Open Access

Mimicking lichens: incorporation of yeast strains together with sucrose-secreting cyanobacteria improves survival, growth, ROS removal, and lipid production in a stable mutualistic co-culture production platform

  • Tingting Li1,
  • Chien-Ting Li1,
  • Kirk Butler2,
  • Stephanie G. Hays3, 4,
  • Michael T. Guarnieri5,
  • George A. Oyler1 and
  • Michael J. Betenbaugh1Email author
Biotechnology for Biofuels201710:55

https://doi.org/10.1186/s13068-017-0736-x

Received: 13 August 2016

Accepted: 17 February 2017

Published: 21 March 2017

Abstract

Background

The feasibility of heterotrophic–phototrophic symbioses was tested via pairing of yeast strains Cryptococcus curvatus, Rhodotorula glutinis, or Saccharomyces cerevisiae with a sucrose-secreting cyanobacterium Synechococcus elongatus.

Results

The phototroph S. elongatus showed no growth in standard BG-11 medium with yeast extract, but grew well in BG-11 medium alone or supplemented with yeast nitrogen base without amino acids (YNB w/o aa). Among three yeast species, C. curvatus and R. glutinis adapted well to the BG-11 medium supplemented with YNB w/o aa, sucrose, and various concentrations of NaCl needed to maintain sucrose secretion from S. elongatus, while growth of S. cerevisiae was highly dependent on sucrose levels. R. glutinis and C. curvatus grew efficiently and utilized sucrose produced by the partner in co-culture. Co-cultures of S. elongatus and R. glutinis were sustained over 1 month in both batch and in semi-continuous culture, with the final biomass and overall lipid yields in the batch co-culture 40 to 60% higher compared to batch mono-cultures of S. elongatus. The co-cultures showed enhanced levels of palmitoleic and linoleic acids. Furthermore, cyanobacterial growth in co-culture with R. glutinis was significantly superior to axenic growth, as S. elongatus was unable to grow in the absence of the yeast partner when cultivated at lower densities in liquid medium. Accumulated reactive oxygen species was observed to severely inhibit axenic growth of cyanobacteria, which was efficiently alleviated through catalase supply and even more effectively with co-cultures of R. glutinis.

Conclusions

The pairing of a cyanobacterium and eukaryotic heterotroph in the artificial lichen of this study demonstrates the importance of mutual interactions between phototrophs and heterotrophs, e.g., phototrophs provide a carbon source to heterotrophs, and heterotrophs assist phototrophic growth and survival by removing/eliminating oxidative stress. Our results establish a potential stable production platform that combines the metabolic capability of photoautotrophs to capture inorganic carbon with the channeling of the resulting organic carbon directly to a robust heterotroph partner for producing biofuel and other chemical precursors.

Keywords

CyanobacteriaYeastsCo-cultureSucroseROSArtificial lichenHydrogen peroxideLipid production

Background

Most microbes live in complex interacting heterogeneous populations in nature, including the gut microbiota of insects and animals, waste sludge, and lichens. Lichens, composed of photoautotrophs and heterotrophs, provide an extremely stable and self-supporting symbiosis [1, 2]. These autotroph–heterotroph symbionts thrive by performing a wide variety of functions that combine the attributes and metabolic pathways of multiple microbes. Carbon needed for biosynthesis is typically provided by the photobiont (cyanobacteria or green algae) and supplied to the heterotroph. In turn, the heterotrophic symbionts, typically fungi, provide minerals and nutrients, as a result of the vast array of metabolites in their secondary metabolism, as well as carbon dioxide, water, and protection from the environment [35]. Furthermore, these relationships offer a division of labor to each participant by allowing the photobiont to perform carbon dioxide (CO2) and energy capture and the heterotroph to accumulate biomass and produce complex metabolites. This differentiation allows the community to function most efficiently by allowing each organism to perform specific tasks. However, the cultivation of natural lichens remains challenging and the exact nature of these complex interactions among lichens is not well known or characterized.

Phototrophs and heterotrophs exhibit mutualistic, competitive, and allelopathic interactions. Heterotrophic bacteria related to Pseudomonas and isolated from cyanobacterial mat led to an eightfold increase in the cyanobacterial biomass accumulation when co-cultured together with the cyanobacterium Synechocystis PCC6803 [6]. A marine cyanobacterium Prochlorococcus has been pair-wise co-cultured with hundreds of marine heterotrophic bacteria, some of which enabled Prochlorococcus to grow faster and reach higher final culture chlorophyll fluorescence. In contrast, some strains showed antagonistic interactions with Prochlorococcus and several displayed strong growth inhibition on Prochlorococcus [7]. Differential interactions have also been reported in green microalgal consortia [810]. Co-culture of the green algae Chlorella with isolates from a microbial consortium resulted in 0.5-3 times greater algal growth than that of algal cells alone, and the growth of the microbial isolates was also promoted in co-culture with algae [8]. On the other hand, the interactions between Chlorella and heterotrophic microbes can exhibit variable patterns, in which some co-cultured bacteria inhibited algal growth [10], and some strains had no influence [9]. Therefore, the physiological behaviors and interactions between phototrophs and heterotrophic partners in microbial consortia can vary and the exact interaction is not always evident.

