Preliminary Development of 3D-Printed Custom Substrata for Benthic ...

Original Article

Preliminary Development of 3D-Printed Custom Substrata for Benthic Algal Biofilms Kamran Kardel,1 Andres L. Carrano,1 David M. Blersch,2 and Manjinder Kaur2 Abstract Due to their fast rates of growth and regeneration, algae are a promising source of biomass for biofuels, aquatic pollution recovery, and a source of protein nutrients, among others. Cultivation of benthic algal biofilm communities, in particular, shows promise for these functions, yet control of quality and yield are strongly dependent on substrata characteristics that affect algal attachment and growth. No previous research efforts have taken advantage of the recent developments in additive technology to support algal biofilm development. Additive manufacturing allows for the design and control of surface features and provides a platform for developing substrata with surface topographies customized for algal colonization. This article seeks to establish the feasibility of colonizing 3D-printed custom substrata with algal biomass. Three exploratory experiments on algal biofilm colonization of printed surfaces were conducted under a variety of laboratory and natural environments, and all printed substrata showed various degrees of colonization success. The preliminary results seem to indicate that (1) 3D-printed substrata can be successfully colonized by algal communities; (2) there is a roughness effect on the colonization rate of benthic algae; (3) substratum roughness can be designed for optimal interstitial spacing between surface asperities, providing refugia for regenerative growth that allows shorter lifecycles of the next algae crop; and (4) increased efficiencies in the packing of biomass can be achieved by complex 3D-printed geometries that provide very high surface area in compact volumes. Future research will seek to quantify these effects as well as to establish substrata conditions that optimize attachment, colonization, and regeneration rates.

Introduction Additive manufacturing (AM) or 3D printing technologies are mostly associated with applications in product design and development and small batch manufacturing. Due to the ability to produce complex geometries at relatively high speeds, AM technologies are increasingly being employed in various nonmanufacturing applications. Applications in domains such as tissue engineering and osteopathy have been actively researched in recent years.1 The extension of AM into environmental

engineering applications has been very limited, however, despite the many potential applications to mass transfer and biological growth processes that are mediated by spatial and topological relationships. A primary environmental application that might be suitable for AM technologies is the manufacture of designer surface topographies for phototrophic biofilm production. Phototrophic biofilms are composed of benthic algae and bacteria that attach to solid substrata surfaces in a flow environment.2 Naturally occurring

in all fresh and marine water flow environments, phototrophic biofilms have been investigated under controlled cultivation scenarios for various environmental engineering applications. Benthic algal biofilm cultivation has been investigated for aquatic pollution recovery from natural waters and wastewaters, water quality improvements in natural waters and groundwaters, as well as biomass production for various economic uses, including protein production and biofuels feedstocks.3–6 At - scale applications are typically open systems subject to mixed algal

Departments of 1Industrial and Systems Engineering and 2Biosystems Engineering, Auburn University, Auburn, Alabama.

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MARY ANN LIEBERT, INC. • VOL. 2 NO. 1 • 2015 • DOI: 10.1089/3dp.2014.0024

3D-Printed Custom Substrata for Benthic Algal Biofilms

communities, and the types and abundance of various attached algal species that colonize a particular surface substratum determine, in part, the overall yield of the particular biotic process or product of interest. Control of the colonization and growth process through operational parameters such as substratum quality (e.g., feature and roughness design) can be an important determinant to overall system performance. In natural flow environments such as streams, the topographical characteristics of the substratum can be a controlling factor in the development of a colonizing biofilm.7 Heterogeneity of the substratum morphology determines the flow turbulence characteristics at the fluid boundary layer, affecting the kinetics of cell colonization, growth, and metabolism. Boundary layer flow characteristics also affect advection and diffusion of limiting nutrients to colonizing biofilms, which can strongly influence growth kinetics. Therefore, it is hypothesized that manipulation of the substratum roughness characteristics can be used to control the structural and functional characteristics of the colonizing algal biofilm. The objective of this preliminary work is to demonstrate the feasibility of using AM technologies to engineer substratum surface characteristics that are suitable for colonization of algal biofilms. The specific objectives for this investigation are as follows: (1) to demonstrate that algal biofilms can colonize 3D-printed substrata both in laboratory-based bioreactors as well as in natural settings; (2) to observe the influence of 3D-printed surface topography on the rates of attachment and growth of algal biofilms; and (3) to demonstrate the role of 3Dprinted topographical features to provide refugia during sacrificial removal of biomass, thereby enabling faster regenerative harvesting. In addition to these objectives, several promising research directions are proposed based on the potential of using AM technologies to support novel applications in bioenvironmental systems.

