Physiology of microalgal biofilm: a review on prediction of adhesion on substrates

Figures & data

Table 1. Functions of related EPS components. (Adapted from [17,61])

EPS components	Functions
Polysaccharides	Enhances biofilm-substrate adhesion
	Aggregates microalgal cells
	Supplies nutrient for biofilm utilization
	Exhibits flow and elastic recovery to the matrix shape
	Forms a hydrated polymer network
	Enhances the tolerance of microenvironment in water-deficient environments
	Offers host defenses toward infection
	Stores excess carbon under unbalanced carbon to nitrogen ratios
	Accumulates enzymes
Proteins	Enhances biofilm-substrate adhesion
	Forms a hydrated polymer network
	Allows cell-cell communication
	Enhances the tolerance of microenvironment in water-deficient environments
	Offers host defenses toward infection
	Digests exogenous macromolecules for nutrient acquisition
	Degrades structural EPS, releasing cells from biofilms
Lipids	Bio-surfactant and bio-emulsifier
Nucleic acids (DNA)	Regulates the biofilm formation and structure
	Enhances biofilm-substrate adhesion
	Facilitates horizontal gene transfer between biofilm cells
	Aids metabolic turnover by exporting cell components
Uronic acids	Interact with cations to increase metal ions concentration for cells in oligotrophic condition
Humic substances	Permits redox activities in the biofilm
Sulfates	Impart EPS hydrophilicity to have gel-like consistency
Cations (Ca^2+ and Mg^2+)	Cross-links different polysaccharide chains

Figure 1. Schematic diagram of the microalgal biofilm and extracellular polymeric substance (EPS) structure. (Modified from [117])

Figure 2. Schematic designs of the microalgal biofilm systems: (a) constantly submerged biofilms, (b) partially submerged biofilms and (c) permeated biofilms (adapted and modified from [83])

