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. Author manuscript; available in PMC: 2021 Apr 22.
Published in final edited form as: Sci Total Environ. 2020 Dec 1;758:143969. doi: 10.1016/j.scitotenv.2020.143969

Dual-function oleaginous biocatalysts for non-sterile cultivation and solvent-free biolipid bioextraction to reduce biolipid-based biofuel production costs

Myung Hwangbo a, Jason J Gill b,c, Ry Young c,d, Kung-Hui Chu a,*
PMCID: PMC8061307  NIHMSID: NIHMS1688626  PMID: 33333303

Abstract

Triacylglycerols (TAGs) are starting materials for the production of biolipid-based fuels such as biodiesel and biojet fuel. While various microorganisms can produce TAGs from renewable resources, the cultivation of TAG-producing microorganisms under sterilization conditions to avoid microbial contamination and application of solvent to extract TAGs from the TAG-filled microorganisms are costly. To overcome these challenges, this study reports the feasibility of a non-sterile cultivation of an oleaginous bacterium Rhodococcus opacus PD631SpAHB under saline conditions, followed by the use of a solvent-free, phage-lysis-protein-based bioextraction approach for TAGs release. The engineered strain PD631SpAHB was developed by introducing a recombinant plasmid carrying a phage lytic gene cassette (pAHB) into Rhodococcus opacus PD631 via transformation, followed by adaptive evolution under saline conditions. This newly developed strain is a salt-tolerant strain with the inducible plasmid pAHB to enable TAGs release into the supernatant upon induction. Cell lysis of PD631SpAHB was confirmed by the decrease of the optical density of cell suspension, by the loss of cell membrane integrity, and by the detection of TAGs in the culture medium. Up to 38% of the total TAGs accumulated in PD631SpAHB was released into supernatant after the expression of the lytic genes. PD631SpAHB strain is a promising candidate to produce TAGs from non-sterile growth medium and release of its TAGs without solvent extraction - a new approach to reduce the overall cost of biolipid-based biofuel production.

Keywords: Triacylglycerols, Rhodococcus opacus PD631SpAHB, Non-sterile, Salt-tolerance, Cell lysis, Biolipid extraction

Graphical Abstract

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1. Introduction

Triacylglycerols (TAGs), one type of biolipids, are starting materials for producing biolipid-based biofuels such as biodiesel and biojet fuel (EPA, 2018; Kim et al., 2017). TAGs can be produced from various microorganisms including microalgae, yeast, and bacteria (Amara et al., 2016; Gujjala et al., 2019; Lee et al., 2017; Patel et al., 2018; Tanimura et al., 2014). Mostly, the production of bacterial TAGs is easy and sustainable, since high TAG yields from various inexpensive and renewable organic wastes are feasible (Davila Costa et al., 2017). For example, Rhodococcus opacus PD630 (designated PD630 hereafter), a model strain capable of accumulating TAGs up to 76% of the cell dry weight (Alvarez et al., 2000; Wältermann et al., 2000), has been shown to produce TAGs from hydrolysates of renewable lignocellulosic biomass (Wang et al., 2014). However, the production cost of lipid-based biofuels remains high, in part, due to the two additional obstacles: the high cost of cultivation under sterilized conditions to avoid microbial contamination and the expensive downstream process for extracting TAGs from the TAG-filled microorganisms.

Sterilization of growth medium accounts for 16.4% of the total energy consumption of biolipid-based biofuel production (Soccol et al., 2017). Accordingly, the non-sterile cultivation of biolipid-producing microorganisms is attractive and has been received considerable attention (Santamauro et al., 2014). One promising strategy to implement non-sterile cultivation is to leverage or create growth advantages for the desired strains to outgrow non-desired ones during the cultivation. For example, cultivation of salt-tolerant polyhydroxyburyrate (PHB)-accumulating strains in non-sterilized salt media for production of PHB, a type of biolipids, has been successfully demonstrated (Asiri et al., 2020; Tan et al., 2011; Wu et al., 2019; Yue et al., 2014). The success of these studies relied on the fact that these salt-tolerant PHB-accumulating microbial strains can accumulate osmolytes such as glutamate, ectoine, or trehalose to survive under high saline conditions and outgrow those non-salt tolerant and non-PHB-accumulating strains. Accordingly, a similar strategy will be applicable to oleaginous strains for non-sterile cultivation in the saline growth medium, if the oleaginous strains become salt-tolerant. However, no study has explored this aspect – non-sterile saline cultivation of oleaginous bacterial strains for TAG production.

The high cost of extraction of biolipids from oleaginous microbes is another major obstacle, making the price of biolipid-based biofuels less competitive to those of petroleum-based fuels. While solvent extraction is a common method for biolipid extraction (Hwangbo and Chu, 2020), this extraction method is considered as a bottleneck for commercial production of biolipid-based biofuels due to the toxicity of chlorinated solvent used in extraction, the requirement of additional energy input to separate biolipids from the solvent mixture, and the treatment cost for the spent solvent (Harris et al., 2018; Madkour et al., 2013; Ren et al., 2017).