Specific synergistic relationships between photoautotrophs and heterotrophs have been observed. Exchange of CO2 and O2 gas between phototrophs and their heterotrophic partners often occurs. For example, dissolved oxygen in a pure culture of yeast increases dramatically when microalgae is supplied [11, 12], and the oxygen generated by microalgae benefits the co-cultured heterotrophic microorganisms for biomass and lipid production [13, 14]. Alternatively, oxygen removal by a heterotrophic partner can facilitate photosynthesis and lipid production in microalgae. High oxygen accumulation imposes an impediment on algal growth by inhibiting photosynthesis, which can be especially problematic in closed photobioreactors [15]. Minimizing dissolved oxygen in the culture medium has been shown to enhance the lipid production of the green alga Chlorococcum littorale under photoautotrophic conditions [16]. In turn, the heterotrophic partner supplies the microalgae with additional CO2 derived from several metabolic processes.

The members present in phototrophic–heterotrophic symbiosis, such as lichens, sometimes include cyanobacteria, which can convert CO2 and sunlight energy into useful metabolites. These cyanobacteria can produce soluble organic carbon, including glucose and sucrose as a result of photosynthesis [1719]. Sucrose accumulation in some cyanobacteria occurs as a cellular response to salt stress. Previously, the cyanobacterium Synechococcus elongatus PCC7942 has been engineered to effectively secrete intracellular sucrose in the culture medium by over-expressing the Escherichia coli gene cscB, which encodes a proton and sucrose symporter [20]. Addition of 200 mM NaCl in the culture medium induced about 80% of biomass generated as extracellular sucrose. Furthermore, both cell expansion and sucrose production occurs simultaneously, and the ratio of cellular biomass to sucrose is adjustable via changing the osmotic pressure in the culture medium [20]. This characteristic makes sucrose-secreting S. elongatus a promising candidate for providing carbon to a co-cultured heterotrophic partner in a mutualistic co-culture pairing as shown in previous experiments [21, 22].

One potential benefit of photobiont–heterotroph co-culture is the production of potential biofuel precursors. Oleaginous yeasts such as Cryptococcus curvatus and Rhodotorula glutinis, with their ability to channel various carbon feeds into complex secondary metabolites, have proven to be generators of biofuel precursors [2325], with C. curvatus and R. glutinis accumulating up to 69 and 72% lipid, respectively [26, 27]. However, in order to synthesize advanced biofuels products, oleaginous yeasts must be fed with organic carbon feedstocks, which adds processing steps and increases costs. The wide abundance of cellulosic materials represents one alternative feedstock, but these often require additional pre-treatment steps [28, 29] and can lead to toxic culture environments [30, 31].

Co-culture fermentation of cyanobacteria generating sugar as substrate represents a potentially viable alternative to cellulosic feedstocks. Such an approach provides a division of labor and symbiotic interactions between the photobiont and heterotrophs, in which the chemical precursors for biofuel production could be generated. Therefore, the goal of this study was to establish a synthetic system mimicking lichens by pairing phototrophic sucrose-secreting S. elongatus together with yeast strains capable of producing metabolic byproducts, including fatty acid biofuel precursors. Equally important was an examination to determine if the presence of the yeast heterotrophic partner could be beneficial to and symbiotic with the phototrophic partner. In this way, we can begin to establish stable phototrophic–heterotrophic partnerships which represent a potentially sustainable production platform for biofuels and other products by combining the light and CO2 harvesting capability of a phototroph with the robust metabolic performance and varied capabilities of heterotrophic yeast.

Methods

Growth conditions and assays

Inoculum culture of sucrose-secreting cyanobacterium, cscB+ S. elongatus PCC 7942 [20], was prepared in 250 mL Erlenmeyer flasks containing 100 mL BG-11 medium supplemented with 1 g/L HEPES (pH 8.9) and 100 mM NaCl. Flasks were incubated at 28 °C agitated with filter-sterilized air that was enriched with CO2 to 1% (v/v). Illumination was provided by cool-white fluorescent lamps to give a light intensity of 65 μmol/m2 s with 16:8 h light/dark cycle. For experimental cultures (pure cultures of S. elongatus and co-cultures of S. elongatus together with yeasts), exponentially growing inoculum was transferred into each flask containing BG-11 medium (1.6 × 107/mL in initial culture, if no specific statement), which was supplemented with 3 g/L HEPES (pH 8.9), 1.2 g/L yeast nitrogen base without amino acids (YNB w/o aa), 4 mM ammonium chloride, 1 mM IPTG, and 100 mM NaCl (denoted BG-11[co]). Flasks were weighed at the setup of the time course experiments, and water was added prior to each sampling to correct for evaporation. Cultures were maintained under illumination of 100 μmol/m s with 16:8 h light/dark cycle.

Yeast strains C. curvatus (ATCC 20509), R. glutinis (ATCC 204091), and Saccharomyces cerevisiae (ATCC 204508) were grown overnight in the YPD medium containing 10 g/L yeast extract (YE), 20 g/L peptone, and 20 g/L glucose. YPD cultures were then inoculated (2%, v/v) into 20 g/L sucrose supplemented BG-11[co] medium for seed preparation, maintaining within log phase growth under 65 µmol m2 s‒1 constant light at 28 °C and 200 rpm in 150 mL flasks. The yeast seeds were washed twice with BG-11 medium before inoculation, and the initial inocula were 0.01 OD600 for C. curvatus and R. glutinis, and 0.03 OD600 for S. cerevisiae.

Quantification of cyanobacteria and yeasts

For axenic culture of yeasts, optical density was measured spectrophometrically at 600 nm (OD600). In co-culture, serial dilutions were plated on YPD plates to determine yeast viability. Colony-forming units (CFU) were counted after 2 days’ incubation at 25 °C.