Background There has been much interest in controlled cultivation of algal biofilms

for various environmental engineering applications. Algal biofilms comprise mixed microbial communities dominated by photosynthetic algae that grow and thrive attached to a surface (or substratum) in a flow environment, typically occurring in nature within shallow aquatic environments where light and water flow are available.7,8 Controlled cultivation of algal biofilms using various reactor design configurations has been investigated for pollution recovery from natural waters 9–11; nutrient recovery from wastewater streams3,12,13; and biomass production for biofuels.11,14,15 Studies in both the field and laboratory have shown that the ecological characteristics of a colonizing algal biofilm can be influenced by the physical and chemical characteristics of the substratum.7 Physically, the heterogeneity of roughness characteristics of a substratum affects the colonization, attachment, and growth characteristics of viable microbial spores or cells that act as seed colonizers for biofilm establishment. 16 – 18 Typically, the mechanisms of substratum control over the biofilm characteristics have been investigated only at the macroscopic level for the design of reactors around a process or function, employing a uniform substratum characteristic across an entire reactor bed.11 With regard to benthic algae, the influence of physical substrata features encompasses a long-standing topic of research. It was found that substrata topographies with netlike or weblike configuration reduce local fluid velocities and enable high rates of algal cell settling and attachment.19 Similarly, substrata with heterogeneous surface microrelief, such as microcrevices in rock, provide depressions where velocities are low and spores can settle. 16,17 Definitive experimental research focusing on the spores of marine macroalgae has demonstrated the importance of microtopography in algal settlement and germination.20–22 Because of boundary- layer effects, substratum surface roughness affects the rate of retention of microorganisms on that surface. However, this effect is observed only for certain roughness


feature dimensions. If features are considerably larger than the microbial cells, then cell retention is not significant, whereas if features are on the order of microbial dimensions, then cell retention can increase.23 Also, a set of topographical parameters was proposed as a standard for surface roughness characterization in bacterial adhesion studies to improve the likelihood of identifying direct relationships between substratum topography and bacterial adhesion.24 It has been shown that smooth substrata are the least favorable for propagule settlement, and that increased settlement on substrata would occur if surface relief were increased up to an optimum roughness with an average depression depth of 800 μm.21 Recent research efforts to design surfaces for algal and single-cell/spore attachment have been pursued, and various methods have been employed to produce designed surfaces. Settlement and adhesion of zoospores from the green algae Ulva linza were investigated within defined topographies.25 In this research, four topographic size scales (Rz peak to valley varying between 25 and 100 μm) were manufactured by molding a plankton net between two PMMA microscope slides in a press while applying heat. It was found that fewer spores were removed from the smallest topographic structure tested (Rz = 25 μm) than from the larger ones. Other research shows that algae growth on a textured surface is several times higher than that on a smooth surface. The surface texturing was accomplished by an Nd:YVO4 laser, which created dimple features of 6–8 μm in diameter, 2–3 μm in depth, and spaced every 40 μm on stainless steel.26 Research has also been pursued to investigate the effects of surface roughness and shear on the attachment of Oscillatoria sp. algal filaments onto stainless steel coupons in a spinning cylindrical environment.27 The surfaces in this study were manufactured with traditional abrasive processes. Six coupons with average roughness (Ra) increasing from 0.801 to 1.309 μm were utilized. It was found that the amount of algae strands deposited in the coupons increased with the Ra. More recently, the impact of surface texture on microalgal cell attachment to solid nylon and polycarbonate carriers with ridges, pillars, and groove features

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was investigated.28 These features and textures were produced with laser and micromilling methods. It was found that algal cells could fully penetrate into the designed textures, but the adhesion behavior would be dependent on the size and shape of the cell.

uses a polyjet technology to deposit a layer (28 μm thick) of UV-light-cured acrylic polymer. In general, these designs were subjected to various conditions of algal culture and observed for colonization and growth of algae.

To date, little research effort has taken advantage of the recent developments in additive technology to support algal attachment and colonization. The technology has been investigated for use in direct placement of microbial colonies for spatial arrangement to enhance biofilm metabolic processes.29 Additionally, research has demonstrated the use of 3D printing for the enhancement of antimicrobial chemical interactions on a surface.30 No studies have been performed, however, using 3D printing to enhance biofilm characteristics on a surface by influencing the selective colonization via transport processes within a mixed community. AM can allow for design and control of surface features and provide a platform for testing the feasibility of colonization on customized substrata surface topographies, and, as such, the technology can serve as a valuable tool to perform replicable research in biofilm colonization kinetics. This article attempts to establish the foundation for such promising research area.

