Nature-Based Solution to Eliminate Cyanotoxins in Water Using Biologically Enhanced Biochar

Climate change and high eutrophication levels of freshwater sources are increasing the occurrence and intensity of toxic cyanobacterial blooms in drinking water supplies. Conventional water treatment struggles to eliminate cyanobacteria/cyanotoxins, and expensive tertiary treatments are needed. To address this, we have designed a sustainable, nature-based solution using biochar derived from waste coconut shells. This biochar provides a low-cost porous support for immobilizing microbial communities, forming biologically enhanced biochar (BEB). Highly toxic microcystin-LR (MC-LR) was used to influence microbial colonization of the biochar by the natural lake-water microbiome. Over 11 months, BEBs were exposed to microcystins, cyanobacterial extracts, and live cyanobacterial cells, always resulting in rapid elimination of toxins and even a 1.6–1.9 log reduction in cyanobacterial cell numbers. After 48 h of incubation with our BEBs, the MC-LR concentrations dropped below the detection limit of 0.1 ng/mL. The accelerated degradation of cyanotoxins was attributed to enhanced species diversity and microcystin-degrading microbes colonizing the biochar. To ensure scalability, we evaluated BEBs produced through batch-scale and continuous-scale pyrolysis, while also guaranteeing safety by maintaining toxic impurities in biochar within acceptable limits and monitoring degradation byproducts. This study serves as a proof-of-concept for a sustainable, scalable, and safe nature-based solution for combating toxic algal blooms.

The microcystin concentrations were monitored by UPLC-PDA-MS/MS to assess the rate of BEB biodegradation, using coconut shell biochar synthesized using a continuous scaled-up production methodology.The time taken for 50 % of the microcystins to be degraded was then calculated.450, 550 & 700 refers to the HTT pyrolysis temperature (℃) on synthesis of the coconut biochar.
Error bars represent the standard deviation, n=3.

Fig. S1 -
Fig. S1 -Biologically enhanced biochar nature-based solution for microcystin removal from contaminated water.Lab scale proof-of-concept study displaying BEBs in lake water spiled with 5 µg/ml MC-LR.

Fig. S4 -
Fig. S4 -Mass spectra of cyanopeptolin 1012 extracts of M. aeruginosa B2666.The top spectra displays the high energy and the bottom spectra the low energy mass spectra of cyanopeptolin 1020 in extracts of M. aeruginosa B2666.

Fig. S5 -
Fig. S5 -Mass spectra of putative aeruginosin extracts of M. aeruginosa B2666.The top spectra displays the high energy and the bottom spectra the low energy mass spectra putative aeruginosin in extracts of M. aeruginosa B2666.

Fig. S6 -
Fig. S6 -Box plots of degradation half-lives of BEB 450, BEB 550 and BEB 700 produced from batch-scale pyrolysis unit for challenge 0-7 with p-values obtained from One-way ANOVA tests.

Fig. S10 -
Fig.S10-Microcystin biodegradation half-life, in the presence of biologically enhanced biochar, using a scaled-up coconut biochar production methodology.The microcystin concentrations were monitored by UPLC-PDA-MS/MS to assess the rate of BEB biodegradation, using coconut shell biochar synthesized using a continuous scaled-up production methodology.The time taken for 50 % of the microcystins to be degraded was then calculated.450, 550 & 700 refers to the HTT pyrolysis temperature (℃) on synthesis of the coconut biochar.Error bars represent the standard deviation, n=3.

Fig. S12 -
Fig. S12 -Microcystin biodegradation half-life, in the presence of biologically enhanced biochar challenge 12: Cyanobacterial Extract.The microcystin concentration was monitored by UPLC-PDA-MS/MS to assess the rate of biodegradation by the biologically enhanced coconut biochar.The time taken for 50 % of the each microcystin to be degraded was then calculated.BEB 450, 550 & 700 refers to the HTT pyrolysis temperature on synthesis of the coconut shell biochar.Error bars represent the standard deviation n=3.

Fig. S16 -
Fig. S16 -BEB microcystin biodegradation half-life, challenge 8 and challenge 10.The microcystin concentration was monitored by UPLC-PDA-MS/MS to assess the rate of biodegradation by the biologically enhanced coconut biochar.The time taken for 50 % of the each of microcystins to be degraded during a. challenge 8: MC-LR, -RR, -YR & -WR and b. challenge 10: MC-LF, -LA, -LY & -LW was then calculated.BEB 450, 550 & 700 refers to the HTT pyrolysis temperature on synthesis of the coconut shell biochar.Error bars represent the standard deviation n=3.

Table S2 -Chemical Analysis of Rescobie Loch Water.
Collected 13 January 2021.Water chemical analysis performed by the James Hutton Institute.Error represents the standard deviation n=3.

Table S3 -Summary of Sample Flask Set-up for each Challenge Assay 1-7
* All purified microcystins purified as per Enzo Life Sciences

Fig. S2 -Mass spectra of MC-LR extracts of M. aeruginosa B2666.
The top spectra displays the high energy and the bottom spectra the low energy mass spectra for MC-LR in extracts of M. aeruginosa B2666.
Fig. S3 -Mass spectra of MC-LA extracts of M. aeruginosa B2666.The top spectra displays the high energy and the bottom spectra the low energy mass spectra for MC-LA in extracts of M. aeruginosa B2666.14Oct21 B2666 cell extract 1

Table . S5 -Properties of coconut shell biochar obtained from a batch scale pyrolysis unit.
EC-Electrical conductivity, PAHs-Polycyclic Aromatic Hydrocarbons, SSA-Specific Surface Area, FC-Fixed Carbon, VM-Volatile Matter.Errors represent the standard deviation, 1 -n=4, 2 -n=3, 3 -n=2, FC-Fixed Carbon, VM-Volatile Matter, , % wt.d.b:Yields and composition of coconut shell biochar were calculated as a proportion of the mass of dry feed.

Table . S6 -Properties of coconut shell biochar obtained from a continuous-scale (Stage 2) pyrolysis unit.
FC-Fixed Carbon, VM-Volatile Matter, Errors represent the standard deviation n= 4 , % wt.d.b:Yields and composition of coconut shell biochar were calculated as a proportion of the mass of dry feed FC-Fixed carbon, VM-Volatile matter

Table S7 -Microcystin biodegradation half-life, in the presence of biologically enhanced biochar.
The microcystin concentration was monitored by UPLC-PDA-MS/MS to assess the rate of biodegradation by the biologically enhanced coconut biochar.The time taken for 50 % of the microcystins to be degraded was then calculated.BEB 450, 550 & 700 refers to the HTT pyrolysis temperature on synthesis of the coconut shell biochar.Error represents the standard deviation n=3.