Bioresource Technology 169 (2014) 462–467

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Bioresource Technology

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Boosting D-lactate production in engineered cyanobacteria usingsterilized anaerobic digestion effluents

http://dx.doi.org/10.1016/j.biortech.2014.07.0030960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Address: Campus Box 1180, One Brookings Drive, St.Louis, MO 63130, USA. Tel.: +1 314 935 3441.

E-mail address: yinjie.tang@seas.wustl.edu (Y.J. Tang).1 W.D.H. and A.M.V. have equal contributions to this work.

Whitney D. Hollinshead a,1, Arul M. Varman a,b,1, Le You a, Zachary Hembree a, Yinjie J. Tang a,⇑a Department of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130, USAb Biological and Materials Science Center, Sandia National Laboratories, Livermore, CA 94550, USA

h i g h l i g h t s

� Anaerobic digestion effluents provide N/P nutrients for cyanobacterial cultivation.� Acetate-rich effluents enhance D-lactate synthesis from engineered cyanobacteria.� Alkaline pH culture condition is important for cyanobacterial D-lactate secretion.

a r t i c l e i n f o

Article history:Received 19 April 2014Received in revised form 30 June 2014Accepted 1 July 2014Available online 10 July 2014

Keywords:D-lactate dehydrogenaseMunicipal wastePhotomixotrophicSynechocystis 6803

a b s t r a c t

Anaerobic digestion (AD) is an environmentally friendly approach to waste treatment, which can gener-ate N and P-rich effluents that can be used as nutrient sources for microalgal cultivations. Modificationsof AD processes to inhibit methanogenesis leads to the accumulation of acetic acid, a carbon source thatcan promote microalgal biosynthesis. This study tested different AD effluents from municipal wastes ontheir effect on D-lactate production by an engineered Synechocystis sp. PCC 6803 (carrying a novel lactatedehydrogenase). The results indicate that: (1) AD effluents can be supplemented into the modified BG-11culture medium (up to 1:4 volume ratio) to reduce N and P cost; (2) acetate-rich AD effluents enhance D-lactate synthesis by �40% (1.2 g/L of D-lactate in 20 days); and (3) neutral or acidic medium had a dele-terious effect on lactate secretion and biomass growth by the engineered strain. This study demonstratesthe advantages and guidelines in employing wastewater for photomixotrophic biosynthesis using engi-neered microalgae.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

High feedstock costs and environmental burdens remain majorobstacles in the development of industrial-scale biorefineries. Toovercome these problems, microalgae-based biorefineries havebeen proposed as an economical and environmentally friendly pro-cess that can be potentially integrated into CO2 sequestration andwastewater treatment processes. Numerous wastewaters (live-stock waste, poultry waste, and municipal slurries) contain signif-icant levels of nitrate (N), phosphate (P), and other nutrients thatcan be used to support algal cultivations (Patil et al., 2010;Olguín, 2012; Cho et al., 2013). In addition, P and N stripping fromwaste water are often necessary to avoid eutrophication and envi-ronmental damage to local ecological systems. Therefore, it is ideal

if the N and P in wastewater are consumed by photo-biorefineries,serving both bio-production and bioremediation (Cho et al., 2011;Cho et al., 2013).

Anaerobic digestion (AD) is an effective method for wastetreatment, which involves four major conversions: organicwastes ? simple sugars ? organic acids ? acetic acid ? methane(Chen et al., 2008). The effluents from anaerobic digestion of muni-cipal and agricultural wastes may provide cheap N and P sourcesfor microalgal cultivations. In addition, AD can accumulate aceticacid at high concentrations by blocking methanogenesis usingeither acidic conditions or chemical inhibitors (Wilkes, 2008).The resulting effluents have higher N & P levels as well as abundantamounts of acetic acid and other organic acids. These acetate-richeffluents have previously been successfully used for biodiesel fer-mentation (Liu et al., 2013).