A novel bioextraction method using bacteriophage (phage) to lyse cells to release biolipids like PHB (Hand et al., 2016) and TAGs (Gill et al., 2018) has been recently reported. Bacteriophage, also called phage, is a host-specific bacterial virus. Once its DNA has invaded the bacterial host cell, the phage can enter a lytic cycle to degrade the cell envelope using phage-encoded lytic proteins, leading to release of the intracellular components upon cell lysis (Cahill and Young, 2019; Pires et al., 2016; Young, 2014). However, achieving synchronous infection of the culture under the conditions of biomass growth is problematic.

In this study, we explored the development of a salt-tolerant TAG-accumulating bacterium with an ability to express phage lysis proteins by induction to achieve non-sterile cell cultivation and solvent-free bioextraction of TAGs from the TAG-filled bacterium (as shown in Fig. 1). First, an engineered TAG-producing bacterium with an ability to lyse cell by induction was developed by introducing an inducible phage lytic gene cassette into a known TAG-producing R. opacus PD631 (designated PD631 hereafter). Then, the engineered strain (designated PD631pAHB hereafter) was further developed via an adaptive evolution strategy to become a high-salt tolerant strain, PD631SpAHB. This PD631SpAHB strain was characterized for its ability to outgrow non-TAG-producing microorganisms in non-sterile saline growth media and to release of its TAGs into the supernatant upon induction.

Fig. 1.

Fig. 1.

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Proposed experimental approach in this study.

2. Materials and methods

2.1. Bacterial strain, culture conditions, and chemicals

The strain PD631, a domesticated derivative of PD630, was used in this study. PD631 was developed by subculturing on Reasoner’s 2A (R2A) agar plates for six months and showed a similar TAG-accumulating ability to PD630 (Gill et al., 2018). The strain PD631 was maintained aerobically in R2A or Luria-Bertani (LB) broth medium at 200 rpm at 30 °C. Amido black, hexane, methanol, and Imperial™ protein stain were purchased from Fisher Scientific (Hampton, NH, USA). R2A broth was purchased from Teknova (Hollister, CA, USA). Difco LB broth, Difco LB agar, Difco R2A agar, Difco Nutrient Agar, polypepton, and beef extract powder were purchased from VWR (Radnor, PA, USA). Diethyl ether, glyceryl trioleate (TL), chloroform, thiostrepton, chloramphenicol (CAM), and all other chemicals used in this study were purchased from Millipore Sigma (Burlington, MA, USA).

2.2. Construction of a plasmid with a phage lytic gene cassette under inducible control

To express phage lysis proteins in TAG-accumulating Rhodococcus strains, the lytic genes of mycobacteriophage D29 were selected because Mycobacterium and Rhodococcus have similar cell envelope structure. The phage D29 lytic gene cassette consists of the genes lysA-holin-lysB (Table 1a). Phage D29 was provided from Center for Phage Technology at Texas A&M University (College Station, TX). The genomic DNA of phage D29 was prepared, purified by Promega Wizard DNA cleanup kit (Promega, Madison, WI), and then used for PCR amplification. The lytic gene cassette was amplified using 2 primers as listed in Table 1b. The PCR product was cloned into a pTipQC2 plasmid (AIST Japan, Tokyo, Japan) using the NdeI-NcoI cloning site. All detailed description of PCR reaction, ligation, and transformation is available in SI.

Table 1.

(a) Names and sizes of phage lysis enzymes and associated phage lytic genes of mycobacteriophage D29. (b) Primer sets used for traditional cloning of the phage lytic gene cassette (lysA-holin-lysB) into the plasmid pTipQC2 as plasmid pAHB.

(a)
Target gene Enzyme Size (kb) # of amino acids MW (kDa)
lysA Endolysin A 1.5 493 55
holin Holin 0.5 141 15
lysB Mycolate esterase (Endolysin B) 0.8 254 29
(b)
Primers Sequence Primer length (bp) Tm (°C)
AHB_NdeIFW 5′-TAAGGGCATATGATGACGCTCATAGTCACACGC-3′ 33 64
AHB_NcoIRV 5′-TGCTACCATGGTCAGATCTGTCGTAGGAA-3′ 30 62
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2.3. Development of PD631pAHB by transformation of recombinant plasmid

The verified recombinant plasmid (pAHB) was transformed into PD631 via electroporation to obtain a new engineered strain, PD631pAHB. Transformation was done as described previously (Gill et al., 2018). Briefly, the recombinant plasmid (about 300 ng) was mixed with electrocompetent cells of PD631 and preincubated at 40 °C for 5 min. The electroporation was performed in electrocuvettes (gaps of 2 mm) by micropulser (Bio-Rad, Hercules, CA, USA) with the following settings: ec2: 2.5 kV, 600 Ω, and 25 μF. The electroporated cells were recovered outgrown in Nutrient Broth (NB) medium at 30 °C for 4 h, and then plated on an NB agar plate with 35 mg/L of CAM as a selective marker. The transformant colonies were visible after 3 to 5 days incubation at 30 °C. About 45 colonies were screened and transformation efficiency was 3.8 × 102 cfu/μg.

