Boosting d-lactate production in engineered cyanobacteria using sterilized anaerobic digestion effluents

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  • Bioresource Technology 169 (2014) 462467Contents lists available at ScienceDirect

    Bioresource Technology

    journal homepage: www.elsevier .com/locate /bior techBoosting D-lactate production in engineered cyanobacteria usingsterilized anaerobic digestion effluents 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: (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,aDepartment of Energy, Environmental and Chemical Engineering, Washington University, St. Louis, MO 63130, USAbBiological 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 6803a 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;Olgun, 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 idealif 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) 462467 463metabolic 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 Lius 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 (1015 mL working volume) under 80100 lmol ofphotons m2 s1. 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 theirTable 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)

  • 464 W.D. Hollinshead et al. / Bioresource Technology 169 (2014) 462467initial 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 (SigmaAldrich, St. Louis, MO) in tetrahydrofu-ran (SigmaAldrich, St. Louis, MO) at 70 C. The samples wereanalyzed via GCMS, 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 (day1) 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 mediumwas 0.7 g/L or below (i.e.,

  • 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.

    W.D. Hollinshead et al. / Bioresource Technology 169 (2014) 462467 465However, 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 ofFig. 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-11Fig. 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 GCMSmeasurement.

  • 466 W.D. Hollinshead et al. / Bioresource Technology 169 (2014) 462467medium. 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 impr...


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