Experiment 1: 3D-Printed Growth Plates in Controlled Bioreactor Cultivators

Materials and Methods Three separate experiments were conducted to demonstrate the potential of using 3D-printed technologies for the production of surfaces for algal biofilm colonization. For these experiments, various surfaces were designed and fabricated in an Objet 30 machine (Stratasys Ltd., Eden Prairie, MN), which

The first experiment involved designing, fabricating, and placing two different growth plates in a laboratory bioreactor cultivator. The purposes of this experiment were to demonstrate that algal biofilms can indeed attach to and colonize 3D-printed polymer surfaces and to investigate the feasibility of using surface feature sizes to support regenerative harvesting. The growth plates were designed in Solidworks (Dassault Syst è mes SolidWorks Corp., Waltham, MA) and fabricated in the Objet 30 printer. A rectangular plate (90 mm × 100 mm) with four parallel channels and 5 mm collimating walls was designed with hemispherical surface features of increasing scale. The first channel was ideally smooth (Ra = 0.198 μm), while the remaining three channels had a pattern of adjacent hemispheres of diameters 500, 1000, and 2000 μm, respectively. The second plate was circular in shape (diameter of 100 mm) with each quadrant containing the same scale of features as in the rectangular plate.20 Anchor points were designed into the plates for ease of attachment to the bottom mesh in the bioreactor. Figure 1 shows the computer models with the corresponding fabricated versions of the two different growth plates used in this experiment.

The plates were placed in the same bench-scale benthic algal cultivator in the laboratory.31 The cultivator is a shallow trough in which attached benthic filamentous algae are typically grown on polypropylene screen substratum placed in the bottom of the trough. Water pumped from a reservoir continuously flows over the substratum in a thin layer (1–2 cm deep) and returns to the reservoir where a submersible pump recirculates it. A tipping bucket mechanism provides a periodic wave surge that helps stimulate the growth of benthic algae and disperse the nutrients. The cultivator was operated continuously at a flow rate of 45 L min−1 with a wave surge frequency of 4 min−1. Light was provided continuously by two 400 W metal halide grow lamps (Virtual Sun, La Verne, CA) located directly above the center of the cultivator. The plates were fixed to the substratum screen at the anchor points and remained in place for 45 continuous days. The cultivator was inoculated with rocks containing a mixed algal community that were collected from local streams. The dominant species in these rocks was Spirogyra communis. The cultivator was dosed daily with commercial F/2 media at a loading concentration rate of nitrogen (0.1 mg NO3-N d−1) and phosphorus (0.01 mg PO43− d−1). The flow velocity over the surfaces was estimated about 5 cm s−1 and the irradiance over the biofilm surface ranged between 175 and 500 μmol m−2 s−1. Following colonization, tiles were removed and imaged using optical microscopy at low magnification (10× to 40×) for topographical attachment and higher magnification (400×) for algal species identification. Biomass was sampled in three locations from each channel and quadrant, respectively; mixed and homogenized; and

Figure 1. CAD models and 3D-printed (rectangular and circular) growth plates. Color images available online at www.liebertpub.com/3dp

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subsampled for species identification using optical microscopy. Biomass was then removed using vacuuming (at approximately −15 psi) and rinsing with water.32 Following this, the surfaces of the two plates were scanned with 3D axial chromatic (noncontact) profilometer (Nanovea ST400, Irvine, CA). The optical pen provided a 1.4 mm Z-range with 20 nm resolution and over an area of 150 mm × 150 mm. Pseudoimages (photo simulations) were collected from the new plates (before placement in the cultivators) and after vacuum harvesting.

Experiment 2: Natural Environment Species Recruitment on 3D-Printed Growth Surfaces The second experiment focused on investigating the recruitment of colonizing algal species from natural freshwater environments. To accomplish this, multiple sets of 48 mm by 48 mm 3D-printed tiles and paired unglazed ceramic tiles (commonly used for field benthic investigations33,34 and to serve as a standard for comparison) were deployed in several local rivers and

streams in eastern Alabama. All printed tiles were manufactured as smooth as possible with Ra values ranging from 0.198 to 0.932 μm. The ceramic tiles used in the experiment had a measured Ra ranging from 1.144 to 1.855 μm. Figure 2 shows the tile arrangement on plastic mesh mats as well as the location in one of the natural streams. The tiles were placed in three different streams and monitored on a weekly basis for integrity and algal growth. The tiles were left on location for 30 days and then removed for measurement and analysis in the laboratory. After removal, tiles were imaged at low magnification (10× to 40×) via digital microscopy. Also, the biofilm was subsampled in three locations, and subsamples were mixed and stored in 10% buffered formalin solution for species identification using optical microscopy.