Synechococcus elongatus concentration was analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) after adding with BD Liquid Counting Beads (BD Biosciences, San Jose, CA). The absolute cell numbers in samples were determined by comparing cellular events to the beads events measured by the flow cytometer using the equation provided in the kit’s TDS document. S. elongatus viability was determined by staining with dye solutions and adding with fluorescent counting beads in Cell Viability Kit (BD Biosciences, San Jose, CA) prior to flow cytometry, following the manufacturer’s instructions.

Measurements and analysis

After pelleting cells, sucrose was determined directly from the supernatant fraction with a Biochemistry Analyzer YSI 2700 Select (YSI Incorporated, Yellow Springs Instrument Co., Inc., Ohio) equipped with a sucrose (YSI 2703) membrane and standardized with 5.0 g/L standard sucrose (YSI 2780) solutions. The pH of the samples was determined using a pH meter (Accumet Model 15, Fisher Scientific) using a combination pH electrode with silver/silver chloride reference (Fisher Scientific), which had been previously standardized to pH 4, 7, and 10. Hydrogen peroxide concentration in the culture was quantified with the Fluorimetric Hydrogen Peroxide Assay Kit (MAK165, Sigma-Aldrich, St. Louis, MO, US) according to manufacturer’s instruction. To determine biomass dry weight (DW), cell suspension sample was centrifuged at 4000 rpm for 5 min, washed twice with distilled water, and then lyophilized by freeze dry vacuum (LGJ-25, Xiangyi, China). Total lipids and lipid profile were analyzed with gas chromatography, using heptadecanoic acid (C17:0) as an internal standard. The corresponding chromatographic conditions and the extraction/transesterification method are described in Dong et al. [32].

Statistics

Three biological replicates were performed for all data collection, and the statistical tests for significance were determined via a two-tailed t test, with a significance level of 0.05, unless stated otherwise.

Results and discussion

Axenic growth of different yeast strains

Previously, sucrose permease, cscB, expressing S. elongatus cells were engineered to efficiently secrete sucrose into the culture medium. The sucrose production is dependent upon osmotic pressure, alkaline environment, and supply of IPTG inducer [20]. To examine if various yeasts could grow well with sucrose as the carbon source, and under the same media conditions in which S. elongatus cells grow and produce sucrose, yeast strains R. glutinis, C. curvatus, and S. cerevisiae were cultured individually in modified BG-11 medium.

The yeast C. curvatus could not grow in BG-11[co] medium supplemented with sucrose only (Fig. 1a). Alternatively, R. glutinis and S. cerevisiae cells grew in these conditions only after a long lag phase (Fig. 1b, c). However, all the three yeast strains performed well in 2 g/L of sucrose complemented BG-11[co] medium containing either yeast extract (YE) or yeast nitrogen base without amino acids (YNB w/o aa). YE yielded the best growth of each strain, with the highest cell density over the duration of the culture.
Fig. 1

Axenic growth of different yeast strains. ac Effect of medium components on monoculture of yeasts; “BG” indicates BG-11 added with 2 g/L sucrose, 4 mM ammonium chloride, 1 mM IPTG, and 100 mM NaCl; “BG + YNB w/o aa” indicates “BG” supplied with YNB w/o aa; “BG + YE” indicates “BG” supplied with YE. df Effect of glucose/sucrose and light/dark on monoculture of yeasts; Medium used here was BG-11[co] supplied with 2 g/L sucrose or 2 g/L glucose, as indicated in the legends. gi Effect of sucrose concentration on monoculture of yeasts; Medium used here was BG-11[co] supplied with various concentration of sucrose, as indicated in the legends. jl Effect of NaCl concentration on monoculture of yeasts. Medium here was BG-11[co] supplied with 2 g/L sucrose, but with adjusted NaCl concentration in each condition. a, d, g, j for C. curvatus; b, e, h, k for R. glutinis; c, f, i, l for S. cerevisiae. Light condition was used if no specific statement. All data are averages of biological triplicates ± standard deviation

Typically, wild-type yeast cells require YE for growth, which provides nitrogenous compounds, carbon, sulfur, trace nutrients, vitamins, and other important growth factors. However, the proposed partner strain S. elongatus showed no growth in BG-11 medium with added YE (see Additional file 1), but could grow well in BG-11 medium supplemented with or without YNB w/o aa. The growth of a diverse array of cyanobacteria is inhibited by exogenous metabolites such as amino acids [3335], although endogenous synthesis is obligatory. For example, glutamine, lysine, as well as histidine inhibited the growth of Synechocystis PCC6803 [35]. Therefore, YNB w/o aa instead of YE was adopted as component supplement in BG-11[co] medium for yeast growth.

The above results in Fig. 1a–c indicated that the three wild-type yeast strains can consume sucrose as a sole carbon source, and no sucrose remained in the final culture medium (data not shown). As glucose is the most ubiquitous carbon source for yeast in laboratories, growth on sucrose was compared to this carbon source. C. curvatus exhibited similar growth with sucrose as a substrate compared with glucose; while R. glutinis and S. cerevisiae exhibited a prolonged lag phase with sucrose (Fig. 1d–f). Additionally, illumination had little influence on the growth of the three yeast strains, which indicates compatibility of yeast strains with phototrophs under light condition.

