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HomeMicrobiologyA droplet-based microfluidic platform allows high-throughput combinatorial optimization of cyanobacterial cultivation

A droplet-based microfluidic platform allows high-throughput combinatorial optimization of cyanobacterial cultivation

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On this examine we introduce a microfluidic methodology that permits us to quickly display screen one- and two-dimensional (1D and 2D) tradition medium parameters to optimize the expansion situations of industrially related cyanobacteria. Our outcomes present that our droplet-based microfluidic method is properly suited to display screen tradition media situations in a quick and environment friendly approach, with low materials enter and a lowered quantity of incubator area. As well as, we noticed that generally used medium formulations are usually not optimized for optimum development and small modifications may end up in considerably larger biomass outputs. This methodology needs to be extensively relevant to quite a lot of freshwater and saltwater strains and has the potential to facilitate high-throughput pressure optimization.

Each freshwater and marine unicellular cyanobacteria may be efficiently cultivated in microdroplets

To check the applicability of microdroplet cultivation to cyanobacteria we selected three biotechnologically related species. PCC 6803 is a freshwater mannequin cyanobacterium that has been extensively characterised and for which many molecular instruments exist. UTEX 2973 is a freshwater fast-growing relative of Synechococcus elongatus PCC 7942. As a result of their genetic proximity, a median nucleotide id (ANI) > 99.8%32, the instruments developed for Synechococcus elongatus PCC 7942 are largely transferable to UTEX 2973. UTEX 2973 is of explicit curiosity for biotechnological purposes due to its quick development phenotype with doubling occasions reported as quick as 1.5 h33. As a 3rd pressure, we selected a saltwater pressure. A just lately remoted pressure, Synechococcus sp. PCC 11901 (hereafter PCC 11901), is genetically tractable and has clear biotechnological potential because of its reported doubling time of roughly 2 h and its capability to develop at excessive gentle intensities and a variety of salinities. Below optimized situations, PCC 11901 can accumulate as much as 33 g of dry cell weight per liter. Curiously, it has an ANI of 96.76% when in comparison with the generally used Synechococcus sp. PCC 7002 pressure26. Due to this fact, it might be doable to make the most of instruments beforehand developed for Synechococcus sp. PCC 7002 in PCC 11901. Nevertheless, this pressure is a vitamin B12 auxotroph. Therefore, we determined to make use of a intently associated, spontaneous mutant of this pressure, UTEX 3154, that may develop with out an exterior provide of vitamin B12.

First, we proceeded to check whether or not these three cyanobacterial strains might develop within the droplet-based microfluidic setup. For the preliminary screening, sensors detecting autofluorescence (excitation: 405 nm, emission: 425 nm) and optical density (OD) at 470, 505, 615 and 750 nm had been examined over a interval of seven days. In our microfluidic setup, OD displays the discount of the depth of transmitted gentle by use of a microflow-through photometer. OD usually correlates properly with the ultimate cell quantity. Nevertheless it doesn’t enable to differentiate between alive and lifeless cells. Development will also be monitored by measuring the endogenous mobile autofluorescence with a micro flow-through fluorimeter. The fluorescence can be utilized to guage the approximate variety of physiological lively cells and, usually, is a extra delicate parameter than OD. The noticed improve of the indicators in all sensor channels clearly demonstrates cell development (Supplementary Fig. 2, Supplementary Knowledge Desk S1). Nevertheless, the best intensities had been noticed within the OD470 (Fig. 3a) and the autofluorescence (Fig. 3b) channels. Due to this fact, in additional experiments, we determined to make use of the autofluorescence channel to guage biomass accumulation. In some instances, biomass accumulation within the droplet storage coils was seen by the bare eye already after 4 days (Fig. 3c). The excessive cell density was confirmed by the commentary of chosen particular person droplets by gentle microscopy (Fig. 3d). The usually reported doubling occasions for PCC 6803 are within the vary of 10 to 12 h34,35 and roughly 2 h for UTEX 297332 and UTEX 315426. In step with this, our knowledge present that PCC 6803 grows considerably slower than UTEX 2973 and UTEX 3154 (Fig. 3a,b). Between UTEX 2973 and UTEX 3154 we noticed related exponential development charges and closing biomass values regardless of the longer lag part of UTEX 2973. Total, the person development profiles match with printed literature26,32,36 and present that microdroplet cultivation is appropriate for unicellular freshwater and saltwater strains.

