus obliquus. Acta Scientiae Circumstantiae 31, 2303-2311.
    [19]

    Liu, S., Qin, F.C., Yu, S.X., 2018b. Eucalyptus urophylla root-associated fungi can counteract the negative influence of phenolic acid allelochemicals. Appl. Soil Ecol. 127, 1-7. doi: 10.1016/j.apsoil.2018.02.028
    [20]

    Liu, T., He, Z., Hu, H., Ni, Y., 2011b. Treatment of APMP pulping effluent based on aerobic fermentation with Aspergillus niger and post-coagulation/flocculation. Bioresour. Technol. 102, 4712-4717. doi: 10.1016/j.biortech.2011.01.047
    [21]

    Liu, T., Hu, H., He, Z., Ni, Y., 2011c. Treatment of poplar alkaline peroxide mechanical pulping (APMP) effluent with Aspergillus niger. Bioresour, Technol., 102, 7361-7365. doi: 10.1016/j.biortech.2011.04.043
    [22]

    Ma, H.Y., Wu, Y.L., Gan, N.Q., Zheng, L.L., Li, T.L., Song, L.R., 2015. Growth inhibitory effect of Microcystis on Aphanizomenon flos-aquae isolated from cyanobacteria bloom in Lake Dianchi, China. Harmful Algae 42, 43-51. doi: 10.1016/j.hal.2014.12.009
    [23]

    Men, Y.J., Hu, H.Y., Li, F.M., 2007. Effects of the novel allelochemical ethyl 2-methylacetoacetate from the reed (Phragmitis australis Trin) on the growth of several common species of green algae. J. Appl. Phycol. 19, 521-527. doi: 10.1007/s10811-007-9165-8
    [24]

    Meng, P., Pei, H., Hu, W., Liu, Z., Li, X., Xu, H., 2015. Allelopathic effects of Ailanthus altissima extracts on Microcystis aeruginosa growth, physiological changes and microcystins release. Chemosphere 141, 219-226. doi: 10.1016/j.chemosphere.2015.07.057
    [25]

    Nakai, S., Inoue, Y., Hosomi, M., 2001. Algal growth inhibition effects and inducement modes by plant-producing phenols. Water Res. 35, 1855-1859. doi: 10.1016/S0043-1354(00)00444-9
    [26]

    Ni, L., Acharya, K., Hao, X., Li, S., 2012. Isolation and identification of an anti-algal compound from Artemisia annua and mechanisms of inhibitory effect on algae. Chemosphere 88, 1051-1057. doi: 10.1016/j.chemosphere.2012.05.009
    [27]

    Paerl, H.W., Xu, H., McCarthy, M.J., Zhu, G., Qin, B., Li, Y., Gardner, W.S., 2011. Controlling harmful cyanobacterial blooms in a hyper-eutrophic lake (Lake Taihu, China):the need for a dual nutrient (N & P) management strategy. Water Res. 45, 1973-1983. doi: 10.1016/j.watres.2010.09.018
    [28]

    Pereira, A.L., Santos, C., Azevedo, J., Martins, T.P., Castelo-Branco, R., Ramos, V., Vasconcelos, V., Campos, A., 2018. Effects of two toxic cyanobacterial crude extracts containing microcystin-LR and cylindrospermopsin on the growth and photosynthetic capacity of the microalga Parachlorella kessleri. Algal Res. 34, 198-208. doi: 10.1016/j.algal.2018.07.016
    [29]

    Popa, V.I., Dumitru, M., Volf, I., Anghel, N., 2008. Lignin and polyphenols as allelochemicals. Ind. Crop. Prod. 27, 144-149. doi: 10.1016/j.indcrop.2007.07.019
    [30]

    Smallwood, I.M., 1996. Handbook of organic solvent properties. Amsterdam:Elsevier, 15-17.
    [31]