Table 2 Comparison of microalgal biofilm cultivation on different substrates

Culture system	Cultivation area (m^2)	Substrate	Temp (°C)	pH	Light attenuation (µ mol photons /m^2/s) [light/dark (hr)]	Time (days)	Flow rate (L/min)	Rotation speed (rpm)	Culture medium	Species	Biomass productivity (g /m^2/day)	Ref.
Constantly submerged biofilms
Petri-dish biofilm	0.0004	Cotton	24 ± 2	6.5	90 ± 10 [24:0]	16	N/A	70	Bold’s basal	S. vacuolatus	[^b >60% at 2^nd hours]	[37]
		Jute fabrics									[^b >80% at 2^nd hours]
Petri-dish biofilm	0.002	Polyvinylidene fluoride commercial membrane	25 ± 2	N/A	20.25 [12:12]	4	N/A	40	F/2	Amphora coffeaeformis	(^a ~2.84 x 10^8 cells /m^2 at 96^th hours)	[38]
										Cylindrotheca fusiformis	(^a ~1.33 x 10^8 cells /m^2 at 96^th hours)
										Navicula incerta	(^a ~3.11 x 10^8 cells /m^2 at 96^th hours)
Petri-dish biofilm	0.0004	Titanium	24 ± 1	6	60 [12:12]	2	N/A	40	Modified Chu 10	Chlorella vulgaris	(^a ~7.8 x 10^3 cells /cm^2 at 48^th hours)	[8]
										Nitzschia amphibia	(^a ~20 x 10^3 cells /cm^2 at 24^th hours)
										Chroococcus minutus	(^a ~8 x 10^3 cells /cm^2 at 24^th hours)
Curtain-biofilm in aquarium	N/A	Plastic net	20-22	N/A	260	5	N/A	N/A	F/2	Navicula sp.	(^a 1.24 x 10^9 cells /m^2 at 5^th days)	[9]
Multi-layers photobioreactor	0.007	Polyethylene foam	26 ± 2	N/A	100 ± 10 [12:12]	20	0.003	N/A	Modified basal	Botryococcus braunii	1.50	[13]
		Glass fiber reinforced plastic									3.20
		Polyethylene foam							Piggery farm wastewater		3.71
		Glass fiber reinforced plastic									2.91
Flat plate photobioreactor (PBR)	0.004	Glass	26 ± 2	7	160 [16:8]	20	0.013-0.023	N/A	Modified Chu 10	Nitzchia palea	2.8	[74]
										Scenedesmus obliquus	2.1
Membrane biofilm reactor	0.0017	Mixed cellulose esters membrane	26 ± 2	6.8-7.5	50 [14:10]	8	0.001	N/A	Culture medium (2 g/L glycerol)	Chlorella vulgaris	9.27	[39]
Attached cultivation system	0.005	Glass fibre-reinforced plastic	26 ± 2	7	150 [14:10]	14	N/A	N/A	Artificial seawater (18 mM glycine)	Nannochloropsis oculata	15.76	[40]
Attach cultivation system	N/A	Filter paper	20 ± 2	N/A	100	8	0.01	N/A	Swine wastewater	Chlorella pyrenoidosa	5.03	[41]
Algal biofilm photobioreactor	0.28	Flexible fiber bundles	25-28	6.8-7.5	8000 lux	20	0.07	N/A	Simulated secondary effluent	Chlorella vulgaris	0.05	[42]
Partially submerged biofilms
Flat plate algal biofilm photobioreactor (FPBR)	0.015	Rice husk	25 ± 2	6.8	300	20	0.003	N/A	BG 11	Scenedesmus obliquus, Chlorella vulgaris, Oscillatoria tenuis mixed culture	7.32	[114]
		Pine sawdust									10.92
		Oak sawdust									8.67
		Sugarcane bagasse									9.54
Rotating algal biofilm (RAB)	0.045	Cotton duct	25	N/A	110-120 [24:0]	7	N/A	4	Bold’s basal	Chlorella vulgaris	3.51	[43]
RAB	0.186	Cotton rope	19	N/A	170 [14:10]	12	N/A	4.8	Logan WWTP wastewater	Mixed culture of algal-bacterial	5.5	[7]
Rotating flat plate photobioreactor	0.036	PVC	22 ± 2	N/A	139 [12:12]	24		2.8	Bold’s basal	Chlorella vulgaris SAG 211-12	0.42	[12]
Algadisk rotating biological contactor	0.09	Rough stainless steel mesh	38 ± 1	6.75	422	7	N/A	11	M8-a	Chlorella sorokiniana	20.1	[44]
		Smooth stainless steel mesh									~18
		Poylcarbonate									~14.5
Rocking cultivation reactor	0.019	Glass-reinfroced plastic	26 ± 2	8	100 [24:0]	11	N/A	8	Modified Basal	Chlorococcum sp.	4.26	[30]
		Stainless steel		7		15				Scenedesmus dimorphus	0.39
										Chlorella protothecoides	0.11
										Chlorella vulgaris	0.04
										Scenedesmus obliqnus	0.18
										S. dimorphus	0.32
										Chlorococcus sp.	0.53
Rocking cultivation reactor	0.006	Polystyrene foam	20	N/A	110-120 [24:0]	15	N/A	15	Manure wastewater	Chlorella sp.	2.57	[45]
		Cardboard									1.47
		Polyethylene landscape fabric									0.58
		Loofah sponge									1.28
Algal turf scrubber	30	Landfill liner & nylon netting	< 32	7-7.5	outdoor	May and June	93	N/A	Manure wastewater	WW consortium	25	[46]
RAB	0.186	Braid cotton rope	20	8	230 ± 15 [24:0]	3months	N/A	4.8	Petroleum refining wastewater	Mixed culture of algal-bacterial	4.11	[47]
Photorotating biological contactor (PRBC)	1.6	PVC	19-23	N/A	756 [12:12]	60	0.01	2-5	Simulated acid mine drainage	Microbial consortium dominated by Ulothrix sp.	0.42	[48]
Permeated biofilms
Filtration photobioreactor	0.0113	5 µm pore size membrane	35	N/A	100	4	0.005	N/A	Synthetic medium	Chlorella sp.	13.56	[49]
Single layer vertical plate attached photobioreactor	0.002	0.45 µm cellulose acetate/nitrate membrane	25	N/A	100	8	N/A	N/A	BG 11	Botryococcus braunii	6.25	[50]
										Scenedesmus obliquus	10.46
									F/2	Nanochloropsis	~5
										Cylindrotheca fusiformis	~5
Twin layer biofilm photobioreactor (TL-PBR)	0.0005	0.4µm polycarbonate membranes	26 ± 2.5	N/A	1023 [14:10]	31	0.004	N/A	Bold’s basal	Halochlorella rubescens	31.2	[51]
Tube-type-twin-layer PBR	0.002	Plain printing paper	26	N/A	67 [15:9]	25	0.003	N/A	Modified F/2	Isochrysis sp.	0.60	[120]
										Nannochloropsis sp.	0.80
										Tetraselmis suecica	1.50
										Phaeodactylum tricornutum	1.80
Twin-layer photobioreactor (outdoor)	0.67	Plain printing paper	29	N/A	4-320	25	0.1-0.17	N/A	Modified F/2	Isochrysis sp.	4.20	[120]
										Tetraselmis suecica	5.1
										Phaeodactylum tricornutum	6.1
Porous substrate bioreactor	0.0004	Glass fiber filter paper	25	N/A	110 [24:0]	3	N/A	N/A	BG 11	Anabaena variabilis	2.88	[52]
Attached cultivation reactor	0.06	0.45 µm cellulose acetate/nitrate membrane	25	N/A	100 [24:0]	7	0.06	N/A	BG 11	Aucutodesmus obliquus	9.16	[53]
Phototrophic biofilm reactor	0.125	Polyethylene woven geotextile	21	7	180 [24:0]	40	0.177	N/A	Synthetic wastewater	Wastewater consortium	7	[54]
Horizontal flow lane reactor	0.028	Polyethylene woven geotextile	24	7	200 [24:0]	40	0.2044	N/A	Synthetic wastewater	Wastewater consortium	~7	[54]
Biofilm cultivation reactor	0.09	0.45 m cellulose µ acetate/nitrate membrane	17	N/A	200 flashing light	3	0.03	N/A	BG 11	Scenedesmus dimorphus	9.13	[55]
Algal biofilm photobioreactor	0.275	Concrete	25 ± 1	8.3	55 [24:0]	35	0.15	N/A	BG 11	Botryococcus braunii	0.71	[99]