Determine 3
figure 3

Development of cyanobacteria in microdroplet setup. Development kinetics of cyanobacterial strains (UTEX 3154, UTEX 2973 and PCC 6803) within the microfluidic coils measured with multi-channel photo-fluorimeter gadget displaying (a) absorbance (optical density OD) at 470 nm and (b) normalized autofluorescence within the microfluidic setup over a 7 day interval. Knowledge factors are averages of fifty ± 5 droplets. Error bars signify commonplace deviation. Normalized autofluorescence is proven as NFU. (c) Picture of a microfluidic coil carrying UTEX 2973 droplets. (d) Mild microscopy pictures of cyanobacteria PCC 6803, UTEX 2973 and UTEX 3154 in droplets after 4 days of development (scale bar: 100 µm).

Microdroplet expertise facilitates high-throughput, high-resolution dose response screening

Following the profitable droplet-based cultivation of PCC 6803, UTEX 2973 and UTEX 3154, we proceeded to use the methodology to research the response of the cyanobacteria to quite a lot of medium parameters. Due to this fact, we designed 1D screening experiments for media optimization. We used the usual development medium for the respective strains (BG-11 for PCC 6803 and UTEX 2973, AD7 for UTEX 3154) and assorted one parameter at a time by microfluidic means. The various parameters included the nitrogen (N) supply, phosphorus (P) supply in addition to medium salinity and sodium bicarbonate concentrations (Desk 1). Moreover, we proceeded to take a look at combinatorial results utilizing 2D screening the place each the N and P concentrations had been assorted concurrently. An summary of the focus vary of all examined effectors for 1D and 2D screening experiments is proven in Desk 1. A abstract of the optimum values for the medium parameters examined within the 1D screening is proven in Desk 2.

Desk 1 Vary of concentrations for 1D and 2D screening experiments of chosen medium elements used on this examine.
Desk 2 Abstract of the focus ranges the place the best development was noticed within the 1D screening.

We first began 1D testing of various concentrations of NaNO3 starting from 2 to 50 mM. The info present that the freshwater PCC 6803 and UTEX 2973 achieved most biomass values at roughly 10 and 20 mM NaNO3, respectively (Fig. 4a, Supplementary Knowledge Desk S2). For the saltwater UTEX 3154, the utmost biomass worth was achieved at roughly 30 mM NaNO3 (Fig. 4a). The standard focus of NaNO3 current within the freshwater cyanobacterial development medium BG-11 is 17.6 mM. Evaluating this worth with the limiting NaNO3 concentrations for PCC 6803 and UTEX 2973 (10 and 20 mM, respectively), we will conclude that N will not be usually the limiting nutrient in BG-11 medium. Accordingly, an earlier examine confirmed that in batch cultivation of PCC 6803 in BG-11 medium one of many main medium limitations could also be sulfate ions37. Concerning the saltwater medium AD7, the focus of NaNO3 is 12 mM. Contemplating that the biomass accumulation of UTEX 3154 solely peaked at round 30 mM NaNO3, our knowledge recommend that N could also be a limiting nutrient in AD7. That is supported by the unique PCC 11901 pressure publication the place the authors decided the best NaNO3 focus to be between 24 and 48 mM26. Total, these knowledge present that, given sufficient N, all three cyanobacteria are quickly restricted by different vitamins. Due to this fact, we proceeded to use our microfluidic method to check the impact of various concentrations of phosphate (Okay2HPO4 for UTEX 2973 and PCC 6803 and KH2PO4 for UTEX 3154) as much as a most focus of 5 mM. The info present that the utmost biomass values had been achieved between 2.5 and three.5 mM phosphate for PCC 6803 and UTEX 2973 and 0.75 mM phosphate for UTEX 3154 (Fig. 4b, Supplementary Knowledge Desk S3). Wanting on the formulations of the bottom medium, phosphate is current at a focus of 0.175 mM Okay2HPO4 in BG-11 and 0.37 mM KH2PO4 in AD7. Due to this fact, our microfluidic development knowledge recommend that each media are P poor. That is significantly the case for BG-11 which has a Okay2HPO4 focus 5 to six occasions decrease than the degrees at which we noticed the best biomass accumulation. It has been advised that media designed for the expansion of algae and cyanobacteria are sometimes P restricted because of an absence of compatibility with the Redfield ratio38. This ratio describes the quantity of carbon (C), N and P usually current in each phytoplankton biomass and in dissolved nutrient swimming pools and has been decided to be 106 C:16 N: 1 P. Primarily based on the media formulation used within the examine, AD7 presents a N:P ratio of 32:1 and BG-11 100:1. Thus, supporting our earlier speculation that each media, and BG-11 particularly, could also be P restricted.