Previous wastewater studies on facilitating algal bioprocessesmainly focus on the search for new algal species, optimization ofalgal cultivations, and pre-treatment methods to avoid contamina-tion (Cho et al., 2011; Huang et al., 2012; Ho et al., 2013). Recently,

W.D. Hollinshead et al. / Bioresource Technology 169 (2014) 462–467 463

metabolic engineering and synthetic/systems biology tools havebeen employed to create novel microalgal strains to produce biofu-els and other commodity chemicals (Angermayr et al., 2012; Lanand Liao, 2012; Berla et al., 2013). Among these studies, cyanobac-teria are a promising chassis because of their fast growth and effec-tive photosynthetic production (Wang et al., 2012). Therefore, it isof great interest to develop economical bioprocesses by integratingwaste treatment with cyanobacterial biorefinery. In this study, wehave investigated the feasibility of using different anaerobic diges-tion effluents for cultivation of wild-type Synechocystis sp. PCC6803 and its engineered variant (AV10 strain with a novel D-lactatedehydrogenase) (Wang et al., 2011; Varman et al., 2013). We mon-itor both cyanobacterial growth and D-lactate biosynthesis (achemical important in food, pharmaceutical, and plastic industries)under the influence of wastewater supplementation. By studyingthis model cyanobacterial system, we can obtain knowledge onapplying waste streams to promote engineered microalgalbioprocesses.

2. Methods

2.1. Anaerobic digestion

Effluents from anaerobic digestion processes were generatedand provided to us by Professor Yan Liu’s group at Michigan StateUniversity. The municipal sludge was obtained from the East Lan-sing Wastewater Treatment Plant (East Lansing, MI, USA). Thesludge was subjected to anaerobic digestion under three condi-tions (Rughoonundun et al., 2010): (1) AD1 – Under normal condi-tion; (2) AD2 – Under acidic condition (pH = �5) to promoteacetogens and inhibit pH-sensitive methanogens; (3) AD3 – Treat-ment with iodoform solution to inhibit methanogenesis (Liu et al.,2013). The compositions of the different AD effluents are shown inTable 1. The AD effluents were filtered then autoclaved before eachexperiment (stored at �20 �C).

2.2. Strains and growth conditions

The wild type Synechocystis 6803 cells were transferred fromBG-11 agar plates into shaking flasks containing BG-11 mediumand grown at 30 oC (Wang et al., 2011; Varman et al., 2013). Duringthe mid-log growth phase, aliquots of culture were withdrawn andthen resuspended in their respective media (combinations ofBG-11 medium and AD effluents) to a starting biomass equivalentto OD730 of 0.1. The cultures were cultivated in 50 mL shakeflasks (10�15 mL working volume) under 80–100 lmol ofphotons m�2 s�1. The engineered strain of Synechocystis 6803(AV10) employs a novel D-lactate dehydrogenase GlyDH⁄ (mutatedfrom glycerol dehydrogenase) and a soluble transhydrogenase tobalance the cofactors (Wang et al., 2011; Varman et al., 2013).The seed culture for AV10 was grown in BG-11 media with20 lg/mL of kanamycin. During the mid-log growth phase, aliquotsof culture were withdrawn and then resuspended in their

Table 1Composition of anaerobic digestion sludges.

AD conditions AD 1 Normal AD 2 L

Total phosphorus (mg/L) 183 100Total nitrogen (mg/L)a 280 420Chemical oxygen demand (g/L) 4.5 15.3Butaric acid (g/L) 0.02 1.28Propionic acid (g/L) 0.49 1.29Acetic acid (g/L) 0.41 3.85

D-Lactate (g/L) <~0.025 ~0.025

a Most nitrogen in AD sludge is in the form of ammonia (Sheets et al., 2014).

respective media (with 1 mM IPTG) to a starting biomass equiva-lent to OD730 of 0.4.