^aThis parameter indicates the biomass productivity in terms of cell counts.

^bThis parameter indicates the biomass productivity in terms of percentage of colonization area.

N/A: non-available.

All the biomass productivities were reported at the best performing combinations.

Table 3. Classification of monosaccharides in microalgal EPS. (Adapted from [63])

Monosaccharides	Example
Neutral sugars	arabinose (Ara), ribose (Rib), xylose (Xyl), glucose (Glc), galactose (Gal), mannose (Man), quinovose (Qui), fucose (Fuc), rhamnose (Rha)
Uronic acids	glucuronic acid (GlcA), galacturonic acid (GalA), mannuronic acid (ManA)
Amino sugars	Glucosamine (GlcN), galactosamine (GalN), mannosamine (ManN)
Uncommon sugars	3-deoxy-D-manno-2-octulosonic acid (Kdo), Neuraminic acid (Neu)

Figure 3. Application of the interaction forces during biofilm formation. (Modified from [106,107])

Flemming HC, Wingender J. The biofilm matrix. Nat Rev Microbiol. 2010;8(9):623–633.

 PubMed Web of Science ®Google Scholar

Lembre P, Lorentz C, Di P. Exopolysaccharides of the biofilm matrix: a complex biophysical world. In: Karunaratne DN, editor. The complex world of polysaccharides. Germany: Books on Demand; 2012. p. 371–392.

 Google Scholar

Seviour T, Derlon N, Dueholm MS, et al. Extracellular polymeric substances of biofilms: suffering from an identity crisis. Water Res. 2019;151:1–7.