Determine 4
figure 4

Extremely resolved dose–response screenings of key macronutrients in UTEX 3154, UTEX 2973 and PCC 6803. (a) Normalized autofluorescence after 7 days in response to various concentrations of NaNO3. (b) Normalized autofluorescence after 7 days in response to various concentrations of Okay2HPO4 (UTEX 2973, PCC 6803) or KH2PO4 (UTEX 3154). (c) Normalized autofluorescence after 7 days in response to various concentrations of bicarbonate (NaHCO3). Knowledge factors are averages of 10 ± 3 droplets. Error bars signify commonplace deviation. Normalized autofluorescence is proven as NFU.

The autotrophic development of cyanobacteria requires a supply of inorganic C which is often offered as CO2. CO2 may be provided by air (0.04% CO2), CO2 enriched air (usually 1–5%) or within the type of bicarbonate salts. Putting in a gasoline provide to cyanobacterial cultures may be pricey and presents a logistical problem for parallel experimentation. Due to this fact, sodium bicarbonate is a well-liked low-cost inorganic C supply for cyanobacterial medium. Right here, we examined the addition of NaHCO3 to BG-11 and AD7 within the vary of 0 to 100 mM (Fig. 4c, Supplementary Knowledge Desk S4). The addition of bicarbonate improved the expansion of PCC 6803 from 35 mM and achieved a most biomass accumulation at roughly 50 mM. For UTEX 2973 and UTEX 3154 there was no vital distinction in biomass accumulation within the examined vary. In our earlier experiments we now have proven that BG-11 is P restricted (Fig. 4b). This might clarify the dearth of response of UTEX 2973 to the various bicarbonate concentrations. In regard to UTEX 3154, a earlier examine in Synechococcus sp. PCC 7002 confirmed that vital biomass accumulation was solely seen with bicarbonate concentrations larger than 500 mM39. This implies that the vary examined right here could not comprise the best bicarbonate values for this cyanobacterium. Nevertheless, Synechococcus sp. PCC 7002 and UTEX 3154 current vital genetic variations due to this fact it’s tough to make particular conclusions. Lastly, one other issue could possibly be gentle limitation. In our experiments we used 20 ± 5 µmol photons m–2 s–1, whereas the utmost development charges of UTEX 2973 had been reported at 1500 µmol photons m–2 s–1, 42 °C and 5% CO233. Nevertheless, this isn’t straight comparable as the sunshine path in our microfluidic setup is 1.0 mm versus the 27 mm used to find out the utmost development charges of UTEX 2973 within the literature. Additional checks can be wanted to ascertain a strong comparability.