    Sun, R., Sun, P., Zhang, J., Esquivel-Elizondo, S., Wu, Y., 2018. Microorganisms-based methods for harmful algal blooms control:a review. Bioresour. Technol. 248, 12-20. doi: 10.1016/j.biortech.2017.07.175
    [32]

    Turlings, T.C., Tumlinson, J.H., Heath, R.R., Proveaux, A.T., Doolittle, R.E., 1991. Isolation and identification of allelochemicals that attract the larval parasitoid, Cotesia marginiventris (Cresson), to the microhabitat of one of its hosts. J. Chem. Ecol. 17, 2235-2251. doi: 10.1007/BF00988004
    [33]

    Wang, W., Meng, B., Lu, X., Liu, Y., Tao, S., 2007. Extraction of polycyclic aromatic hydrocarbons and organochlorine pesticides from soils:a comparison between Soxhlet extraction, microwave-assisted extraction and accelerated solvent extraction techniques. Anal. Chim. Acta. 602, 211-222. doi: 10.1016/j.aca.2007.09.023
    [34]

    Whittaker, R.H., Feeny, P.P., 1971. Allelochemics:chemical interactions between species. Science 171, 757-770. doi: 10.1126/science.171.3973.757
    [35]

    Wu, J.T., Chiang, Y.R., Huang, W.Y., Jane, W.N., 2006. Cytotoxic effects of free fatty acids on phytoplankton algae and cyanobacteria. Aquat. Toxicol. 80, 338-345. doi: 10.1016/j.aquatox.2006.09.011
    [36]

    Wu, Y., Kerr, P.G., Hu, Z., Yang, L., 2010. Removal of cyanobacterial bloom from a biopond-wetland system and the associated response of zoobenthic diversity. Bioresour. Technol. 101, 3903-3908. doi: 10.1016/j.biortech.2009.12.144
    [37]

    Xiao, X., Huang, H., Ge, Z., Rounge, T.B., Shi, J., Xu, X., Li, R., Chen, Y., 2014. A pair of chiral flavonolignans as novel anti-cyanobacterial allelochemicals derived from barley straw (Hordeum vulgare):characterization and comparison of their anti-cyanobacterial activities. Environ. Microbiol. 16, 1238-1251. doi: 10.1111/1462-2920.12226
    [38]

    Yang, X., Zhang, L.H., Shi, C.P., Shang, Y., Zhang, J.L., Han, J.M., Dong, J.G., 2014. The extraction, isolation and identification of exudates from the roots of Flaveria bidentis. J. Integr. Agric. 13, 105-114. doi: 10.1016/S2095-3119(13)60495-5
    [39]

    Zhang, C., Yi, Y.L., Hao, K., Liu, G.L., Wang, G.X., 2013. Algicidal activity of Salvia miltiorrhiza Bung on Microcystis aeruginosa:towards identification of algicidal substance and determination of inhibition mechanism. Chemosphere 93, 997-1004. doi: 10.1016/j.chemosphere.2013.05.068
    [40]

    Zhang, T., Wu, A.P., He, M., Chen, C.P., Nie, L.W., 2007. The allelopathy and its mechanism of phenolic acids on water-bloom algae. China Environmental Science 27, 472-476.
    [41]

    Zou, W.S., Wang, Z., Song, Q.S., Tang, S.X., de Peng, Y., 2018. Recruitment-promoting of dormant Microcystis aeruginosa by three benthic bacterial species. Harmful Algae 77, 18-28. doi: 10.1016/j.hal.2018.05.008
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(3) / Tables(1)

Article Metrics

Article views(100) PDF downloads(5) Cited by()

Related
Proportional views

Extraction of Allelochemicals from Poplar Alkaline Peroxide Mechanical Pulping Effluents and Their Allelopathic Effects on Microcystis aeruginosa

    Corresponding author: Tingzhi Liu, liutz@tust.edu.cn
  • Tianjin Key Laboratory of Pulp and Paper, Tianjin University of Science and Technology, Tianjin 300457, China
Fund Project:  National Undergraduate Innovation Program 2016100570039Tianjin Research Program of Application Foundation and Advanced Technology 14JCZDJC40500