To further evaluate if yeast cells could grow on a sucrose substrate with a phototrophic partner in a co-culture system, the effect of varying sucrose concentrations was investigated using pure cultures of the three yeast strains in BG-11[co] medium. Growth of S. cerevisiae demonstrated dependence on sucrose concentration in the medium as previously reported [21, 22] (Fig. 1i). However, both C. curvatus and R. glutinis showed similar growth profiles regardless of the sucrose concentrations, although R. glutinis exhibited an overall longer lag phase (Fig. 1g, h). A similar delay in the growth phase was observed for the R. glutinis when comparing growth on sucrose to growth on glucose (Fig. 1e).

The sucrose production by cscB + S. elongatus cells can be tuned via the osmotic pressure imposed on the culture system, with up to ~80% of total mass generated as sucrose at higher salinities [20]. Therefore, we investigated yeast growth with increasing NaCl concentrations (0, 100, 150 and 200 mM) in BG-11[co] medium (Fig. 1j–l). The three yeast strains exhibited similar growth patterns in which the lag phases were extended and the growth rates delayed with increasing salt concentrations (see Additional file 2). Lag phases appeared approximately two times longer for growth of the three yeast strains with 200 mM NaCl compared with no supplemental NaCl supply. To ensure robust growth of the three yeast strains, 100 mM NaCl was used with BG-11[co] medium in co-culture with S. elongatus in subsequent studies.

Axenic growth of S. elongatus

Sucrose export by cscB + S. elongatus is dependent upon medium pH, with maximal sucrose export in an alkaline environment [20]. To ensure that sucrose was continuously and efficiently secreted, three different Good’s buffers, HEPES, HEPPSO, and TAPS, were tested for their capability in buffering the axenic growth of cyanobacteria S. elongatus cells. As shown in Fig. 2, S. elongatus cells did not grow and gradually died in BG-11[co] medium without any buffering agent, in which the pH dropped from an initial value of 8.8 to below 6 after 4 days. For the pH buffered groups, however, the S. elongatus cells exhibited similar growth curves and exported the sucrose at comparable rates, with the sucrose level in the medium gradually increasing up until 9 days. Additionally, the three buffers displayed similar pH buffering capabilities and could all maintain pH values above 7.2 over the culture duration. Based on these findings, HEPES buffer was chosen as the buffering agent to be used in following co-culture experiments.
Fig. 2

Axenic growth of S. elongatus with different pH buffer in the medium. a Cell numbers (per mL) of S. elongatus; b Sucrose (mg/L) secreted by S. elongatus; c pH value. 3 g/L of different buffers were used in the medium. All data are averages of biological triplicates ± standard deviation

Co-culture of S. elongatus with different yeast strains

Following the initial mono-culture experiments, the CscB-expressing S. elongatus was next grown in co-culture medium BG-11[co] with the different yeast strains. Shown in Fig. 3 are the cell counts for S. elongatus and the three yeast strains, along with sucrose levels. The S. elongatus cells grown in co-culture with yeast cells exhibited slightly higher cell densities, on average, than cells in mono-culture in the middle to late exponential phase (6 and 9 days). Among yeast strains, wild-type S. cerevisiae displayed no growth in the co-culture consistent with previous findings [22], and actually decreased in cell numbers over the time period, even though the initial CFU numbers inoculated were 20 times larger compared with the other two yeast species. C. curvatus showed a modest enhancement in cell numbers from 1.8 × 105 to 5.7 × 106 CFU over the 11 day culture duration. Finally, R. glutinis cells grew efficiently in co-culture, reaching cell densities five times higher than the other two cultures. Surprisingly, R. glutinis and C. curvatus cells utilized similar amounts of sucrose with sucrose levels kept below 200 mg/L in the medium compared to levels above 500 mg/L in the mono-culture of S. elongatus. Sucrose levels in the co-culture medium of S. cerevisiae with S. elongatus showed an accumulation pattern more similar to the mono-culture of S. elongatus, with final sucrose levels around 500 mg/L after 11 days, indicating the yeast S. cerevisae used in this study could not utilize sucrose at these levels, and likely require higher levels of sucrose for expansion as indicated in Fig. 1 and as demonstrated in the literature [21, 22].
Fig. 3

Co-culture of S. elongatus with different yeast strains. a Cell numbers of S. elongatus; b CFU numbers of yeasts; c Sucrose secreted by S. elongatus. All data are averages of biological triplicates ± standard deviation

In order to evaluate sustainability of a co-culture system composed of cyanobacteria and yeast, long-term batch and semi-continuous cultures of the cyanobacteria S. elongatus together with the yeast R. glutinis were carried out. After 4 days in batch, the growth of S. elongatus plateaued but remained stable for more than 1 month with a marginal increase in densities (Fig. 4a). Meanwhile, the CFU of the yeast R. glutinis increased rapidly for 8 days and then increased more gradually in the batch cultures (Fig. 4b). In semi-continuous culture, cyanobacterial and yeast cell numbers were reduced by half through replacement of the original cultures with fresh BG-11[co] medium at 10 and 21 days, respectively, shown in Fig. 4 as arrows. Nonetheless, cell numbers rapidly recovered and eventually attained similar levels to the batch culture. Phototrophic cyanobacteria and heterotrophic yeasts possess different resource acquisition traits, so less competition is observed between members compared with either co-cultures of phototrophs or co-cultures of heterotrophs. For example, acetate-secreting cyanobacterium Synechococcus sp. overtook a culture when growing together with the acetate-consuming algae Chlamydomonas reinhardtii, and the cyanobacterial cells needed to be encapsulated in alginate beads to slow their growth [36].
Fig. 4