2.4. Development of a salt-tolerant engineered strain (PD631SpAHB) via adaptive evolution

Ectoine and glutamate are the two most common osmolytes in salt-tolerant bacteria (Burg and Ferraris, 2008; Ikeuchi et al., 2003; Kuhlmann and Bremer, 2002). An ectoine synthesis gene cluster (ectA, ectB, and ectC) encoding ectoine synthesis proteins were found in PD630 genome based on Blast-P results against to the genome of a known salt-tolerant strain Zobellella denitrificans ZD1 (Wu et al., 2019). Additionally, six possible glutamate synthases genes encoding glutamate synthesis proteins were directly identified in the genome of PD630 (CP003949.1 as an accession number of GenBank databases). Table S1 lists names of these enzymes and genes. Since these osmolyte-producing genes are present in strain PD630, we hypothesized that both the domesticated strain PD631 and the engineered strain PD631pAHB also contain these genes.

Accordingly, an adaptive evolution approach was used to develop PD631SpAHB, a salt-tolerant version of PD631pAHB. Briefly, PD631pAHB was successively subcultured on LB medium agar containing 35 mg/L of CAM and 3% (w/v) NaCl and incubated at 30 °C. After 3 days of incubation, colonies were observed and re-streaked on a new plate containing LB agar medium with 35 mg/L of CAM and 3% NaCl. This process was repeated ten times over a month. Similarly, PD631S, a salt-tolerant version of PD631, was developed by ten times of successive subculturing of PD631 on LB medium agar containing 3% NaCl. These newly developed salt-tolerant strains PD631S and PD631SpAHB were allowed to adjust to the high saline liquid medium.

2.5. Assessment of cell lysis after induction of pAHB in the engineered strains

The success of cell lysis after induction of pAHB in the engineered strains (i.e., PD631pAHB and PD631SpAHB) was determined by observation of decrease of optical density and detection of lactate dehydrogenase (LDH) in the supernatant. LDH is a cytosolic enzyme in cells. When cell membranes are damaged and/or cells are lysed, LDH will be released into growth medium. As LDH remains stable extracellularly in solution, presence of LDH in the supernatant can be used as an indication of cell lysis (Henein et al., 2016). Also, produced phage lysis proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All detailed description of these analyses is available in SI.

2.6. Expression levels of genes encoding enzymes responsible for producing known osmolytes

Experiments were designed to quantify the expression of genes encoding osmolytes-producing enzymes in PD631S grown in medium with or without salts. Briefly, the strain was grown on 10 g/L of glucose in the presence or absence of 3% NaCl aerobically in 50 mL of ammonium mineral salts (AMS) medium (Gill et al., 2018) with a minor modification (i.e., replacing ammonium chloride with ammonium sulfate at the same molarity) at 200 rpm at 30 °C. When the cell density reached an OD600 of 2.0, cells were harvested for total RNA extraction, followed by cDNA synthesis using the extracted RNA and quantification of targeted genes using the synthesized cDNA. A detailed description of the growth medium, total RNA extraction, and cDNA synthesis is available in SI.

2.7. Non-sterile cultivation of PD631S for TAG production

Experiments were conducted to examine whether TAGs can be produced from non-sterile cultivation of PD631 and PD631S. Non-sterile cultivation of both strains for TAG production were conducted via two-stage cultivation process as described previously (Gill et al., 2018), except that non-sterile saline (3% NaCl) growth medium was used. Strains (PD631 and PD631S) were cultured with 10 g/L of glucose in AMS medium at 200 rpm at 30 °C until stationary growth phase (above OD600 of 2.0). These saturated cultures were used as inocula for non-sterile cultivation experiments. An aliquot of the saturated culture was added to 50 mL of sterile or non-sterile AMS medium containing 10 g/L of glucose with or without 3% NaCl, resulting an initial OD600 ~0.02. After the inoculated cultures were grown at 200 rpm at 30 °C until early stationary phase, the cell suspension was pelleted, resuspended in sterile or non-sterile nitrogen-free mineral salts medium (MS) containing 10 g/L of glucose, and then incubated at 30 °C without shaking for 24 h. This process was to allow TAG accumulation while minimize TAG utilization. After 24 h of incubation, 50 mL of cell culture was pelleted by centrifugation at 10,000g for 10 min and the cell pellet was used for TAG measurement using thin layer chromatography (TLC) analysis as described previously (Wang et al., 2014). All description of TLC analysis is available in SI.

2.8. Non-sterile cultivation of PD631SpAHB for TAG production and release

TAG production under non-sterile conditions and TAGs release were investigated using PD631SpAHB along with PD631pTipQC2 used as a positive control. The experiments were conducted similarly as described in the above section with some modification. Most protein expression from cloning vector are generally induced by adding an inducer at OD600 of 0.6–1.0 (Berrow et al., 2006; Rahmanpour and Bugg, 2013). However, PD631 can maximize TAGs accumulation ability during stationary phase under nitrogen limited conditions (Alvarez et al., 2000). Due to this limitation, the previous study cultivated TAG-filled PD631 under nitrogen-limit conditions first, followed by promoting cell growth through nitrogen source addition before infecting with Toil phages because most phages propagate on the growing cells (Gill et al., 2018). The infected cells were then able to produce lysis enzymes that resulted in cell lysis to release of TAGs to supernatant. Similarly, in this study, after accumulation of TAGs from the strain under nitrogen limited conditions, the cell cultures returned to the active growth by adding additional nitrogen content (0.78 g/L of (NH4)2SO4) and 1 μg/mL of thiostrepton to induce phage lytic genes in PD631SpAHB. Following overnight induction, the cell pellet and suspension were collected respectively by centrifugation at 10,000g for 10 min and used for TAG measurement using TLC analysis.