Abstract: In this study, allelochemicals were extracted from pulping effluents rather than from the raw material of plants. Herein, five organic solvents (ethyl acetate (EAC), methyl tert-butyl ether (MTBE), dichloromethane (DCM), carbon tetrachloride (CTC), and petroleum (PE)) were applied to separately extracting the allelochemicals from alkaline peroxide mechanical pulp (APMP) effluents. The results from the algal density, inhibition ratio, and optical density of 446 nm (OD446nm) concluded that the extractives from the APMP effluents can act as effective allelochemicals and showed noticeable allelopathic inhibition effects on Microcystis aeruginosa growth. The results indicated that organic solvent extraction could be a practical approach to isolate the allelochemicals from the APMP effluents, which would broaden the potential application of the APMP effluents in the production of antimicrobial agents and other value-added materials.

1.   Introduction
  • The Microcystis aeruginosa Kützing (MA), as one of the most common and harmful freshwater algae (Zou et al., 2018), can cause serious cyanobacterial blooms in lentic aquatic ecosystems, such as lakes or rivers, that are polluted by an excess of nutrient elements (e.g., N, P, K) all over the world (Ma et al., 2015; Sun et al., 2018). Such harmful algae blooms (HABs) would pose expanding threats to the environment by blocking sunlight and depleting oxygen in the water, killing other aquatic plants and animals; thus, decreasing the biodiversity and sustainability of freshwater ecosystems (Wu et al., 2010; Paerl et al., 2011; Liu et al., 2018a).

    Allelochemicals, which belongs to a class of allelochemic that can induce chemical interactions between organisms or species to affect their growth, health, behavior, or even population biology (Whittaker and Feeny, 1971). Allelochemicals so far have at least several hundred different organic compounds, such as 2-propyl phenol (Jiang et al., 2014), ethyl 2-methyl acetoacetate (EMA) (Li and Hou, 2007), and other lignin and polyphenols related organics (Popa et al., 2008). It has been reported that allelochemicals released from plants and microbes can generate an effective allelopathic inhibition on the growth of cyanobacteria strains and control the HABs (Li and Hu, 2005; Zhang et al., 2013). For example, some anti-algal allelochemicals (mainly including indoles, ketones, esters, alcohols, etc.) had been extracted from Arundo donax L. through organic solvent extraction, the methanol extract was fractioned into neutral and acidic fractions, and both these fractions were found to have an allelopathic inhibition on the growth of the MA (Hong et al., 2010). Linoleic acid (LA) sustained-release microspheres were used for anti-algal growth and the optimal dose of the LA microspheres was 0.3 g/L with an inhibitory ratio over 90% (Ni et al., 2012).

    It is well known that the process to extract allelochemicals from bioresources is as follow according to the literatures (Coll et al., 1982; Barnes et al., 1987; Turlings et al., 1991; Li and Hu, 2005): 1) pre-extraction of raw materials with hot-water or steam to obtain a coarse aqueous solution of allelochemicals; 2) further purification of allelochemicals from the above solution by organic solvents extraction for several times to obtain an aqueous solution of allelochemicals with a highly purified concentration; and 3) rotary evaporation and subsequential vacuum drying in an N2 atmosphere to obtain dried allelochemicals for further application. However, there are two issues for the extraction of allelochemicals from biomass materials directly, such as rye herbage (Barnes et al., 1987) and Eucalyptus urophylla (Liu et al., 2018b): 1) large amounts of solid waste residues would be generated, thus increasing the environmental load; and 2) some treasured biomass species for isolation of allelochemicals would increase the production cost (Gahukar, 2012). Alkaline peroxide mechanical pulp (APMP) effluents are derived from a newer pulping process for the pulp and papermaking industries (Liu et al., 2011c). The APMP effluents contain dissolved organics with high levels of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) (Freitas et al., 2009), in which the content of the extractives is 50–800 mg/L and approximately 30%–70% of the extractives are organic acids (Li, 2016). These acids (e.g., abietic acid, gallic acid, catechinic acid, shikimic acid) mainly come from lignin or lignin derivatives and have been proven to have allelopathic inhibition on microorganism growth (Nakai et al., 2001; Ni et al., 2012; Liu et al., 2018b). Interestingly, a typical pretreatment process for the production of the APMP is that the wood chips are treated with hot water at 70 ℃–95 ℃ for 30–90 min from which the APMP effluents are mainly released (Hideno, 2017), which is similar to the main extraction process of allelochemicals from the bioresources stated above. That is why the extractives (i.e., organic acids) from the APMP effluents can be regarded as allelochemicals. In addition, no solid wastes are generated from the APMP effluents during the extraction process. Furthermore, the extraction of organic acids from the APMP effluents will decrease the BOD and biological toxicity of effluents simultaneously (Liu et al., 2011b). Therefore, it is feasible to extract allelochemicals (organic acids) from the APMP effluents for the allelopathic inhibition study of allelochemicals on the MA.