Long-term batch and semi-continuous co-culture of S. elongatus and R. glutinis. a Cell numbers of S. elongatus; b CFU numbers of yeast R. glutinis. Arrows indicate the time when 50 mL of original culture was withdrawn and 50 mL of fresh BG-11[co] medium was added. All data are averages of biological triplicates ± standard deviation

Interestingly, the final biomass DW in the batch co-culture was much higher than the batch mono-culture of S. elongatus (Table 1). We also examined lipid content in the co-culture versus monoculture. The total fatty acids (TFA) in the batch co-culture was 54% higher than in mono-culture, in part because of the increased biomass from the yeast partner. In semi-continuous culture, the biomass DW and TFA in the final harvested biomass were 650 and 35 mg/L, respectively. With addition of the 50 mL of culture that was removed both at 10 and 21 days, respectively, the total biomass yield finally in semi-continuous culture was 721 mg/L of medium. The TFA was 39 mg/L, which was about 60% above the levels for S. elongatus mono-culture. Also, the cultures could be manipulated by nitrogen or other stresses for even higher levels of lipids as desired.
Table 1

Biomass DW and total fatty acids (TFA) in batch mono-culture, batch co-culture, and semi-continuous co-culture

 

Mono-culture (mg/L)

Co-culture (mg/L)

Semi-continuous culture

Final harvest (mg/L)

10st day withdraw (mg in 50 mL)

21st day withdraw (mg in 50 mL)

Total harvest (mg/L)

DW

570.37 ± 3.49

792.36 ± 0.98

650.00 ± 3.93

28.90 ± 0.52

50.30 ± 0.33

721.00 ± 1.02

TFA

24.61 ± 1.49

37.86 ± 0.77

34.66 ± 3.77

1.64 ± 0.02

2.69 ± 0.07

39.02 ± 2.13

All data are averages of biological triplicates ± standard deviation

A fatty acid profile analysis revealed that the principal fatty acids produced in the mono- and co-cultures were palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), and linoleic acid (C18:2) (Fig. 5). While the mono-cultures of S. elongatus contained significantly higher levels of palmitic, stearic, and oleic acids, the co-cultures of both batch and semi-continuous culture showed enhanced levels of palmitoleic and linoleic acids. These oils could serve as potential oil feedstocks for biodiesel production that require long-chain fatty acids with 16 and 18 carbon atoms [37].
Fig. 5

Comparison of fatty acid profiles in different cultures. All data are averages of biological triplicates ± standard deviation. *Significant difference in paired comparisons (p < 0.05)

Chemical interaction involved in cultures of cyanobacteria with/without yeast

While the yeast R. glutinis is clearly dependent on the cyanobacterium for a supply of sucrose for growth, we wanted to explore any impact the yeast may have on the growth and maintenance of S. elongatus in HEPES buffered BG-11[co] medium.

Hydrogen peroxide (HOOH) and related reactive oxygen species (ROS) can arise from a number of sources, including light exposure [38, 39]. Further, chemicals present in cultural systems are known to produce HOOH [40]. Previously, HEPES with 1-10 mM concentration in culture could generate enough HOOH to kill the axenic phytoplankter Prochlorococcus strain [40]. Indeed, in our cultures, BG-11[co] medium with various concentrations of HEPES produced increasing levels of HOOH (Fig. 6), with HOOH concentrations approaching an asymptotic level after several days. S. elongatus cells at various inoculum concentrations were tested to check the influence of HOOH on their axenic growth and to explore any role of yeast heterotrophs on the growth of the cyanobacterium under these conditions. At higher cell inoculum concentrations (1.6 × 107 and 3.2 × 106 cells/mL), the axenic growth of S. elongatus was robust (Fig. 7a, b), with negligible fractions of dead cells (data not shown). With a lower inoculum concentration of 1.6 × 106 cells/mL, however, no growth of axenic S. elongatus culture was observed, and the fraction of live cells rapidly dropped to less than 10% after 2 days, and eventually all cells died (Fig. 7c, d). Correspondingly, the HOOH levels were kept below 1 µM in the cultures with higher cell inoculum concentrations (data not shown), but rapidly increased in the cultures with the lowest inoculum concentration, and reached 30 µM at the end of culture, showing a similar value as in BG-11[co] medium (Fig. 8). Although the unicellular cyanobacterium S. elongatus PCC 7942 possesses enzymes including ascorbate peroxidase and catalase to help to breakdown HOOH [41], the S. elongatus culture at the lowest cell inoculum was unable to prevent HOOH accumulation, likely resulting in the death of all S. elongatus cells.
Fig. 6

HOOH formation in illuminated BG-11[co] medium containing various concentration of HEPES. All data are averages of biological triplicates ± standard deviation

Fig. 7

Growth of S. elongatus with/without yeast R. glutinis. a Initial cell number of S. elongatus was 1.6 × 107/mL; b Initial cell number of S. elongatus was 3.2 × 106/mL; c Initial cell number of S. elongatus was 1.6 × 106/mL; d Live fraction of S. elongatus cells with initial cell number of 1.6 × 106/mL. All data are averages of biological triplicates ± standard deviation

Fig. 8

HOOH formation in HEPES buffered culture. Initial cell number of S. elongatus and R. glutinis was 1.6 × 106 and 2 × 105/mL, respectively. 1.25 mg/L of catalase was daily supplied. All data are averages of biological triplicates ± standard deviation

Catalase was added in order to limit the impact of HOOH in pure cultures of S. elongatus. Contrary to BG-11[co] medium and in the pure cultures of S. elongatus, HOOH was kept at minimal concentrations following addition of catalase (Fig. 8). Importantly, S. elongatus grew better in the cultures with catalase, especially at an initial inoculum of 1.6 × 106 cells/mL, while maintaining high viabilities (Fig. 7c, d). Also, catalase supplied to the axenic cultures of S. elongatus with higher inoculum concentrations of 1.6 × 107 and 3.2 × 106 cells/mL, exhibited a slightly enhancement in the growth and cell density during experimental growth.