Cell optical density OD730 was used to monitor biomass growthin the BG11 medium culture. For culture media with AD effluents,Chlorophyll a was used as an indicator for cyanobacterial biomassto avoid the interference of light absorption by wastewater. Inbrief, the culture samples were centrifuged, and then Chlorophylla was extracted from the biomass with 1 mL of methanol by vor-texing for 5 min. After centrifugation to remove solids, Chlorophylla was measured for its absorbance at 663 nm via a UV–Vis spectro-photometer (Agilent Cary 60, USA) as adapted from (Meeks andCastenholz, 1978). Biomass correlation between OD730 andOD663 (Chlorophyll a) can be estimated using equation:OD730 = 0.38 � OD663 (R2 = 0.99).

2.3. D-Lactate quantification

D-Lactate concentrations in the supernatant were measuredusing an enzyme assay (D-Lactic acid/L-Lactic acid Enzyme Kit, R-Biopharm, Germany) on a 96 well plate reader (Infinite 200 PROmicroplate photometer, Tecan, Switzerland). The intracellular D-lactate concentration in the engineered strain was estimated usinga isotopomer-based approach as described in a standard protocol(Bennett et al., 2008). In brief, the engineered strain was grownin BG-11 medium with 4 g/L of fully labeled 13C-sodium bicarbon-ate (Sigma–Aldrich, St. Louis) in a closed glass bottle for three days.The labeled cultures (OD730 = 1.9, 20 mL) were filtered at 4 �C andwashed with fresh BG-11 medium (pH = 7) to remove the residualextracellular lactate. The biomass-containing filter paper was thenplaced in a mixture of methanol and chloroform (1:2 ratio, with0.01 lg/mL unlabeled lactate in the extraction solution as theinternal standard) to extract the intracellular lactate. The sampleswere lyophilized (FreeZone Freeze Dry System, Labconco, MO) andderivatized using methoxyamine hydrochloride and N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The samples wereanalyzed via a Gas Chromatography-Mass Spectrometry (GC–MS)(Hewlett Packard 7890A and 5975C, Agilent Technologies, USA).The MS peak [M-117] of the trimethylsilyl-derivatized lactic acidwas used for analysis of D-lactate labeling, and the presence of nat-ural isotopes were corrected (You et al., 2012). The intracellularconcentration of lactate was approximately estimated based onthe ratio of the 13C lactate abundance to the 12C lactate abundance.

2.4. Validation of organic carbon utilization by the engineered strain

The seed culture for AV10 was prepared in BG-11 media with20 lg/mL of kanamycin. During the mid-log growth phase, aliquotsof culture were withdrawn and resuspended in BG-11 mediawith 4 g/L of NaH13CO3 (initial OD730 < 0.01) and 20 lg/mL ofkanamycin. Grown for four days, this labeled seed culture was thenused to inoculate a control culture (BG-11 media with 2 g/L ofNaH13CO3) and a culture with a mixture of AD2 and BG-11 media(1:4 volume ratio, 2 g/L NaH13CO3). All labeled cultures started at an

ow pH AD 3 Chemical Inhibition BG11 Medium

185 30590 149524.9 ND2.691.417.30~0.025

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initial OD730 of 0.3 with 1 mM IPTG. After two days, additionalNaH13CO3 was added to restore the concentration level of labeledbicarbonate to 2 g/L. Biomass samples were taken at Day 0 andDay 4 for analysis of 12C incorporation from AD2 into proteinogenicamino acids (aspartate and glutamate). Biomass pellets werewashed twice with 0.9% w/v NaCl solution then hydrolyzed in6 M HCl at 100 �C overnight. Hydrolyzed amino acids weredried then derivatized with N-Methyl-N-[tert-butyldimethyl-silyl]trifluoroacetamide (Sigma–Aldrich, St. Louis, MO) in tetrahydrofu-ran (Sigma–Aldrich, St. Louis, MO) at 70 �C. The samples wereanalyzed via GC–MS, as described in the published protocol (Youet al., 2012).