    The common technique to isolate organics from organic-contained mixtures is the organic solvent extraction, which is an energy-efficient process (Wang et al., 2007). Ethanol, acetone, chloroform, carbon tetrachloride, ethyl acetate, methyl tert-butyl ether, and petroleum are the main organic solvents applied in the chemical engineering process (Smallwood, 1996; Covington et al., 2012). It is well known that polar organic solvents have a better extraction ratio for polar organic materials, and vice versa (Dachuri et al., 2016). In the literature, Chen et al. (2017) successfully screened the most promising extractant with high extraction efficiency and good physical properties by extracting phenol from its aqueous waste solutions via the model of conductor-like screening model segment activity coefficient (COSMO-SAC) from 40 organic solvents, during which, ketones were found to be promising solvents for extracting phenol from wastewater. Yang et al. (2014) obtained the allelochemicals (phthalate n-octyl ester and phthalate 2-ethylhexyl ester) from the root of Flaveria bidentis culturing solution by dichloromethane (DCM) extraction and found that the obtained allelochemicals from the culturing solution of root exudates had an effective allelopathic inhibitionon seed germination.

    In this study, five organic solvents, EAC, methyl tert-butyl ether (MTBE), DCM, carbon tetrachloride (CTC), and petroleum (PE) were used separately to extract allelochemicals from APMP effluents. Hence, the allelopathic effects of the extracted allelochemicals on rhe MA were further studied. In addition, the extract yields of the allelochemicals by the five organic solvents at different solvent concentrations were discussed via gas chromatography-mass spectrometry (GC-MS) analysis. The growth and inhibition ratio of the MA influenced by the five solvent-extracted allelochemicals were also studied.

2.   Materials and Methods
  • The poplar APMP effluent sample was obtained from Sun Paper in Shandong Province, China. The effluent sample was a combination of several wastewater streams from the APMP process, including chip washing, hot water impregnation, chemical impregnation, pressing, and mechanical refining, to name a few. The effluent sample was passed through a 200-mesh screen to remove fibers, fines, and debris, and then stored in a refrigerator until its use.

    Organic solvents, including EAC, MTBE, DCM, CTC, PE, and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich (Shanghai, China). Deionized water was utilized throughout the experiments. All of the reagents in the experiments were of analytical grade and used without further purification.