Next, the impact of co-culturing with R. glutinis on S. elongatus growth and HOOH was investigated. The presence of the yeast R. glutinis partner clearly facilitated the growth of S. elongatus in co-cultures consistent with effects seen in dilute co-culture of cyanobacteria with other heterotrophs including E. coli, B. subtilis, and S. cerevisiae [22]. Cell numbers of S. elongatus with higher inoculum concentrations were significantly augmented in co-culture with R. glutinis compared with the axenic culture (Fig. 7a, b), even more than catalase alone. The growth enhancement was superior to the addition of catalase alone, suggesting that R. glutinis may be providing factors beyond HOOH reduction to the S. elongatus in co-culture. Even at the lowest inoculum concentration, the S. elongatus gradually grew in co-culture, albeit with a prolonged lag phase (Fig. 7c). Initially, the live fraction of S. elongatus cells decreased to approximately 60% by 3 days, but then gradually recovered to nearly 100% (Fig. 7d). At the same time, the HOOH content was progressively reduced and efficiently minimized to a similar low level as in BG-11[co] medium with catalase (Fig. 8).

The ability of pure cultures of S. elongatus to grow was strongly dependent upon the initial cell density, yet there was effectively no density dependence when catalase was supplied. These results along with the corresponding HOOH measurements indicated that HOOH accumulation in the illuminated cultures is likely to be a major cause of the toxicity effect during axenic growth of S. elongatus. However, S. elongatus in co-culture with R. glutinis could grow even at a dilute initial cell concentration, and the HOOH accumulation in the culture was efficiently reduced, suggesting that the eukaryotic heterotroph R. glutinis plays a key role in enabling its phototrophic partner’s growth by scavenging extracellular HOOH. Previous results have shown that dilute cultures of the marine cyanobacterium Prochlorococcus can be supported when co-cultured with certain marine heterotrophic bacteria [42, 43].

Furthermore, R. glutinis significantly boosts the growth of S. elongatus at high initial cell densities (Fig. 7), and the augmentation was higher than that obtained with addition of catalase. This increase indicates that the eukaryotic heterotroph may provide additional benefits to phototrophic partners in addition to HOOH elimination in order to support the growth of cyanobacteria even more. Previous studies have shown that the addition of thiosulfate, vitamin B12, biotin, and thiamine can be used to enhance and elevate the growth of the cyanobacterium Synechococcus leopoliensis CCAP1405/1 [44].

Conclusions

Culture media formulations were obtained by evaluating the impact of medium components on axenic growth of the three yeast strains and the sucrose-secreting cyanobacterium S. elongatus. The yeasts R. glutinis and C. curvatus could efficiently take up sucrose secreted from S. elongatus and expand their cell numbers in co-culture, while S. cerevisiae cells failed to grow in co-culture with S. elongatus likely due to inefficient sucrose incorporation [21]. Furthermore, robust co-cultures of R. glutinis and S. elongatus could last for more than 1 month, with higher biomass and lipid yield than the axenic culture of S. elongatus in batch culture. These findings demonstrate the potential of a stable sustainable phototrophic-heterotrophic co-culture system as an emerging bioenergy platform, taking the advantage of synthesis of organic carbon from CO2 by photobionts and channeling the carbon to oleaginous heterotrophs without the need for exogenous supplies of carbon. Equally important, the phototroph and heterotroph both exhibited symbiotic interactions with their respective partner species. The cyanobacteria provide essential organic carbon nutrients to the yeast and perhaps other components, such as oxygen. Concomitantly, the yeast cells efficiently limit the generation of toxic ROS in the culture which enable and encourage survival and expansion of S. elongatus at low cell densities, significantly facilitating the growth of S. elongatus in co-cultures. Dealing with ROS may be a common feature defining community interaction between phototrophs and heterotrophs [45]. The finding that cyanobacterial growth in co-culture with yeast was superior to cultures containing catalase suggests additional factors gained from the heterotroph in co-culture. Two of the major constituents of some lichens include cyanobacteria and eukaryotic species [1, 2]. The results from the co-culture of cyanobacteria and fungal partner constructed in this study provide strong evidence that mutual interactions can be achieved between phototrophs and eukaryotic heterotrophs. While the phototroph provides an organic carbon source to heterotroph, the heterotroph can play an equally important role by assisting in phototroph survival and robust growth by protecting the cultures against oxidative stress. In this way, we have established a successful artificial lichen system composed of photobionts and eukaryotic heterotrophs yielding symbiotic and synergistic interactions that represent a potential sustainable production platform for biofuels from sunlight and CO2.

Abbreviations

YNB w/o aa: 

yeast nitrogen base without amino acids

YE: 

yeast extract

ROS: 

reactive oxygen species

CO2

carbon dioxide

CFU: 

colony-forming units

HOOH: 

hydrogen peroxide

DW: 

dry weight

TFA: 

total fatty acids

Declarations

Authors’ contributions

TL participated in the study design, data collection and analysis, and drafted the manuscript. CTL performed the fatty acid analysis and the manuscript revision. KB assisted the laboratory work and the manuscript revision. SGH contributed strains and to the conception of this study; MTG contributed to the conception of semi-continuous culture. GAO contributed to experimental design; MJB conceived the experimental design, participated, and supervised manuscript preparation and editing. All authors read and approved the final manuscript.