3. Results and discussion

3.1. Cultivation of wild type Synechocystsis 6803 using dilutedwastewater

This study utilized three different AD wastewaters: AD1 wasproduced through the normal anaerobic digestion conditions,while AD2 and AD3 were modified for acetate accumulation eitherby pH or chemical inhibition of methanogens, respectively. Table 1details the difference in the composition among the three waste-water effluents. AD1 had the lowest chemical oxygen demand.AD2, digestion under acidic condition, generated moderate acetatedue to the inhibition of methanogenesis (�4 g/L). AD3, addition ofchemical inhibitor, contained the highest nitrogen (0.59 g/L) andorganic acids (e.g., acetate �7.3 g/L) concentrations because ofincomplete digestion of organic waste into CH4. All wastewatersamples contained low background D-lactate (~25 mg/L or below).

Cyanobacterial medium supplemented with AD1 (20% AD1 and80% BG-11 medium) was revealed to enhance the growth rate ofwild type Synechocystis 6803 (Fig. 1). However, higher wastewaterloading ratios (>20%) appeared to inhibit algal growth. This occursbecause the cultivation medium becomes too murky for adequatelight penetration as well as potential toxins in AD wastewater(such as heavy metals) (Anderson and McIntosh, 1991; Chenet al., 2008). Although algal consortium (a community system)can be prosperous in wastewater (Chinnasamy et al., 2010), multi-ple algal studies have demonstrated that AD wastewater needs tobe diluted and pre-treated for both optimization of nutrient condi-tions and reduction of inhibitors (Cai et al., 2013; Ji et al., 2014;Sheets et al., 2014). In this study, mixtures up to 20% municipalAD wastewater with BG-11 medium may benefit cyanobacterialgrowth.

Fig. 1. The final optical density measurement and maximal growth rate (day�1) forwild type Synechocystis 6803 grown on mixtures of AD 1 wastewater (0%, 10%, 20%,50%; v/v) with BG-11 medium (60 mL culture) for 12 days. ⁄denotes no growth.Error bars represent standard deviation of two biological samples.

Nitrogen and phosphorus are major contributors to the cost ofalgal medium. The industrialization of algal bioprocesses couldhave a serious impact on the global demand for nitrates and phos-phates (Hannon et al., 2010). Therefore, we investigated the use ofdiluted AD3 to reduce N and P usage in the BG-11 media. Betweenthe three different effluents, AD3 contains the highest concentra-tions of N & P (Table 1). With AD3 supplementation, the nitratein the BG-11 medium could be reduced to 50% of its original levelswhile Synechocystis 6803 maintained a similar growth compared tocultures with the original concentrations of nitrate and phosphatein BG-11 medium (Fig. 2). If total nitrate concentration (NaNO3) inthe culture medium was 0.7 g/L or below (i.e., <50% of original levelin BG11 medium), biomass growth was reduced and chlorosis wereobserved after two week cultivations (a bleaching of the cells dueto nutrient deficiency) (Collier and Goodman, 1992). On the otherhand, the AD3 supplementation could significantly reduce phos-phorus usage in the BG-11 medium (by up to 75%) without aneffect on cyanobacterial growth.