3. Results

3.1. Demonstration of cell lysis of PD631pAHB via induction of its phage lytic gene cassette

An inducible plasmid pAHB for expressing phage lysis proteins was constructed by cloning the phage D29 lytic gene cassette (consisting of genes lysA-holin-lysB) into the thiostrepton-inducible expression plasmid pTipQC2. The pAHB was then used to construct strain PD631pAHB for cell lysis by induction. Cell lysis of PD631pAHB was confirmed by monitoring changes of cell density after inducing the expression of the phage lytic genes. As shown in Fig. 2a, PD631pTipQC2 and PD631pAHB both grew more slowly than PD631. This might be due to CAM in the growth medium as a selective marker for both plasmids. However, PD631pAHB grew even more slowly than cells carrying an empty vector pTipQC2 (PD631pTipQC2), presumably due to leaky gene expression from the inducible promoter. After thiostrepton induction (1 μg/mL), cells carrying the empty vector continued to grow. However, the induced PD631pAHB culture showed decreased optical density after 6 h. The final optical density of PD631pAHB was ~0.3 after overnight induction.

Fig. 2.

Fig. 2.

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Demonstration of inducible lysis of PD631pAHB. (a) Profile of the optical density before and after induction. Blue arrows indicate the time points (around OD600 = 0.5–0.7) where 1 μg/mL of thiostrepton was added as an inducer. (b) Lactate dehydrogenase (LDH) activity detected in the culture medium of PD631pTipQC2, PD631pAHB after induction, and PD631pAHB maximum. PD631pAHB maximum: designated PD631pAHB lysed by sonication artificially.

Additional experiments were conducted to characterize the lysis of the PD631pAHB cells using the release of a cytosolic enzyme, LDH into the growth medium. After pelleting cell debris and unlysed cells, the supernatant of the induced PD631pAHB cells had about 74% of the total LDH activity (Fig. 2b). Moreover, the expression of phage lytic genes in PD631pAHB was confirmed by visualizing the production of these lysis proteins on the SDS-PAGE gel. The protein profiles obtained from the concentrated whole-cell cultures and supernatant of PD631, PD631pTipQC2, and PD631pAHB were compared on the SDS-PAGE gel (Fig. S1 in SI). Based on the predicted molecular masses of LysA, Holin, and LysB listed in Table 1a, three bands of the appropriate sizes were present in the sample from the induced cells carrying pAHB (lane 3) that were not present in the sample from the control: a thicker band for LysA (55 kDa) and two fainter bands for Holin (15 kDa) and LysB (29 kDa). Also, the sample of PD631pAHB after induction did not need additional cell lysis step (i.e., sonication), suggesting successful expression of the lytic gene cassette upon induction that led to cell lysis. However, we only confirmed the overexpression for LysA. The difference of the Shine-Dalgarno (SD) sequence of these three genes in the plasmid pAHB might explain the low expression levels for LysB and Holin. The SD sequence plays a key role during a translation initiation as a ribosomal binding site, and a positive correlation between SD sequence and gene expression level has been identified in prokaryotes (Ma et al., 2002). The consensus SD sequence (AGGAGG) of E. coli is more effective than the shorter SD sequence, leading to a high level of gene expression (Vimberg et al., 2007). This correlation might be applied to the gene expression characteristics of Rhodococcus (Kan et al., 2020). In the plasmid pAHB, the lysA gene has the SD sequence of pTipQC2 (GAAGGAG), which is strong for the gene expression in Rhodococcus (Nakashima and Tamura, 2004), leading to the high expression level of LysA. However, the lysB and Holin genes possess the shorter and weaker SD sequences derived from phage D29: AGGA and GGA, respectively. This difference of the SD sequence might result in overexpression of lysA than the other two lytic genes, resulting in a thicker band of LysA on the SDS-PAGE gel. In any case, despite the low expression of LysB and Holin, PD631pAHB exhibited cell lysis and release of the intracellular components upon induction.

3.2. Characterization of newly developed salt-tolerant strains PD631S and PD631SpAHB

Two salt-tolerant strains PD631S and PD631SpAHB were developed after ten times of successively subculturing PD631 and PD631pAHB on LB agar containing 3% NaCl or 3% NaCl and 35 mg/L CAM, respectively. The growth rates and doubling times of these strains were compared to those of non-salt-tolerant strains PD631 and PD631pAHB (Fig. S2 and Table S2 in SI). PD631S was able to grow in liquid medium containing 3% NaCl, with a decreasing doubling time of 5.8 h for the 1st subculture to 3.9 h for the 3rd subculture (Table S2 in SI). When grown without salts, PD631S showed a similar growth rate to that of strain PD631, i.e., a doubling time of 2.1 h for PD631S vs. a doubling time of 2.2 h for strain PD631 (Table S2 in SI). This indicated that PD631S can survive under high saline conditions for non-sterile cultivation while producing TAGs. PD631pAHB and PD631SpAHB also grew slower than that of strain PD631, as evident by their doubling times (i.e., 3.2 h for both PD631pAHB and PD631SpAHB and 2.2 h for PD631 as shown in Table S2 in SI). To examine the stability of the plasmid pAHB in the newly constructed strain PD631SpAHB, this strain was successively cultured in LB medium without CAM for five generations before checking their ability to grow in LB medium with CAM. The plasmid pAHB was detected in the 5th generation culture (data not shown), suggesting that pAHB was stable in strain PD631SpAHB in the absence of CAM.