Experiment 3: Colonization on Complex Geometries A third experiment involved the design and manufacturing of 3Dprinted cube structures that presented

Figure 2. (a) Mesh mats with tiles, (b) placement in one of the streams, and (c) ceramic and printed tiles. Color images available online at www.liebertpub.com/3dp

very high surface area for attachment and colonization and that are impossible to manufacture other than using additive methods. The structures were based on a mathematical model for a gyroid, a special case of a triply periodic minimal surface. The gyroid surface can be described by Equation (1). (sin x × cos y) + (sin y × cosz) + (sinz × cosx) = 0

(1)

The dimensions of the gyroid cube were 20 mm on a side with a calculated surface area of 8127 mm2, over three times that available from an identical-sized cube with solid facets and over 19 times that of a square tile of similar base footprint. The gyroid cube was placed in a beaker and immersed in a mixed algal culture inoculated from rocks collected from a local stream dominated by Spirogyra communis. The gyroid was cultured in commercial F/2 media at ambient laboratory temperatures under fluorescent full spectrum grow lights (EnviroGro FLT22; Hydrofarm, Petaluma, CA) with irradiance over the gyroid surface ranging between 200 and 212 μmol m−2 s−1. Culture agitation was supplied by a rocking platform table (VWR International, Radnor, PA) set to 20 oscillations per minute. Culture medium was added every week to replace evaporative losses. For analysis following culturing, the gyroid was removed from the culture after 15 and 45 days and imaged at multiple magnifications. At the 45 - day removal, the biofilm was

Figure 3. 3D-printed collimated growth plates with hemispheres of diameters 2000, 1000, and 500 µm and smooth at (a) time of manufacture, (b) 15 days, (c) 30 days, and (d) at removal after 45 days. Color images available online at www.liebertpub.com/3dp


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subsampled in three locations, and subsamples were mixed and stored in 10% buffered formalin solution for species identification using optical microscopy.

Results and Discussion In the first experiment, the growth plates were removed periodically from the cultivator and inspected for algal colonization. Figure 3 depicts the chronological progression of biofilm coverage on the plate. Attached algal filaments were observed after 15 days of

exposure in the cultivator, with observed apparent increase in algal biomass density with increasing hemisphere diameter (Figure 3b). The distribution of biomass density across different roughness zones was observed to persist as growth continued past 30 days (Figure 3c) to completion of the trial at 45 days (Figure 3d). From direct observation, there appears to be a surface roughness effect on biomass density that could be explained, in part, by the increase in effective surface area with increasing diameter of the hemispheres comprising the roughness elements on the surface. This would be consistent with previous

Figure 4. 3D-printed circular growth plates: (a) prior to harvesting at 45 days; (b) after vacuum and mechanical harvesting; (c) micrograph at 30× magnification of residual algae patches (diameter 500 µm); (d) photosimulation of profilometer height readings for the same area. Color images available online at www.liebertpub.com/3dp

work that indicates the existence of a roughness effect and have hinted at the possibility of an optimal roughness value.20 After 45 days, the plates were removed from the reactor and harvested using mechanical scraping, rinsing, and vacuuming. Results from harvesting procedures demonstrated that the algal biofilm attached firmly to the printed surface in certain locations, resulting in patches of bare and colonized zones (Figure 4). From visual inspection, the amount of residual biomass following harvest appears to be correlated to the size of the surface features, with more biomass remaining for the larger diameter (1000 and 2000 μm) features (Figure 4b). Micrograph analysis of the surface following harvest shows that biomass patches remain in the interstitial spaces (Figure 4c). A pseudo-color height map of the same area using the profilometer demonstrates the extent of residual biomass on the surface, distributed over the entire surface but concentrated in the interstitial regions (Figure 4d). In cultivation reactors, these residual patches of algal biomass would ideally form zones of refugia from which a dense biofilm mat could regrow and regenerate the full algal turf. These observations suggest that the design of surface elements could be optimized to maximize regeneration rate and biomass harvest yield for continuously harvested production scenarios. In the second experiment, smooth printed tiles and unglazed ceramic tiles were placed in local streams to test for colonization and recruitment of species. The purpose was to prospect for those