 PubMed Web of Science ®Google Scholar

Mantzorou A, Ververidis F. Microalgal biofilms: a further step over current microalgal cultivation techniques. Sci Total Environ. 2019;651:3187–3201.

 PubMed Web of Science ®Google Scholar

Dora A, Immacolata G, Gabriele P, et al. Evaluating microalgae attachment to surfaces a first approach towards a laboratory integrated assessment. Chem Eng Trans. 2017;57.

 Google Scholar

Tong CY, Derek CJC. Biofilm formation of benthic diatoms on commercial polyvinylidene fluoride membrane. Algal Res. 2021;55:102260.

 Google Scholar

Sekar R, Venugopalan VP, Satpathy KK, et al. Laboratory studies on adhesion of microalgae to hard substrates. In: Ang PO, editors. Asian pacific phycology in the 21st century: prospects and challenges. Dordrecht: Springer Netherlands; 2004. p. 109–116.

 Google Scholar

Gómez Ramírez A, Enríquez-Ocaña L, Miranda-Baeza A, et al. Biofilm-forming capacity of two benthic microalgae, Navicula incerta and Navicula sp., on three substrates (Naviculales: naviculaceae). Rev Biol Trop. 2019;67:599–607.

 Web of Science ®Google Scholar

Shen Y, Zhang H, Xu X, et al. Biofilm formation and lipid accumulation of attached culture of Botryococcus braunii. Bioprocess Biosyst Eng. 2015;38(3):481–488.

 PubMed Web of Science ®Google Scholar

Schnurr PJ, Espie GS, Allen DG. Algae biofilm growth and the potential to stimulate lipid accumulation through nutrient starvation. Bioresour Technol. 2013;136:337–344.

 PubMed Web of Science ®Google Scholar

Rincon SM, Romero HM, Aframehr WM, et al. Biomass production in Chlorella vulgaris biofilm cultivated under mixotrophic growth conditions. Algal Res. 2017;26:153–160.

 Google Scholar

Shen Y, Chen C, Chen W, et al. Attached culture of Nannochloropsis oculata for lipid production. Bioprocess Biosyst Eng. 2014;37(9):1743–1748.

 PubMed Web of Science ®Google Scholar

Cheng P, Wang Y, Liu T, et al. Biofilm attached cultivation of Chlorella pyrenoidosa is a developed system for swine wastewater treatment and lipid production. Front Plant Sci. 2017;8:1594.

 PubMed Web of Science ®Google Scholar

Gao F, Yang ZH, Li C, et al. A novel algal biofilm membrane photobioreactor for attached microalgae growth and nutrients removal from secondary effluent. Bioresour Technol. 2015;179:8–12.

 PubMed Web of Science ®Google Scholar

Zhang Q, Liu C, Li Y, et al. Cultivation of algal biofilm using different lignocellulosic materials as carriers. Biotechnol Biofuels. 2017;10(1):115.

 PubMedGoogle Scholar

Gross M, Henry W, Michael C, et al. Development of a rotating algal biofilm growth system for attached microalgae growth with in situ biomass harvest. Bioresour Technol. 2013;150:195–201.

 PubMed Web of Science ®Google Scholar

Christenson LB, Sims RC. Rotating algal biofilm reactor and spool harvester for wastewater treatment with biofuels by-products. Biotechnol Bioeng. 2012;109(7):1674–1684.

 PubMed Web of Science ®Google Scholar

Melo M, Fernandes S, Caetano N, et al. Chlorella vulgaris (SAG 211-12) biofilm formation capacity and proposal of a rotating flat plate photobioreactor for more sustainable biomass production. J Appl Phycol. 2017;30(2):887–899.

 Web of Science ®Google Scholar

Blanken W, Janssen M, Cuaresma M, et al. Biofilm growth of Chlorella sorokiniana in a rotating biological contactor based photobioreactor. Biotechnol Bioeng. 2014;111(12):2436–2445.