3.3. Effects of salts on non-sterile cultivation of strain PD631S and its TAG production

Experiments were conducted to determine the enzymes involved in the synthesis of common osmolytes in strain PD631S under high salt conditions. Among the osmolytes, ectoine is commonly used to provide osmotic balance under extreme conditions (Lang et al., 2011). Three genes involved in ecotine synthesis are ectA, ectB, and ectC encoding diaminobutyric acid acetyltransferase, diaminobutyrate-2-oxoglutarate transaminase, and ectoine synthase, respectively (Fig. 3a) (Stoveken et al., 2011). Glutamate is another common osmolyte that can be increased as a counter-ion in cells in response to hyperosmotic stress (Burg and Ferraris, 2008).

Fig. 3.

Fig. 3.

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(a) Ectoine synthesis pathway starts from the conversion of L-asparate-semialdehyde to L-diaminobutyric acid, N-gama-acetyldiaminobutyric acid, and then to ectoine. The genes involved in the synthesis steps in this pathway are: ectA encoding diaminobutyric acid acetyltransferase, ectB encoding diaminobutyrate-2-oxoglutarate transaminase, and ectC encoding ectoine synthesis. (b) Salt effects on expression of three ectoine synthesis genes in strain PD631S. Blue bars: PD631S grown on glucose as a carbon source in non-saline AMS medium. Red bars: PD631S grown on glucose as a carbon source in AMS medium with 3% NaCl. All experiments were conducted in duplicate.

Different transcription levels of ectoine synthesis genes in PD631S grown with or without 3% NaCl were observed. Interestingly, as shown in Fig. 3b, the ectA expression level in saline grown PD631S was two-fold higher than that in the non-saline grown PD631S. The mRNA levels encoding ectB was slightly increased while the ectC expression level was slightly decreased in saline grown PD631S. Unlike those observed for ectoine synthesis genes, the expression levels of glutamate synthesis genes (i.e., mRNA levels encoding glutamate synthesis) in saline-grown PD631S were similar to those in non-saline grown PD631S (Fig. S3 in SI), suggesting that glutamate might not play a key role in PD631S when grown under saline conditions. The gene expression levels for ectoine and glutamate synthesis proteins suggested that ectoine was the key osmolyte produced in PD631S under high salts conditions. However, the reasons for the decreased expression levels of ectC are unclear and need to be further investigated.

The effects of two cultivation variables, sterilization of growth medium and presence of 3% NaCl, on the growth of and TAG production by PD631 and PD631S (Table 2, Fig. S4 in SI) were examined. In sterilized salt-free media, PD631 produced 22.9 mg/L TAG under the conditions of the experiment; the addition of 3% NaCl reduced the production of TAG to 15 mg/L and significantly retarded the growth rate from 0.08 h−1 to 0.04 h−1. The final optical density of PD631 in salt media was about two times lower than that of PD631 in salt-free media (Fig. S4a in SI). Also, in the preliminary experiment, only inoculated PD631 from the saturated culture could grow with 3% NaCl while it could not grow directly from a single colony (data not shown). In contrast, the amounts of TAG production by PD631S in sterilized salt-free media was slightly less (15.5 mg/L) but maintained its growth rate in the high salts as 0.05 h−1 from 0.06 h−1.

Table 2.

TAG production from PD631 or PD631S cultivated with 1% of glucose in AMS medium with or without salts under sterile or non-sterile conditions. All experiments were conducted in duplicate.

Experiment Variables
 Growth rate (h−1) TAG (mg/L) Total TAG mass (mg)
Culture % NaCl (w/v) Sterilization
# a PD631 0 Yes 0.08 22.9 ± 4.6 1.2 ± 0.2
# b PD631 0 No 0.08 0 0
# c PD631 3 Yes 0.04 15.0 ± 0.4 0.8 ± 0.0
# d PD631 3 No 0.07 4.9 ± 1.6 0.2 ± 0.1
# e PD631S 0 Yes 0.06 15.5 ± 0.9 0.8 ± 0.0
# f PD631S 0 No 0.06 0 0
# g PD631S 3 Yes 0.05 9.8 ± 1.6 0.5 ± 0.1
# h PD631S 3 No 0.05 8.0 ± 2.1 0.4 ± 0.1
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The advantage of using PD631S for TAG production is that under non-sterile high salt conditions, which is not necessary to sterilize the growth media to achieve efficient TAG accumulation. PD631S in non-sterile salt media produced 8.0 mg/L TAGs (52% of that achieved in sterile salt-free media) while PD631S in non-sterile salt-free media did not produce any TAGs, suggesting that PD631S in non-sterilie salt-free media might be contaminated. The TAGs produced by PD631S in non-sterile salt media were 3 times lower than those produced by PD631 in sterile salt-free media. The differences in the total TAG yields can be explained by the differences in cell densities in both cultures at the time harvested for TAG measurements. As shown in Fig. S4a and S4c, the optical density of PD631S in non-sterile salt media (#h) was 3 times lower than that of PD631 in sterile salt-free media (#a). In contrast, PD631 in non-sterile salt media only produced 4.9 mg/L TAGs (22% of that achieved in sterile salt-free media) while there was no TAGs production from PD631 in non-sterile salt-free media. This indicates that PD631S showed a better ability to survive in non-sterile salt media than PD631 due to adaptive evolution.

