Whatman filter paper

The preparation of a Citrusfusion extract involved obtaining leaves from Citrus limon and Citrus medica for green synthesis of nanoparticles, leveraging their bioactive compounds. Fresh leaves were carefully harvested, sterilized, and ground into a fine paste. This paste was then macerated in autoclaved distilled water overnight, utilizing a specific weight-to-volume ratio. Subsequently, the macerated extract underwent triple filtration using centrifugation and Whatman filter paper to eliminate fibres and small leaf sediments, ensuring a pure extract. The Citrus limon and Citrus medica extracts were mixed into Citrusfusion extract with an equal 1:1 ratio. The clarified extract was then stored at 4 °C for preservation.
The phytochemical analysis of Citrus limon and Citrus medica leaf extracts involved a comprehensive assessment of their bioactive constituents, including phenols, tannins, flavonoids, saponins, anthocyanins, alkaloids, steroids, and carotenoids. The evaluation followed established protocols as outlined by [12 (link)], ensuring meticulous identification and characterization of these phytochemicals.
Citrus limon (lemon) and Citrus medica (citron) used in study was identified and authenticated by Dr. D. Narasimhan, Botanist, Chennai. A specimen of the plant was deposited at School of Life Sciences, BSACIST, Chennai (Accession number: SLS-BSAU-23,008).

The dairy wastewater (DWW) and paper-and-pulp wastewater (PWW) utilized in this research was supplied by local dairy industry and paper-and-pulp industry located in KwaZulu-Natal Provine, South Africa. The DWW was collected from the storage tank containing both processing and cleaning wastewaters generated during dairy production. While the PWW sample was obtained from the secondary effluent treatment plant. Each wastewater sample (20 L) was filtered separately using Whatman filter paper to get rid of the solid particles. Subsequently, these filtered samples were combined in various proportions to make a wastewater mixture formulation ratio for the cultivation of Chlorococcum sp. A comprehensive analysis of the physicochemical characteristics of the DWW and PWW is presented in Table 1.Table 1

Physico-chemical parameters of DWW and PWW.

Table 1

Parameter	Unit	Dairy wastewater	Paper and pulp wastewater
pH	–	2.87	6.94
Colour	–	White	Brown
COD	mg/L	876	955
TN	mg/L	736.25	562.25
TP	mg/L	27.07	1.20
Na	mg/L	237.73	1153.73
K	mg/L	27.73	68.40
Ca	mg/L	50.80	48.00
Mg	mg/L	5.47	18.13
Fe	mg/L	0.24	0.04
Cu	mg/L	0.01	0.004
Zn	mg/L	0.13	0.04
Mn	mg/L	0.04	0.76
Al	mg/L	0.15	0.52

TN-Total nitrogen, TP- Total phosphorous.

Thereafter the wastewater collection, a preliminary assessment was performed to evaluate the viability of DWW and PPW as a sole or combined medium, as well as treated or untreated for the cultivation of Chlorococcum sp. The pH of the treated wastewater was adjusted to pH 7.1 and autoclaved at 121 °C for 15 min before the wastewater was used for the cultivation. Then the cultivation experiments in an Erlenmeyer flask (100 mL working volume) were conducted under growth conditions of pH 7.1, ambient temperature, agitation speed (150 rpm) and cultivation duration of three weeks.
Furthermore, varied amount of DWW and PWW with or without BG11 medium supplementation was utilized to cultivate Chlorococcum sp with the most favourable condition (Table 2) then modelled and optimized.Table 2

Selected mixtures for the optimization model.

Table 2

Mixture	BG11 (%)	PWW (%)	DWW (%)	BG11+PWW + DWW (%)
DWBG25 (A)	25	0	75	100
DWBG50 (B)	50	0	50	100
DWPWBG25 (C)	25	25	50	100
DWPWBG50 (D)	50	25	25	100
A + B + C + D	150	50	200	400

DWBG25: Dairy wastewater (75 %) and blue-green algae 11 (25 %); DWBG50: Dairy wastewater (50 %) and blue-green algae 11 (50 %); DWPWBG25: Dairy wastewater (50 %), paper and pulp wastewater (25 %) and blue-green algae 11 (25 %); DWPWBG50: Dairy wastewater (25 %), paper and pulp wastewater (25 %) and blue-green algae 11 (50 %).