 PubMed Web of Science ®Google Scholar

Shen Y, Xu X, Zhao Y, et al. Influence of algae species, substrata and culture conditions on attached microalgal culture. Bioprocess Biosyst Eng. 2014;37(3):441–450.

 PubMed Web of Science ®Google Scholar

Johnson MB, Wen Z. Development of an attached microalgal growth system for biofuel production. Appl Microbiol Biotechnol. 2010;85(3):525–534.

 PubMed Web of Science ®Google Scholar

Mulbry W, Kondrad S, Pizarro C, et al. Treatment of dairy manure effluent using freshwater algae: algal productivity and recovery of manure nutrients using pilot-scale algal turf scrubbers. Bioresour Technol. 2008;99(17):8137–8142.

 PubMed Web of Science ®Google Scholar

Hodges A, Fica Z, Wanlass J, et al. Nutrient and suspended solids removal from petrochemical wastewater via microalgal biofilm cultivation. Chemosphere. 2017;174:46–48.

 PubMed Web of Science ®Google Scholar

Orandi S, Lewis DM, Moheimani NR. Biofilm establishment and heavy metal removal capacity of an indigenous mining algal-microbial consortium in a photo-rotating biological contactor. J Ind Microbiol Biotechnol. 2012;39(9):1321–1331.

 PubMed Web of Science ®Google Scholar

Zhang D, Fung KY, Ng KM. Novel filtration photobioreactor for efficient biomass production. Ind Eng Chem Res. 2014;53(33):12927–12934.

 Web of Science ®Google Scholar

Liu T, Wang J, Hu Q, et al. Attached cultivation technology of microalgae for efficient biomass feedstock production. Bioresour Technol. 2013;127:216–222.

 PubMed Web of Science ®Google Scholar

Schultze LKP, Simon M-V, Li T, et al. High light and carbon dioxide optimize surface productivity in a twin-layer biofilm photobioreactor. Algal Res. 2015;8:37–44.

 Google Scholar

Naumann T, Çebi Z, Podola B, et al. Growing microalgae as aquaculture feeds on twin-layers: a novel solid-state photobioreactor. J Appl Phycol. 2012;25(5):1413–1420.

 Web of Science ®Google Scholar

Murphy TE, Berberoglu H. Flux balancing of light and nutrients in a biofilm photobioreactor for maximizing photosynthetic productivity. Biotechnol Prog. 2014;30(2):348–359.

 PubMed Web of Science ®Google Scholar

Ji C, Wang J, Zhang W, et al. An applicable nitrogen supply strategy for attached cultivation of Aucutodesmus obliquus. J Appl Phycol. 2013;26(1):173–180.

 Web of Science ®Google Scholar

Boelee NC, Janssen M, Temmink H, et al. The effect of harvesting on biomass production and nutrient removal in phototrophic biofilm reactors for effluent polishing. J Appl Phycol. 2013;26(3):1439–1452.

 Web of Science ®Google Scholar

Toninelli E, Wang J, Liu M, et al. Scenedesmus dimorphus biofilm: photoefficiency and biomass production under intermittent lighting. Sci Rep. 2016;6(1):32305.

 PubMedGoogle Scholar

Ozkan A, Kinney K, Katz L, et al. Reduction of water and energy requirement of algae cultivation using an algae biofilm photobioreactor. Bioresour Technol. 2012;114:542–548.

 PubMed Web of Science ®Google Scholar

Van Wychen S, Laurens LML. Total carbohydrate content determination of microalgal biomass by acid hydrolysis followed by spectrophotometry or liquid chromatography. Methods Mol Biol. 2020;1980:191–202.

 PubMedGoogle Scholar

Cheng Y, Feng G, Moraru CI. Micro- and nanotopography sensitive bacterial attachment mechanisms: a review. Front Microbiol. 2019;10:191.

 PubMed Web of Science ®Google Scholar

Carniello V, Peterson BW, van der Mei HC, et al. Physico-chemistry from initial bacterial adhesion to surface-programmed biofilm growth. Adv Colloid Interface Sci. 2018;261:1–14.

 PubMed Web of Science ®Google Scholar