Salinity of the expansion medium is a vital issue as excessive salinities can induce quite a lot of stresses and consequently pose a problem to cell survival40. As well as, future large-scale cultivation of cyanobacteria needs to be achieved in seawater as a result of restricted freshwater assets current on Earth. Due to this fact, there was an elevated curiosity in prospecting for and growing salt-tolerant cyanobacterial chassis. Right here, we used our microfluidic platform to find out the salt tolerance of PCC 6803, UTEX 2973 and UTEX 3154 (Fig. 5, Supplementary Knowledge Desk S5). As anticipated, the 2 freshwater strains exhibit decrease salt tolerances than UTEX 3154. UTEX 2973 reveals the bottom salt tolerance with a decline in biomass accumulation beginning at 0.3 M NaCl. PCC 6803 maintains related ranges of biomass accumulation till 0.65 M with a pointy decline noticed thereafter. Complete inhibition was noticed of each PCC 6803 and UTEX 2973 at 0.7 M NaCl. UTEX 3154 accumulates related biomass ranges till roughly 0.8 M, whereafter a pointy decline can be noticed. It’s value noting right here that the bottom AD7 used on this examine incorporates 308 mM NaCl and eight mM KCl due to this fact the info present that UTEX 3154 can tolerate salt concentrations as much as 1.1 M NaCl. These values are in accordance with the printed literature on salt tolerance in cyanobacteria41. Our knowledge present that this microfluidic setup can be utilized for high-resolution screening for optimum salinity cultivation situations of single-celled cyanobacteria and will function an efficient high-throughput methodology to display screen for strains with elevated salt tolerance in future research.

Determine 5
figure 5

1D screening of NaCl tolerance in cyanobacteria. Normalized autofluorescence after 7 days in response to various concentrations of NaCl proven for 3 totally different cyanobacterial strains, UTEX 3154, UTEX 2973 and PCC 6803. Knowledge factors are averages of 10 ± 3 droplets. Error bars signify commonplace deviation. Normalized autofluorescence is proven as NFU.

Simultaneous screening of phosphorus and nitrogen is important for medium optimization

The above outcomes show that our microfluidic platform can quickly generate droplet screening sequences with a variety of various parameters (i.e. 1D screening). Nevertheless, it’s typically of curiosity to display screen a mixture of parameters. Due to this fact, we took benefit of the capabilities of the droplet-based method to concurrently differ two impartial parameters (2D screening). The outcomes of a 2D medium optimization experiment are 2D response diagrams the place we will simply estimate the impact of the 2 variables on one readout parameter (e.g. biomass accumulation). For the 2D proof of idea experiment we determined to display screen N (NaNO3) and P (Okay2HPO4 or KH2PO4) sources in conjunction (Fig. 6, Supplementary Knowledge Desk S6, S7, S8). These two parameters had been chosen as a result of the N:P ratio is likely one of the key parameters that influences algal development and quickly optimizing N and P concentrations is key for cost-efficient scale-up of microalgal cultures42. Our knowledge present that closing biomass values in PCC 6803 and UTEX 2973 peaked on the most N values examined (0.45 mM Okay2HPO4/30 mM NaNO3 for PCC 6803 and 0.4 mM Okay2HPO4/30 mM NaNO3 for UTEX 2973) (Fig. 6a,b). Curiously, a rise in N at low P values or a rise of P at low N values was not adequate to acquire excessive biomass values. Solely when each parameters had been elevated concurrently was a major improve in closing biomass values noticed. This contrasts with the conclusions from the 1D screening knowledge the place P appeared to be limiting the tradition. This implies that it’s extra environment friendly to discover a good stability between N and P as a substitute of simply growing one parameter. Due to this fact, 2D screening is key to understanding nutrient dynamics in cyanobacterial cultures. Concerning the N:P ratio, each strains achieved their most biomass values at a ratio of roughly 100:1. This is similar ratio as BG-11 which means that the basic cyanobacterial medium has a great N:P ratio however would profit from larger concentrations of each N and P. For UTEX 3154 no clear development was noticed inside the examined N:P vary (Fig. 6c). Along with the 1D screening knowledge, this implies that N often is the limiting nutrient within the AD7. Nevertheless, the likelihood {that a} nutrient apart from N or P limits the tradition stays open.