3.4. Demonstration of cell lysis of PD631SpAHB and its TAGs release

Cell lysis of PD631SpAHB was confirmed based on a decrease of cell density following induction with thiostrepton. As shown in Fig. 4, in the absence of salts, PD631SpAHB grew slower than those of PD631pTipQC2. The strain PD631SpAHB showed a long lag phase of 48 h when grown in salt media from a colony on LB agar containing 3% NaCl and 35 mg/L CAM. The long lag phase might be due to a hyperosmotic pressure imposed on the strain in salt media even the strain has already adapted to the saline conditions on agar plates. After adding thiostrepton (1 μg/mL) at 28 h (Fig. 4), PD631pTipQC2 continued to grow up to an optical density of 2.5. However, 6 h after induction with thiostrepton (1 μg/mL), the optical density of PD631SpAHB (regardless of the cultivation conditions) decreased from 0.8 to 0.3. The decrease of optical density after induction was similar to those observed in the induction experiments using PD631pAHB (Fig. 2a). Also, after overnight induction, cell debris of PD631SpAHB was found in the cell culture (data not shown). Phage lysis proteins in the supernatants of induced PD631SpAHB were also observed on the SDS-PAGE gel (Fig. S5 in SI), providing evidence that phage lysis proteins produced in PD631SpAHB caused cell lysis. Similar to the SDS-PAGE results of induced PD631pAHB, Fig. S5 shows the overexpression of LysA and expression of Holin and LysB in induced PD631SpAHB. Overall, the results showed that cell lysis of PD631SpAHB can be effectively controlled by expressing the phage lytic gene cassette at the desired time, preferably after cells have accumulated a high level of TAGs.

Fig. 4.

Fig. 4.

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Delayed growth of PD631SpAHB in saline. Inducible lysis of PD631SpAHB grown in either non-saline or saline medium was observed, based on decrease of optical density 6 h following addition of thiostrepton (an inducer). PD631pTipQC2 was used as a control. Blue arrows indicate the time point in the OD600 = 0.5–0.7, growth stage where 1 μg/mL of thiostrepton was added to induce expression of phage lytic genes. All experiments were conducted in duplicate.

The effects of phage lytic gene cassette on cell growth, TAG production, and TAG release of PD631SpAHB were examined and summarized in Table 3 and Fig. S6 in SI. PD631pTipQC2 in sterile salt-free media was used as a control. In sterile media, PD631SpAHB produced similar amounts of total TAG with that from PD631pTipQC2, but slightly decreased the growth rate to 0.15 h−1 in salt media and 0.10 h−1 in salt-free media. TAG was produced by PD631SpAHB in non-sterile salt media up to 17.6 mg/L while PD631SpAHB in non-sterile salt-free media did not produce TAG. After adding additional nitrogen source and inducer, decreased optical densities were observed in the samples with PD631SpAHB but not in the samples with PD631SpAHB grown in non-sterile salt-free media, suggesting that cell lysis occurred by the expression of phage lytic genes. As the controls, the addition of nitrogen source and inducer did not lyse PD631pTipQC2, instead, the cells grew up to an optical density of 3.5. Also, 21–38% of the total TAGs were released from PD631SpAHB in sterile media to the supernatant after the induction of phage lysis proteins while only 6% of the total TAGs were released from PD631SpAHB in non-sterile salt media.

Table 3.

Non-sterile cultivation, TAG production, and TAG release from PD631SpAHB with different growth conditions. All experiments were conducted in AMS medium with 1% of glucose as a carbon source.

Experiment Variables
 Growth rate (h−1) Total TAG (mg/L) TAG in pallet (mg/L) Released TAGa (mg/L)
Culture % NaCl (w/v) Sterilization
i PD631pTipQC2 0 Yes 0.19 24.2 24.2 0
ii PD631SpAHB 3 Yes 0.15 24.7 19.4 5.3
iii PD631SpAHB 0 Yes 0.10 24.2 15.1 9.1
iv PD631SpAHB 0 No 0.24 0 0 0
v PD631SpAHB 3 No 0.13 17.6 16.7 0.9
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a

Released TAG were detected in the supernatant of each culture.