3.2. Enhanced D-lactate productivity using acetate rich AD effluents

We have engineered Synechocystis 6803 to produce D-lactate viaa novel D-lactate dehydrogenase. Cultivations of the engineeredstrain (AV10) with different exogenous carbon sources haverevealed that acetate significantly enhances D-lactate productivityas: (1) Acetate provides building blocks for biomass synthesis. (2)Acetate metabolism inhibits pyruvate dehydrogenase and improvethe availability of pyruvate for lactate synthesis (Varman et al.,2013). As the AD effluents contained significant levels of acetate,the effluents were expected to be beneficial for cyanobacterial lac-tate production. Fig. 3 shows the growth of the AV10 in BG-11medium at 20% v/v for the three different AD effluents. All cultureswere started at neutral conditions (pH = �7). AD1 (low level of ace-tate) demonstrated normal growth of the engineered strain, whileAD2 and AD3 appeared to have a negative impact on AV10 growth.Moreover, the experiments found that very little lactate was pro-duced in all cultures (<0.3 g/L). Previous work has suggested thatonly uncharged lactate (i.e., protonated lactic acid) can freely dif-fuse across the cell membrane (Angermayr et al., 2012). By disso-ciating extracellular lactic acid (acidity pKa = 3.9), alkalineculture conditions can ‘‘milk’’ protonated lactic acid out of cells.Thereby, our 13C-experiments roughly estimated that the concen-tration of total intracellular lactate was only �1.6 lg/g dry cellweight, consistent to concentration ranges of other intracellularmetabolites in Synechocystis 6803 (Takahashi et al., 2008).

Fig. 2. Synechocystis 6803 (wild type) growth in nutrient deficient BG-11 mediumsupplemented with AD3. Order from left to right column represents 1:4 mixture ofAD3 with: normal BG-11; BG-11 containing 50% N and P; BG-11 containing 25% Nand P; BG-11 containing 25% N & 50% P; BG-11 containing 50% N & 25% P. Error barsrepresent standard deviation of two biological samples.

Fig. 3. Growth rates of engineered Synechocystis 6803 on a mixture of BG-11medium with different anaerobically digested effluents (4:1 volume ratio). Orderfrom left to right column: BG-11 + AD3; modified BG-11 (with only 50% N and P) +AD3; BG-11 + AD2; modified BG-11 (with only 50% N and P) + AD2; BG-11 + AD1;modified BG-11 (with only 50% N and P) + AD1; and the control (BG-11 withoutwastewater). Error bars represent standard deviation of three biological samples.

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However, extracellular lactate concentrations can be accumulatedover 1 g/L. If cells are under neutral or acidic cultivation conditions,they cannot secrete lactate and thus decrease biosynthesisefficiency.

To verify this hypothesis, the engineered strain was cultivatedin pure (100% v/v) BG-11 medium at different initial pH values of

Fig. 4. Synechocystis 6803 production of D-lactate in different initial pH medium.Biomass growth (4A) and D-Lactate production (4B) in BG-11 medium withdifferent initial pH. Note: during cultivation, culture pH increased because ofconsumption of bicarbonate (up to ~10). Error bars represent standard deviation ofthree biological samples.

7, 8, 8.4 and 9 (Fig. 4). The culture with an initial pH of 8 showedthe highest D-lactate production (0.88 g/L), while the culture withan initial pH of 7 produced less than 0.2 g/L of D-lactate despiteno effect on its growth. Furthermore, as natural AD wastewateris acidic due to the presence of organic acids, mixing of dilutedAD2 with BG11 medium results in slightly acidic medium. Wetested the cultures using AD2 mixed with BG-11 medium at threedifferent initial pH conditions: pH 6.5, pH 8, and pH 9 (Fig. 5A and5B). Under alkaline conditions (pH > 8), engineered cells grew welland produced 1.26 g/L of D-Lactate. This was a 42% increase inD-lactate accumulation compared to accumulation with pure BG-11

Fig. 5. Synechocystis 6803 production of D-lactate using wastewater AD2 (20%supplementation). Biomass growth (A) and D-lactate production (B) with mediaunder initial pH of 6.5 (white bar), 8.0 (gray bar), and 9.0 (black bar). Error barsrepresent standard deviation of three biological samples. (C) Shows the isotopomeranalysis of proteinogenic amino acids in AV10 strain, which reveals cyanobacterialutilization of organic carbon from AD2 effluent. AV10 strain was cultivated in 13C-BG11 medium (NaH13CO3) for 4 days. With the addition of AD2 in the labeledmedium, the increase of 12C-enrichments (fraction of carbons that are unlabeled) inproteinogenic aspartate and glutamate (black bar) depict the incorporation oforganic carbon from AD2. Error bars represent standard errors during GC–MSmeasurement.