  • The EAC, MTBE, DCM, CTC, and PE were utilized as organic solvents to isolate the allelochemicals from the APMP effluents, successively. The detailed extraction processes were shown as follows according to the previous reports (Li, 2016): first, the pH of poplar APMP effluents were adjusted to 2.0 using 1% NaOH solution or 1% H2SO4 solution followed by an extraction process with organic solvent. The extraction was completed with sharp shaking for 5 min followed by a centrifugation operation for 10 min. Therefore, the organic phase was collected from the multiple liquid/liquid systems according to a method described in Wang et al. (2007). The obtained residual aqueous phase was adjusted to a pH of 9.0 and subjected to an extraction process for the second time with a half amount of the original organic solvent according to the same procedures mentioned above. And then, five fractions were obtained: EAC (including allelochemicals), MTBE (including allelochemicals), DCM (including allelochemicals), CTC (including allelochemicals), and PE (including allelochemicals). Next, the obtained allelochemical-contained organic solvent solution was concentrated by rotary evaporation, in which the evaporation temperature is set to be 1 ℃ -3 ℃ higher than the boiling point of each organic solvent followed by a drying process in a vacuum dryer in N2 atmosphere for more than 48 h. The prepared dried allelochemicals were saved in a refrigerator at -20 ℃ for further experiments. The GC-MS analysis was performed using a VARIAN4000us (Varian Medical Systems, Lincolnshire, IL, USA) as described in the literature (Li, 2016).

  • The MA was provided by the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China) (FACHB). The algae were cultured in a BG11 (Blue-Green) medium at (25 ± 2) ℃ under (2800 ± 100) lx. Light was cycled to provide 12 h of light and 12 h of dark conditions in the phytotron. Cultures were grown in a triplicate for a minimum of seven days. The tested organisms were cultivated to exponential growth phase (approximately 5.0 × 105 cells/mL) for further use.

  • The prepared allelochemicals derived from above five organic solvent extraction processes were dissolved and diluted to given concentrations with the DMSO, respectively. Next, 0.2 mL of the diluted allelochemicals solutions was added into the flask, which has been inoculated with 150 mL of the MA suspension with an initial algal density of 5.0 × 105 cells/mL. For comparison, 150 mL MA solution was added into 0.2 mL DMSO without allelochemicals addition. To analyze the growth and inhibition ratio of the MA influenced by the five solvent-extracted allelochemicals with different concentration, allelochemicals with different concentrations (50 mg/L vs. 100 mg/L) were added into the MA solution for culturing study. All the flasks were cultivated at the same conditions as described earlier. The number of the MA cells of each sample was counted every day using microscopy with a hemocytometer (1492; Hausser Scientific, Horsham, PA, USA) (Men et al., 2007).

    During the culturing process, a 500 μL sample of each culture was removed every 24 h under sterile conditions for the measurement of optical density at 446 nm (OD446nm) of the algal suspension using a Bio-Tek Synergy H1 Hybrid Reader (GoIndustry DoveBid, Bethesda, MA, USA) (Geller et al., 2018). Growth curves (concentration vs. time) were plotted using the daily biomass data and biomass productivity rates (mg/(L·d)), which were determined as the slope of a linear regression of the linear phase of these growth curves using Origin software (OriginLab, v.8.5, Northampton, MA, USA).

3.   Results and Discussion
  • Component analyses of the extractives from the APMP effluent isolated by five organic solvents were performed according to the results from the GC-MS technique shown in Table 1. It was clearly found that the number of extractive ingredients from the APMP effluents extracted by five organic solvents were similar and ranged from 18 to 23, although the yields of the five solvents extracts varied from 75 mg/L to 510 mg/L as shown in Table 1. Due to the different polarities of the solvents, the yield of allelochemicals is also different. According to the order of polarity, EAC > MTBE > DCM > CTC > PE. It was evidenced that the extractives isolated from the APMP effluent by the EAC organic solvent had the highest yield of extractives when compared with those of the other four organic solvents. It can be found from Table 1 that the yield of the EAC extract is 510 mg/L, and the yield of the MTBE is 375 mg/L. Though the MTBE has a lower relative polarity, which might be due to the fact that the molecule structures of the extractive isolated by the MTBE are similar to those of the MTBE (Li et al., 2018). As the polarity decreases, the extraction yield also decreases. The extraction yield of the DCM is 330 mg/L, the extraction yield of the CTC is 136 mg/L, and the smallest extraction yield is the PE with relatively low polarity of 75 mg/L.