4. Discussion

Only a few bacterial species are reported as oleaginous strains including Gram-positive species such as Dietzia, Mycobacterium, Nocardia, Rhodococcus, and Streptomyces (Hwangbo and Chu, 2020). Among oleaginous bacteria, Rhodococcus opacus PD630 is recognized as a model TAG-accumulating strain due to its ability to produce up to 76% of its cell dry weight (Alvarez et al., 1996; Wältermann et al., 2000). Our results indicate that the newly constructed Rhodococcus strain PD631SpAHB is a promising candidate to address the two major challenges – high costs associated with sterilization cultivation and TAGs extraction – of current biolipid-based biofuel production. As many microorganisms, except halophilic bacteria, are unable to survive under 3% NaCl conditions (Ikeuchi et al., 2003), the salt-tolerant characteristics of PD631SpAHB is advantageous because it removes the need of the use of sterile medium during the cultivation, leading to reduction of the overall costs and energy inputs for production of bacterial TAGs.

Unlike Gram-negative bacteria that have a thin layer of peptidoglycan (PG) in their cell wall, Gram-positive bacteria are characterized by a thick layer of PG in their cell wall composition. Some Gram-positive bacteria like Rhodococcus opcaus has a complex cell wall structure containing not only a thick PG layer but also a layer of arabinogalactan, and an outer layer of mycolic acid (MA), making breaking the Rhodococcus cells difficult (Mitani et al., 2005). Accordingly, more phage lysis proteins are needed during infection to open the cell envelope of Rhodococcus strain than that for Gram-negative bacteria (Cahill and Young, 2019). Mycobacteriophages are well-studied phages capable of lysing Mycobacterium which has a similar cell wall structure of Rhodococcus (Hatfull, 2018; Mitani et al., 2005). To lyse the cell wall of Mycobacterium, mycobacteriophages use three phage lysis proteins: an endolysin to disrupt PG, a mycolate esterase to digest MA, and a holin to create a hole on the cell membrane to release the endolysin and the mycolate esterase outside of the cell (Catalao and Pimentel, 2018; Hatfull, 2014; Payne and Hatfull, 2012). Accordingly, to lyse Rhodococcus cells, three phage lysis proteins — endolysin, holin, and mycolate esterase — are required. In this study, these corresponding phage lytic genes were successfully constructed as an inducible phage lytic gene cassette plasmid (pAHB) and expressed in the TAG-filled PD631SpAHB for TAGs release. To our best knowledge, this is the first study reporting the successful internal application of phage lysis proteins to lyse Rhodococcus for TAGs extraction.

Our results showed that 38% of the total TAGs were released from PD631SpAHB. This TAG release results were similar to the previous finding that about 30% of the total TAGs were released from phage-infected PD631 cells (Gill et al., 2018). Different from the bacteriophage infection study, the timing of cell lysis can be controlled and there is no need to prepare an additional phage culture for infection in our study. However, the TAG release % observed in this study was lower than those using solvent-based TAGs extraction process from Rhodococcus strain (i.e., over 55% of TAG extraction efficiency) (Amara et al., 2016; Hwangbo and Chu, 2020). In Gill’s study, TLC analysis revealed that most TAGs were still not released into the supernatant but associated with pellets of cell debris, suggesting that TAG granules are trapped with the cell debris or TAGs are attached to the cell membranes (Gill et al., 2018). This phenomenon was also observed in our study. Accordingly, there is a need for further improvement to increase TAGs yields and/or recover attached TAGs on the cell debris or the cell membranes.

The levels of TAG accumulation in oleaginous bacterium like R. opacus PD630 are known to be affected by several factors such as carbon/nitrogen (C/N) ratios, types of carbon and/or nitrogen sources, and different cultivation methods (single-stage or two-stage) (Alvarez et al., 1996; Castro et al., 2016; Wältermann et al., 2000). Accordingly, higher levels of TAG accumulation in PD631SpAHB can be achieved by modifying C/N ratios, carbon, and nitrogen sources in the growth medium. In our study, the newly constructed PD631SpAHB was grown on glucose at a much lower C/N ratio of 28 using two-stage cultivation strategies (i.e., growing up to the stationary phase, followed by TAG accumulation under nitrogen-free conditions). By using gluconate as a carbon source and ammonium chloride as a nitrogen source in the sterile salt medium with a higher C/N ratio of 147, TAG production by PD631SpAHB was enhanced up to 4-fold higher than that of the strain grown on glucose and a low C/N ratio of 28 (observed in our laboratory).

Different time to lyse the cells is likely to result in different levels of TAG release, as cell lysis timing by phage is tightly regulated inside cells (Cahill and Young, 2019) and actively growing host cells might be needed to produce phage lytic proteins for effective cell lysis. Thus, to check the cell lysis timing, we first monitored the cell lysis based on the decrease of optical density after the addition of inducer, thiostrepton and nitrogen source. The addition of nitrogen was to bring stationary stage cells into actively growing cells. As shown in Fig. 2a, we confirmed that overnight induction would bring the maximum cell lysis. Thus, the other cell lysis experiments in this study were conducted overnight. To examine if phage lytic proteins can be effectively produced by non-growing PD631SpAHB, we used thiostrepton as an inducer without an additional nitrogen source and observed an increase of TAG release (from 38% up to 51% of the total TAGs) from the strain PD631SpAHB. This increased TAGs release % is comparable to the amounts of TAGs extracted from Rhodococcus strain via solvent-based extraction process (i.e., ~55%) (Amara et al., 2016). Our results suggested that minimization of TAG utilization in cells before cell lysis is the key to obtain high TAG yields. Future studies to investigate the optimal cell lysis conditions are thus needed.