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medium. The acetate-rich AD effluents clearly enhance D-lactateproduction, which confirmed our previous findings that acetatemay redirect pyruvate flux towards lactate synthesis (Varmanet al., 2013). In addition, when the engineered cyanobacteria wasinoculated into a slightly acidic culture medium (initial culturepH = 6.5), their growth was strongly inhibited (Fig. 5A). This sug-gests that the engineered strain is more sensitive than the wildtype strain to suboptimal cultivation conditions. For example, poorsecretion of lactate under near neutral pH cultivation conditionmay strongly interfere with cell functions.

3.3. Advantages and limitations of using sewage sludge

Light and CO2 are often limiting factors for large scale micro-algal processes. To improve bio-production, high concentration ofCO2 and sufficient light sources must be provided for long-termincubation. Such an operation is economically infeasible sinceboth gas-pumping and lighting require large amounts of electric-ity. The addition of cheap and renewable organic carbon sourceswould be advantageous because it alleviates the dependency onlight and CO2. Recent studies have shown that organic substrates(such as glucose and acetate) can increase both cyanobacterialphotosynthesis and biomass growth compared to photoautotro-phic conditions (Yan et al., 2012). 13C-analysis of AV10 culturesgrown with AD2 mixed with labeled BG-11 medium (2 g/LNaH13CO3) confirmed the incorporation of organic carbon fromAD2 wastewater into biomass (estimated by the labeling mea-surement of proteinogenic aspartate and glutamate) (Fig. 5C).Moreover, AD2 supplementation also increased 12C concentrationin lactate (by �14%) compared to that in the pure 13C-BG11 cul-ture. This result clearly demonstrates the photomixotrophic useof organic carbon sources from the AD effluents by the engi-neered cyanobacterium. Thereby, it is desirable to design anaer-obic digestion processes to produce acetate (instead of CH4),which can be used to promote large-scale algal cultivationsand biochemical productions.

On the other hand, there are several limitations for usingengineered photo-biorefineries. First, product rate and titer bymicroalgae are low compared to sugar fermentation by hetero-trophs (Wang et al., 2011). Second, the dark color, heavy metals,high ammonia levels, and abundance of solids in the sludgeaffect algal growth, and thus require the dilution of wastewaterbefore its use in microalgal cultivations (Azov and Goldman,1982). Third, sterilization of the wastewater is necessary foralmost all algal biorefineries, and contamination by unwantedbacteria can be a serious problem in an open pond system(Cho et al., 2011). Fourth, engineered microalgae may haveunique physiological features, and need more stringent growthconditions than wild type species (e.g., sensitive to pH and car-bon conditions). There is still quite a way to go before wideindustrial applications of algal processes.

4. Conclusion

Nutrient requirements represent a considerable proportion ofthe algal bioprocessing costs (Clarens et al., 2010; Brentner et al.,2011). A waste-to-biorefinery system is desirable to reduce theemissions of greenhouse gases, to strip wastewater of nitrogenand phosphorus, and to sustain the bio-production of chemicalcommodities. This study also demonstrates the use of acetate fromanaerobic digestion effluents to alleviate carbon burdens on bothphotoautotrophic biomass growth and product biosynthesis. Thisconcept can be potentially expanded to other microbial cell facto-ries for converting waste into profitable products.

Acknowledgements

W.H., A.M.V. and Y.J.T. designed this research. W.H., A.M.V., L.Y.and Z.H. performed the experiments. W.H., A.M.V. and Y.J.T. wrotethe paper. Every author revised the manuscript. We are thankful toProfessor Yan Liu’s group at Michigan State University for provid-ing us with anaerobic digestion effluents. We also thank KatrinaLeyden for her help in editing the paper. This work was supportedby funding from the National Science Foundation (MCB0954016).

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