    EAC MTBE DCM CTC PE
    Yield of extractives (mg/L) 510 375 330 136 75
    Number of extractive ingredients 21 18 23 18 20
    Component 2-amino-isobutyric acid
    2-undecenoic acid
    Ethylamine
    Ethanolamine
    2-hydroxypropionic acid (lactic acid)
    Glycolic acid
    Benzoic acid
    Succinic acid
    2, 3, 4-trihydroxybutyric acid
    Butylene acid
    Pimelic acid
    p-hydroxyphenylacetone
    Caprylic acid
    4-hydroxy-3-Methoxybenzoic acid
    3, 4-dihydroxybenzoic acid
    Sebacic acid
    3, 5-dimethoxy4-hydroxy benzoic acid
    2, 3-dihydroxy-5-allyl methyl ether
    2-hydroxysebacic acid
    Palmitic acid
    Stearic acid
    2-undecenoic acid
    Ethylamine
    Ethanolamine
    2-hydroxypropionic acid
    Glycolic acid
    Benzoic acid
    2, 3, 4-trihydroxybut yric acid
    Butylene acid
    Pimelic acid
    p-hydroxyphenylacetone
    Caprylic acid
    P-hydroxybenzoic acid
    4-hydroxy-3-Methoxybenzoic acid
    3, 4-dihydroxybenzoic acid
    2-hydroxysebacic acid Palmitic acid
    Stearic acid
    2-hydroxyheptanoic acid
    2-undecenoic acid
    Ethylamine
    Ethanolamine
    Glycolic acid
    Benzoic acid
    1, 5 - butyl glycol
    4-carbonyl pentanoic acid
    Butylene acid
    p-hydroxyphenylacetone
    Caprylic acid
    4-hydroxy-3-methoxybenzoic acid
    Azelaic acid
    Sebacic acid
    3, 5-dimethoxy4-hydroxybenzoic acid
    2, 3-dihydroxy-5-allyl methyl ether
    Palmitic acid
    9, 12-octadecarboxylic acid
    9-octadecenoic acid
    Stearic acid
    11, 12-dihydroxy-11-methoxy 9-eichenoic acid
    2-hydroxysebacic acid
    2-hydroxyheptanoic acid
    Twenty-two acid
    2-amino-isobutyric acid
    2-undecenoic acid
    Ethylamine
    Polydiimine carbide
    Ethanolamine
    Benzoic acid
    Palmitic acid
    9, 12-octadecarboxylic acid
    9-octadecenoic acid
    Stearic acid
    11- icosaenoic acid
    Twenty acid
    12, 13- dihydroxy-11 - methoxy 18 - carbon -9- methyl enate
    Denoprost
    3-hydroxy-2-decenedioic acid
    2-hydroxyheptanoic acid
    Twenty-two acid
    Cerotic acid
    L leucine
    2-amino-isobutyric acid
    2-undecenoic acid
    ethylamine
    Polydiimine carbide
    ethanolamine
    Glycolic acid
    3-hydroxydecanoic acid
    Fifteen acid
    (9E) -9-hexadecanoic acid
    Palmitic acid
    9, 12-octadecarboxylic acid
    9-octadecenoic acid
    Stearic acid
    3, 4-dimethoxy benzoyl formic acid
    Twenty acid
    12, 13- dihydroxy-11- methoxy 18- carbon -9- methyl enate
    Denoprost
    Twenty-two acid
    Cerotic acid
    Percentage content of organics in extractives (%)
    Organic acids 58 55 79 74 75
    Amines 8 7 13 19 21
    Ketones 32 38 3 0 0
    Esters 0 0 0 7 4
    Others 2 0 5 0 0
    Notes: EAC, ethyl acetate; MTBE, methyl tertiary butyl ether; DCM, dichloromethane; CTC, carbon tetrachloride; PE, petroleum ether.