After cells were lysed by phage lytic proteins, the TAGs of PD631SpAHB were either released into the supernatant or attached to the cell debris. The TAGs in the supernatant can be theoretically separated from water easily because TAGs are immiscible and have a lighter density than water. The lighter density of TAGs will allow TAGs to float on the surface of water, and this effect is expected to be more pounced in high saline solution. In fact, floating lipid droplets on the surface of the supernatant of lysed cells after centrifugation have been observed in our cell lyse experiments.

As a substantial amount of the TAGs was attached to cell debris after cell lysed, it is necessary to recover the TAGs from the cell debris. Previous studies have shown that extraction time and/or the amounts of solvents required for TAGs extraction can be reduced if cell disruption is in advance (Hwangbo and Chu, 2020). Also, a recent study showed that lipids from Nannochloropsis sp. were recovered by only adding nonpolar solvent hexane (which is less toxic than chloroform that is commonly used in TAG extraction), due to the ultrasonication treatment for cell disruption (Yao et al., 2018). Another possible means to recover the TAGs from the lysed cells is through direct transesterification to convert TAGs into biodiesel (i.e., fatty acid methyl esters) (Yao et al., 2018). Additionally, a recent study demonstrated increases in biolipid release % (up to 73%) from microalgae by using a combination of autolysin and trypsin to cleave chloroplasts and lipid bodies inside the cells, and recovery of the released lipids via centrifugation from the medium without using a solvent (Soto-Sierra et al., 2020). All these approaches can potentially be applied to recover the TAGs attached on phage-protein-lysed cell debris to increase TAGs yield, leading another path to reduce lipid-based biofuel production costs. Yet, future research efforts are needed to investigate novel methods to recover the TAGs from the cell debris.

This study focuses on the non-sterile cultivation of engineered oleaginous strain for TAGs production and extraction, addressing important challenges to be overcome in order to reduce the overall biolipid-based biofuel production cost. Firstly, cost reduction in cultivation of oleaginous strain can be realized by using our novel non-sterile cultivation in high salt solution. Additionally, as TAGs density is lower than water, once TAGs are released from cells into liquid medium, TAGs are expected to float to water surface for easy recovery. This phenomenon is expected to occur when our phage-lytic-protein-based approach is applied – i.e., PD631SpAHB lysed and TAGs are released from PD631SpAHB into the supernatant spontaneously, resulting in the reduction of lipid-based biofuel production costs due to a less solvent usage, wet biomass extraction, and/or no additional cell disruption step.

The energy operation cost per liter of biodiesel produced from microbial oil was estimated to be 16.4% for sterilization, 20.6% for harvesting/cell disruption (i.e., decantation and cell disruption), and 6.7% for solvent-based TAGs extraction (Soccol et al., 2017). By using our newly developed PD631SpAHB for TAGs production and release, although centrifugation is still needed for separating cell debris from the supernatant, this process is expected to save at least 37% of total energy costs from sterilization and harvesting/cell disruption and reduced the amounts of solvent. This simple analysis suggests that PD631SpAHB is a promising candidate for the non-sterile cultivation for TAG production and extraction, leading to a reduction of the production costs of biolipid-based biofuel.

Overall, our novel approach using phage lysis enzymes combined with non-sterile cultivation will have a significant impact on biofuel production costs and environmental issues, comparing to the traditional cultivation and solvent extraction processes. Nevertheless, future studies are needed to determine the cultivation condition for maximizing TAG production and to investigate the best induction strategy to minimize TAG utilization by the TAG-filled PD631SpAHB during the induction of cell lysis. To our knowledge, this is the first report of the non-sterile cultivation of Rhodococcus opacus for biolipids production and application of phage lytic proteins to lyse cells for biolipid extraction. The findings of our study provide the groundwork for an efficient biolipid production and extraction from Rhodococcus strain.

5. Conclusions

This study has successfully demonstrated non-sterile TAGs production and release using a newly developed salt-tolerant PD631SpAHB. The inducible plasmid carrying a phage lytic gene cassette (pAHB) enabled lysis of TAG-filled PD631SpAHB to release TAGs into liquid medium. This technique offers a new means for biolipid extraction, potentially leading to a reduction of overall extraction costs of biolipid for biofuel production. The high salt-tolerant characteristics of PD631SpAHB remove the need of using sterile medium for TAG cultivation. Overall, our results indicate that this new strain PD631SpAHB is a promising candidate to address the two major challenges – high costs associated with sterilization cultivation and TAGs extraction – of current biolipid-based biofuel production.

Supplementary Material

supplementary

HIGHLIGHTS.

  • PD631pAHB was engineered with phage lytic genes to enable cell lysis on demand.

  • PD631S and PD631SpAHB accumulated TAGs in non-sterilize saline growth media.

  • PD631SpAHB produced and released its TAGs into supernatant on demand.

  • PD631SpAHB is a proof-of-principle construct to be optimized for biofuel production.

Acknowledgement

This work was partially supported by the National Science Foundation, United States (Award #1134488).

Footnotes

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2020.143969.

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