Figure 5. Mesh mats from natural streams with ceramic and 3D-printed tiles: (a) after removal on day 30; (b) photograph of biofilm covering 3D-printed tile; (c) micrograph at 400× magnification of biofilm colonizing 3D-printed tile; (d) micrograph 40× of Spirogyra. Color images available online at www.liebertpub.com/3dp

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Table 1. Dominant algal genera identified via microscopy on printed tiles in natural streams Location Genus

Stream 1

Stream 2

Stream 3

Cladophora

−

+

−

Microspora

−

/

+

Mougeotia

−

+

/

Oedogonium

−

−

+

Sirogonium

+

−

/

Spirogyra

+

+

−

Diatoms

/

−

/

+, abundant; /, observed but rare; −, not observed.

Figure 6. Photographs (top row) and micrographs (10× magnification bottom row) of the Gyroid cube at (a) time of manufacture, (b) 15 days, and (c) 45 days. Color images available online at www.liebertpub.com/3dp

algal species that might find the printed surfaces favorable for colonization and growth. Results appear to indicate that, in many cases, algae from the stream colonized printed tiles at densities greater than those found on ceramic control tiles (Figure 5a). In some instances, the biofilm coverage on 3D-printed tiles spanned across the entire surface (Figure 5b). Micrograph analysis of the biofilm displays a diversity of algal types and morphologies (Figure 5c), with filamentous and prostrate varieties often dominant. Micrograph analysis allowed identification of the algal varieties to the genus level, and showed a fair diversity of algal types that colonize the biofilm on the surface (Table 1), often dominated by

filamentous genera such as Spirogyra (Figure 5d). These results show that surfaces of printed material allow attachment and colonization for a range of algal species, and demonstrate the potential of printed surfaces for exploratory and experimental investigation into algal colonization processes. In the third experiment, a gyroid was exposed to an algal culture under controlled laboratory conditions. Figure 6 shows the progressive colonization of the gyroid surfaces over a period of 45 days. The high surface area clearly provides ample opportunity for the algae spores to settle and anchor to initiate colonization. Visual inspection


indicates that colonization took place in every leaf deep inside the gyroid, indicating that the delivery of nutrients by flow was appropriate. No bare areas or spots were noticeable anywhere on the gyroid structure. The possibilities that arise from this experiment point to the design of custom surfaces that would induce different flow characteristics and light incidence in various locations in the periphery or inside the gyroid. The ability to control flow boundary conditions will provide a foundation for naturally controlling the growth of predominant species in the different regions with potential connections between them via novel flow pathways designed into the shape. For example, a bacteria that thrives in a darker, anoxic environment with stagnant flow would colonize the inside of a carefully designed gyroid, while other species needing higher levels of light incidence, flow, and nutrient delivery would settle in the outer leafs of the gyroid, and intersecting throughchannels might connect them materially. These observations suggest that AM could be used for the design of novel topographies for high-specificarea media for attached growth reactors (such as trickling filters). The concept of increasing biofilm colonization density by means of creative and complex geometry design has been established here. The potential for controlling biodiversity, with the intention of achieving higher process yields, is presented as a promising hypothesis.

Conclusions This work sought to establish the feasibility of colonization of printed surfaces for enhanced algal biofilm colonization and growth. Three different experiments were conducted under a variety of laboratory and natural environments, and all printed substrata showed various degrees of colonization success. The following hypotheses were teased out of the experiments and are the subject of ongoing research: (1) there is a roughness effect on the colonization rate of benthic algae, suggesting that an optimal value of certain surface roughness

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parameters exists; (2) substratum roughness can be designed for optimal interstitial spacing between surface asperities and microreplicated features to provide regenerative refugia that can shorten the lifecycle of the next algae crop; and (3) increased efficiencies in the packing of biomass can be achieved by complex geometries that provide very high surface area in compact volumes. While the cost implications of using 3D printing ultimately determine the feasibility of such systems, the unmatched ability of this technology to produce designer surfaces offers great potential to increase efficiencies and yields beyond the economic thresholds.

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Acknowledgments The research reported here was partially funded by a grant from the Auburn University Office of the Vice President of Research, providing funds for material and equipment support. Additional funding was provided jointly by the Auburn University College of Agriculture and the Auburn University Samuel Ginn College of Engineering.

Author Disclosure Statement No conflicts of interest exist. References

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Address correspondence to: Andres L. Carrano Department of Industrial and Systems Engineering 3301 Shelby Engineering Center Auburn University 345 W. Magnolia Avenue Auburn, AL 36849 E-mail: [email protected]

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