Acknowledgements

We would like to thank Prof. Ed Bouwer for kindly providing culture space and Pamela A. Silver for her conceptual ideas, support, and collaboration. We also would like to thank Ph.D. candidate Daniel Shea for technical assistance with the flow cytometry.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Funding

This research was financially supported by the U.S. Department of Energy (DE-SC0012658).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Chemical and Biomolecular Engineering, Johns Hopkins University
(2)
Department of Biophysics, Johns Hopkins University
(3)
Department of Systems Biology, Harvard Medical School
(4)
Wyss Institute for Biologically Inspired Engineering, Harvard University
(5)
National Bioenergy Center, National Renewable Energy Laboratory

References

  1. Oksanen I. Ecological and biotechnological aspects of lichens. Appl Microbiol Biotechnol. 2006;73:723–34.View ArticleGoogle Scholar
  2. DePriest PT. Early molecular investigations of lichen-forming symbionts: 1986–2001. Annu Rev Microbiol. 2004;58:273–301.View ArticleGoogle Scholar
  3. Honegger R. Metabolic interactions at the mycobiont-photobiont interface in lichens. In: Carroll GC, Tudzynski P, editors. Plant relationships. New York: Springer; 1997. p. 209–21.View ArticleGoogle Scholar
  4. Palmqvist K. Cyanolichens: carbon metabolism. In: Rai AN, Bergman B, Rasmussen U, editors. Cyanobacteria in symbiosis. Dordrecht: Kluwer Academic; 2002. p. 73–96.Google Scholar
  5. Rai A. Cyanolichens: nitrogen metabolism. In: Rai AN, Bergman B, Rasmussen U, editors. Cyanobacteria in symbiosis. Dordrecht: Kluwer Academic; 2002. p. 97–115.View ArticleGoogle Scholar
  6. Abed RM. Interaction between cyanobacteria and aerobic heterotrophic bacteria in the degradation of hydrocarbons. Int Biodeter Biodegr. 2010;64:58–64.View ArticleGoogle Scholar
  7. Sher D, Thompson JW, Kashtan N, Croal L, Chisholm SW. Response of Prochlorococcus ecotypes to co-culture with diverse marine bacteria. ISME J. 2011;5:1125–32.View ArticleGoogle Scholar
  8. Park Y, Je K-W, Lee K, Jung S-E, Choi T-J. Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga. Hydrobiologia. 2008;598:219–28.View ArticleGoogle Scholar
  9. Watanabe K, Takihana N, Aoyagi H, Hanada S, Watanabe Y, Ohmura N, Saiki H, Tanaka H. Symbiotic association in Chlorella culture. FEMS Microbiol Ecol. 2005;51:187–96.View ArticleGoogle Scholar
  10. Guo Z, Tong YW. The interactions between Chlorella vulgaris and algal symbiotic bacteria under photoautotrophic and photoheterotrophic conditions. J Appl Phycol. 2014;26:1483–92.View ArticleGoogle Scholar
  11. Xue F, Miao J, Zhang X, Tan T. A new strategy for lipid production by mix cultivation of Spirulina platensis and Rhodotorula glutinis. Appl Biochem Biotechnol. 2010;160:498–503.View ArticleGoogle Scholar
  12. Kitcha S, Cheirsilp B. Enhanced lipid production by co-cultivation and co-encapsulation of oleaginous yeast Trichosporonoides spathulata with microalgae in alginate gel beads. Appl Biochem Biotechnol. 2014;173:522–34.View ArticleGoogle Scholar
  13. Cheirsilp B, Kitcha S, Torpee S. Co-culture of an oleaginous yeast Rhodotorula glutinis and a microalga Chlorella vulgaris for biomass and lipid production using pure and crude glycerol as a sole carbon source. Ann Microbiol. 2012;62:987–93.View ArticleGoogle Scholar
  14. Cheirsilp B, Suwannarat W, Niyomdecha R. Mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock. N Biotechnol. 2011;28:362–8.View ArticleGoogle Scholar
  15. Marquez F, Sasaki K, Nishio N, Nagai S. Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis. Biotechnol Lett. 1995;17:225–8.View ArticleGoogle Scholar
  16. Ota M, Kato Y, Watanabe M, Sato Y, Smith RL, Rosello-Sastre R, Posten C, Inomata H. Effects of nitrate and oxygen on photoautotrophic lipid production from Chlorococcumlittorale. Bioresour Technol. 2011;102:3286–92.View ArticleGoogle Scholar
  17. Du W, Liang F, Duan Y, Tan X, Lu X. Exploring the photosynthetic production capacity of sucrose by cyanobacteria. Metab Eng. 2013;19:17–25.View ArticleGoogle Scholar
  18. Hays SG, Ducat DC. Engineering cyanobacteria as photosynthetic feedstock factories. Photosynth Res. 2015;123:285–95.View ArticleGoogle Scholar
  19. Niederholtmeyer H, Wolfstadter BT, Savage DF, Silver PA, Way JC. Engineering cyanobacteria to synthesize and export hydrophilic products. Appl Environ Microbiol. 2010;76:3462–6.View ArticleGoogle Scholar
  20. Ducat DC, Avelar-Rivas JA, Way JC, Silver PA. Rerouting carbon flux to enhance photosynthetic productivity. Appl Environ Microbiol. 2012;78:2660–8.View ArticleGoogle Scholar
  21. Koschwanez JH, Foster KR, Murray AW. Sucrose utilization in budding yeast as a model for the origin of undifferentiated multicellularity. PLoS Biol. 2011;9:8.View ArticleGoogle Scholar