Determine 6
figure 6

2D screening of nitrogen and phosphorus supply in cyanobacterial cultivation utilizing commonplace development media. (a) PCC6803 normalized autofluorescence after 7 days in response to various concentrations of NaNO3 and Okay2HPO4 in BG-11 medium. (b) UTEX 2973 normalized autofluorescence after 7 days in response to various concentrations of NaNO3 and Okay2HPO4 in BG-11 medium. (c) UTEX 3154 normalized autofluorescence after 7 days in response to various concentrations of NaNO3 and KH2PO4 in AD7 medium. Knowledge factors are averages of roughly 5 ± 3 droplets.

Improved N:P ratios considerably improve biomass accumulation in excessive density cultivation

To evaluate whether or not the optimum N:P concentrations advised by the microfluidic 2D experiments may be translated to a bigger scale cultivation setup, we carried out development assays in a laboratory setting. To this finish, PCC 6803 and UTEX 2973 had been grown in 25 mL high-density cultivators the place gaseous CO2 is provided through built-in semi-permeable membranes. With this setup, development was in contrast in commonplace BG-11 medium (0.175 mM Okay2HPO4, 17.6 mM NaNO3) and BG-11 with the optimized N:P concentrations obtained from the 2D N:P microfluidic screening experiment. Extra particularly, 0.45 mM Okay2HPO4 with 30 mM NaNO3 for PCC 6803 and 0.4 mM Okay2HPO4 with 30 mM NaNO3 for UTEX 2973. For each PCC 6803 and UTEX 2973 vital will increase (p < 0.05) of ultimate biomass values of seven.4% (Fig. 7a) and 15.7% (Fig. 7b), respectively, could possibly be noticed. Total, this confirmed that the findings from the microfluidic experiments are transferable to biotechnologically related high-density cultivation setups.

Determine 7
figure 7

Excessive-density cultivation in BG-11 medium containing N:P ratios optimized by 2D microfluidic screening. Development of (a) PCC 6803 and (b) UTEX 2973 in commonplace vs. optimized N/P ratio noticed by measuring optical density (OD) at 750 nm. n = 3 and error bars signify commonplace deviation.

Droplet microfluidics considerably will increase the multiplexing capability of cyanobacterial cultivation

The cyanobacterial cultivation methodology that provides the best multiplex functionality is at present the microtiter plate (MTP). Assuming a 96-well MTP with 200 µL in every properly, and that every microdroplet is the useful equal of a properly on an MTP, we will set up a comparability between a MTP and the microfluidic method established on this work. For instance, for a 1% focus decision of a single substance (1D screening), 100 focus steps are required. If 10 droplets are screened per focus step, it is a whole of 1000 droplets. With a quantity of 0.2 µL/droplet, a complete of 200 µL of medium is required within the microfluidic reactor. Alternatively, for a similar experiment in an MTP, this may require 200 mL of medium throughout 10 MTPs. For a similar 1% decision within the case of a binary combination (2D screening), 10,000 focus steps are required. Due to this fact, a complete of two mL of medium is required within the microfluidic reactor. To display screen the identical quantity of effector combos in a MTP, 2L of medium throughout 100 MTPs can be required. Your complete microfluidic preparation course of, together with cell counting and dilution, medium and effector preparation, sequence technology and measurement requires roughly 4 h. For 1D screening, the preparation of 10 MTPs with 100 focus steps might feasibly be achieved in 4 h, albeit with a a lot larger threat of error. Nevertheless, when shifting to 2D screening, the preparation and cultivation of 100 MTPs turns into unfeasible. With droplet microfluidics, the transfer from 1 to 2D screening requires little or no further preparation time and the experimental footprint stays the identical.

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