    Table 1.  The GC-MS analysis of extractives from alkaline peroxide mechanical pulp (APMP) effluents isolated by different organic solvent

    The data of the top five major components and percentage contents of organics in the extractives shown in Table 1 indicate that the organic acids are the main components of the extractives from the APMP effluents, and the contents varied from 55% to 79% for the five different solvents. In addition, amines and ketones are also the major components of the extractives. The total percentage content of organic acids, amines, and ketones of the five groups of extractives was almost 95% or more, and it was reported that the above three organics could play an effective role in the allelopathic inhibition of algae growth (Ni et al., 2012). As shown in Table 1, the organic acid content of the APMP effluent extractives was approximately 55%–79%. Additionally, many publications have also proven that the organic acids coming from wood plants have effective allelopathic inhibition on algae growth (Wu et al., 2006; DellaGreca et al., 2010).

    Figure 1 shows the results of algal density of the MA in the culturing system for seven days at the existence of allelochemicals extracted by organic solvents from the APMP effluent. It was clearly noted that the addition of allelochemicals effectively impeded the algal growth of the MA, which showed that allelochemicals extracted from the APMP effluents had a good allelopathic inhibition on the MA growth. The allelopathic inhibition from 100 mg/L of the allelochemical concentration was stronger than that from 50 mg/L, compared with the results of Figs. 1a and 1b.

    Figure 1.  Algal density (105 cells/mL) of MA as a function of culture time (d) at allelochemical concentration of (a) 50 mg/L and (b) 100 mg/L extracted by five organic solvents

    Similar conclusions can be obtained from the literature. Xiao et al. (2014) isolated a pair of chiral flavonolignans as allelochemicals against Microcystis sp. from barley straw (Hordeum vulgare) extract using a bioassay-guided isolation procedure and found that the novel anti-cyanobacterial allelochemicals exhibited significant allelopathic inhibition on Microcystis sp. Additionally, the inhibition effects increased along with the concentration of the chiral flavonolignans.

    The results from Fig. 1 also showed that allelochemicals extracted by the MTBE had the strongest allelopathic inhibition in the first two days of the culturing of the MA. Meanwhile, the allelochemicals extracted by the PE had the weakest effect on the MA growth, which might have been due to the distinct difference of extractive yields from the MTBE and PE, as shown in Table 1.

    Figure 2 shows that the inhibition ratio (%) of the MA was affected by the allelochemicals from the APMP effluent with different extractive dosages (50 mg/L vs. 100 mg/L). It was noted that the growth inhibition ratios of allelochemicals extracted by the five organic solvents ranged from 7.0% to 38.2% when the extract dosage was 50 mg/L, while the growth inhibition ratios increased from 23.0% to 39.6% when the dosage was 100 mg/L. It was concluded that the allelochemicals extracted by the organic solvents had effective allelopathic inhibition on the MA growth, which agreed well with the results from Fig. 1.

    Figure 2.  Inhibition ratio (%) of MA as a function of culture time (Day) at allelochemical concentration of (a) 50 mg/L and (b) 100 mg/L extracted by five organic solvents

    Compared with the results from Table 1, Fig. 1 and Fig. 2, it was found that there was no linear relationship between the allelopathic inhibition and the yield of extractives isolated by the organic solvents. Additionally, the inhibition ratios on the MA were close to each other although the yields of the allelochemicals extracted by the five organic solvents varied widely from 75 mg/L to 510 mg/L as shown in Table 1. It may be ascribed to the different species and contents of the extractive ingredients isolated by different solvents that played similar allelopathic inhibition roles in the MA culturing process, which could be discussed in a future study.