  22. Hays SG, Yan LLW, Silver PA, Ducat DC. Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction. J Bio Eng. 2017. doi:10.1186/s13036-017-0048-5.Google Scholar
  23. Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG. Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol. 2011;90:1219–27.View ArticleGoogle Scholar
  24. Chi Z, Zheng Y, Jiang A, Chen S. Lipid production by culturing oleaginous yeast and algae with food waste and municipal wastewater in an integrated process. Appl Biochem Biotechnol. 2011;165:442–53.View ArticleGoogle Scholar
  25. Meesters P, Huijberts G, Eggink G. High-cell-density cultivation of the lipid accumulating yeast Cryptococcus curvatus using glycerol as a carbon source. Appl Microbiol Biotechnol. 1996;45:575–9.View ArticleGoogle Scholar
  26. Iassonova DR, Hammond EG, Beattie SE. Oxidative stability of polyunsaturated triacylglycerols encapsulated in oleaginous yeast. J Am Oil Chem Soc. 2008;85:711–6.View ArticleGoogle Scholar
  27. Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM. Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res. 2009;48:375–87.View ArticleGoogle Scholar
  28. Yu X, Zheng Y, Dorgan KM, Chen S. Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour Technol. 2011;102:6134–40.View ArticleGoogle Scholar
  29. Huang C, Chen XF, Xiong L, Ma LL. Oil production by the yeast Trichosporon dermatis cultured in enzymatic hydrolysates of corncobs. Bioresour Technol. 2012;110:711–4.View ArticleGoogle Scholar
  30. Leiva-Candia DE, Pinzi S, Redel-Macias MD, Koutinas A, Webb C, Dorado MP. The potential for agro-industrial waste utilization using oleaginous yeast for the production of biodiesel. Fuel. 2014;123:33–42.View ArticleGoogle Scholar
  31. Pienkos PT, Zhang M. Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates. Cellulose. 2009;16:743–62.View ArticleGoogle Scholar
  32. Dong T, Yu L, Gao D, Yu X, Miao C, Zheng Y, Lian J, Li T, Chen S. Direct quantification of fatty acids in wet microalgal and yeast biomass via a rapid in situ fatty acid methyl ester derivatization approach. Appl Microbiol Biotechnol. 2015;99:10237–47.View ArticleGoogle Scholar
  33. Chapman JS, Meeks JC. Glutamine and glutamate transport by Anabaena variabilis. J Bacteriol. 1983;156:122–9.Google Scholar
  34. Hall GC, Jensen RA. Enzymological basis for growth inhibition by l-phenylalanine in the cyanobacterium Synechocystis sp. 29108. J Bacteriol. 1980;144:1034–42.Google Scholar
  35. Labarre J, Thuriaux P, Chauvat F. Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803. J Bacteriol. 1987;169:4668–73.View ArticleGoogle Scholar
  36. Therien JB, Zadvornyy OA, Posewitz MC, Bryant DA, Peters JW. Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002. Biotechnol Biofuels. 2014;7:154.View ArticleGoogle Scholar
  37. Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, Del Cardayre SB, Keasling JD. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature. 2010;463:559–62.View ArticleGoogle Scholar
  38. Bogosian G, Aardema ND, Bourneuf EV, Morris PJ, O’Neil JP. Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state. J Bacteriol. 2000;182:5070–5.View ArticleGoogle Scholar
  39. Grzelak A, Rychlik B, Bartosz G. Light-dependent generation of reactive oxygen species in cell culture media. Free Radic Biol Med. 2001;30:1418–25.View ArticleGoogle Scholar
  40. Jeffrey Morris J, Zinser ER. Continuous hydrogen peroxide production by organic buffers in phytoplankton culture media. J Phycol. 2013;49:1223–8.View ArticleGoogle Scholar
  41. Mittler R, Tel-Or E. Oxidative stress responses in the unicellular cyanobacterium Synechococcus PCC 7942. Free Radic Res Commun. 1991;13:845–50.View ArticleGoogle Scholar
  42. Morris JJ, Kirkegaard R, Szul MJ, Johnson ZI, Zinser ER. Facilitation of robust growth of Prochlorococcus colonies and dilute liquid cultures by “helper” heterotrophic bacteria. Appl Environ Microbiol. 2008;74:4530–4.View ArticleGoogle Scholar
  43. Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. Dependence of the cyanobacterium Prochlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean’s surface. PLoS ONE. 2011;6:2.View ArticleGoogle Scholar
  44. Hayashi S, Itoh K, Suyama K. Growth of the cyanobacterium Synechococcus leopoliensis CCAP1405/1 on agar media in the presence of heterotrophic bacteria. Microbes Environ. 2011;26:120–7.View ArticleGoogle Scholar
  45. Beliaev AS, Romine MF, Serres M, Bernstein HC, Linggi BE, Markillie LM, Isern NG, Chrisler WB, Kucek LA, Hill EA, et al. Inference of interactions in cyanobacterial-heterotrophic co-cultures via transcriptome sequencing. ISME J. 2014;8:2243–55.View ArticleGoogle Scholar

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© The Author(s) 2017

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