    In the literature, Meng et al. (2015) reported a comparable result from a 5-day culturing experiment of MA inhibited by allelochemicals from Ailanthus altissima. They found that the inhibition ratios on the MA growth induced by 50 mg/L A. altissima ranged from 16.8% to 30.9%, and the range of inhibition ratios increased from 28.7% to 66.3% when the allelochemical concentration increased to 100 mg/L. In another study, the allelopathic inhibition of ferulic acid and p-hydroxybenzoic acid on Chlorella pyrenoidosa were studied separately and found that the inhibition rate of 100 mg/L ferulic acid and 194 mg/L p-hydroxybenzoic acid on C. pyrenoidosa were 57% and 84%, respectively (Zhang et al., 2007).

    The optical density at 446 nm of algae suspension can be affected by two main factors (Liu et al., 2011a; Geller et al., 2018): 1) a high algae cell density in suspension responds to high optical density when the algal is in the logarithmic growth phase; and 2) more dissolved color compounds released from algae cells that are degraded and lysed in suspension would induce higher optical density. Figure 3 shows the results of OD446nm of the MA culturing system along with the culture time at an allelochemical concentration of (a) 50 mg/L and (b) 100 mg/L extracted by five organic solvents. It is known that the effect of dissolved color compounds released from disintegrated algal cells plays a more important role in increasing the OD446nm of algal suspension than that of the cell density caused by algal growth. It was clearly noted that the OD446nm of the control MA sample was lower than that of the MA samples with the addition of allelochemicals extracted by the five organic solvents, which indicated that allelochemicals from the APMP effluents had a noticeable allelopathic inhibition effect on the MA growth. The OD446nm of the MA sample with the addition of allelochemicals extracted by the CTC solvent was the highest. This is different from the inhibition ratio of algal cells in Fig. 1 and Fig. 2, mainly because the optical density of the algal suspension is affected by many factors, and the reasons need to be further analyzed. We will explore it further in the future experiments.

    Figure 3.  Optical density at 446 nm (OD446nm) vs. culture time (d) at allelochemical concentration of (a) 50 mg/L and (b) 100 mg/L extracted by five organic solvents

    It was concluded from Figs. 3a and 3b that allelochemicals with a 100 mg/L concentration had a higher OD446nm than that of the allelochemicals with a 50 mg/L concentration, which indicated that the allelochemicals with higher concentration would have a stronger allelopathic inhibition on algae growth. This agreed well with the conclusions from Fig. 1 and Fig. 2. In the literature, Pereira et al. (2018) studied the effects of two toxic cyanobacterial crude extracts as allelochemicals containing microcystin-LR and cylindrospermopsin on the allelopathic inhibition of the microalga Parachlorella kessleri, and found that the allelopathic inhibition of allelochemicals with a concentration of 150 μg/L was higher than that of the allelochemicals with a concentration of 55 μg/L.

4.   Conclusions
  • In this study allelochemicals were successfully isolated from the APMP effluents by five common organic solvents, i.e., EAC, MTBE, DCM, CTC, and PE. The conclusions are as follows:

    (1) The GC-MS analyses showed that the EAC had the highest yield, in which the organic acids were the major ingredients in the extractives. The extracted allelochemicals mainly contained organic acids, amines, ketones, and esters, which occupied more than 90% of the extraction.

    (2) The extractives from the APMP effluents can act as effective allelochemicals and showed noticeable allelopathic inhibition effects on the MA growth. The allelopathic effects of the extracts on the growth of the MA were investigated and the growth inhibition ratios of the extracted allelochemicals from the five above solvents ranged from 7.0% to 38.2% when the extract dosage was 50 mg/L, while the growth inhibition ratios increased from 23.0% to 39.6% when the dosage was 100 mg/L.

    (3) The organic solvent extraction method was used to extract the allelochemicals from the APMP effluents, and the results showed that the allelochemicals extracted by the MTBE exhibited the highest algal inhibitory ratio compared with those extracted by the other extracts.

Conflict of Interest
  • There are no conflicts to declare.

Acknowledgements
  • This study was supported by Tianjin Research Program of Application Foundation and Advanced Technology (No. 14JCZDJC40500) and National Undergraduate Innovation Program (No. 2016100570039).

Reference (41)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return