potential application of algicidal bacteria for improved lipid recovery with specific algae

R E S EA RCH L E T T E R

Potential application of algicidal bacteria for improved lipidrecovery with specific algae

Eric M. Lenneman, Ping Wang & Brett M. Barney

Department of Bioproducts and Biosystems Engineering and Biotechnology Institute, University of Minnesota, St. Paul, MN, USA

Correspondence: Brett M. Barney,

Department of Bioproducts and Biosystems

Engineering and Biotechnology Institute,

University of Minnesota, 1390 Eckles Avenue,

St. Paul, MN 55108-6130, USA.

Tel.: 612 626 8751; fax: 612 625 6286;

e-mail: [email protected]

Received 10 December 2013; revised 14

March 2014; accepted 26 March 2014. Final

version published online 17 April 2014.

DOI: 10.1111/1574-6968.12436

Editor: Gerard Wall

Keywords

Dunaliella tertiolecta; Neochloris

oleoabundans; Pseudomonas

pseudoalcaligenes; Aeromonas hydrophila;

algicidal.

Abstract

The utility of specific strains of natural algicidal bacteria isolated from shallow

wetland sediments was evaluated against several strains of algae with potential

immediate or future commercial value. Two strains of bacteria, Pseudomonas

pseudoalcaligenes AD6 and Aeromonas hydrophila AD9, were identified and

demonstrated to have algicidal activity against the microalgae Neochloris oleo-

abundans and Dunaliella tertiolecta. These bacteria were further evaluated for

the potential to improve lipid extraction using a mild solvent extraction

approach. Aeromonas hydrophila AD9 showed a nearly 12-fold increase in lipid

extraction with D. tertiolecta, while both bacteria showed a sixfold improve-

ment in lipid extraction with N. oleoabundans.

Introduction

The dependence of advancing societies on fossil fuels, the

high cost associated with these fuels and the eventual

depletion of fuel sources have resulted in a strong and

recently renewed interest in alternative biofuels. Several

next-generation biofuels and biomass feedstocks, includ-

ing algae, show potential benefits vs. current seed oil-

based biodiesel and corn-derived ethanol (Chisti, 2007,

2008; Hu et al., 2008). Further, many algae are already

utilized as sources of unique bioproducts important to

nutrition, pharmaceuticals and as specialty chemicals

(Borowitzka, 1999; Raja et al., 2007; Hu et al., 2008).

The cycling of organic compounds in nature involves

both processes that synthesize complex biologic com-

pounds and processes that degrade these compounds back

to simple molecules. In this manner, photosynthetic

organisms utilize carbon dioxide, water, and sunlight to

produce carbohydrates and more complex biomolecules,

which are later used by heterotrophic organisms for their

own growth, thus comprising the various components of

the carbon cycle. Important in this cycle are organisms

involved in the decomposition of decaying matter in

aquatic systems, including various microorganisms that

might assist in breaking down larger organisms and mic-

roalgae.

Previous reports of algicidal bacteria have focused on

bacteria acting against bloom-forming algae or cyanobac-

teria that are known to produce various toxins that can

affect human and animal health (Lee et al., 2000; Kim

et al., 2007, 2008, 2009; Mu et al., 2007; Jung et al., 2008;

Kang et al., 2008; Paul & Pohnert, 2011; Cho, 2012).

While these reports have identified a range of different

bacteria that can be detrimental to the growth of these

algae that are undesirable, less information is available

about organisms that degrade algae cells that might serve

as sources of specific high-value compounds and biofuels.

Many algae are viewed as an ideal future feedstock for

biologic oils, although the costs associated with extraction

and conversion of these fuels are often a primary barrier

to economic feasibility of algal fuels (Chisti, 2007; Hu

et al., 2008; Mercer & Armenta, 2011; Halim et al., 2012).

In this report, we investigated the potential of two algi-

cidal bacteria isolated from shallow wetland sediments in

FEMS Microbiol Lett 354 (2014) 102–110ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

MIC

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OLO

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LET

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Minnesota to be utilized as a potential means of improv-

ing lipid extraction under mild conditions, with a com-

mon solvent often utilized to extract seed oils in

agriculture. The potential improvement in quantities of

lipid extracted and the effect these strains had on the cell

structure of several strains of algae important in aquacul-

ture or as a source for unique bioproducts including bio-

fuels is presented.

Materials and methods

Algal and bacterial strains and growth

conditions

All algal strains were obtained from the UTEX culture

collection (Austin, Texas) unless otherwise specified.

Dunaliella tertiolecta CCAP19/6b was obtained from the

Dunaliella Culture Collection at Brooklyn College, and

was grown in artificial seawater media (1 mL trace ele-

ment solution, 18 g NaCl, 500 mg KCl, 200 mg

MgSO4�7H2O, 20 mg CaCl2�2H2O, 200 mg K2HPO4,

50 mg Na2SiO3�9H2O, 600 mg NaNO3, 300 mg Na2SO4,

10 mg ferric ammonium citrate, 50 mg silt, and 100 lLvitamin stock solution, all per L and adjusted to pH 7.8).

Neochloris oleoabundans UTEX 1185, Scenedesmus dimor-

phus UTEX 417, and Chlorella sorokiniana UTEX 1602

were all grown in a freshwater media based on Bristol

media (1 mL trace element solution, 300 mg K2HPO4,

80 mg MgSO4�7H2O, 20 mg CaCl2�2H2O, 200 mg

Na2SO4, 25 mg NaCl, 15 mg ferric ammonium citrate,

600 mg NaNO3, all per L and adjusted to pH 7.6). The

stock vitamin solution contained 5 mg mL�1 thiamine

HCl, 0. 1 mg mL�1 biotin, and 0.1 mg mL�1 cyanocobal-

amin. The trace element stock solution contained 1 g

boric acid, 1 g sodium ethylenediaminetetraacetic acid

(EDTA), 200 mg MnCl2�4H2O, 20 mg ZnCl2, 15 mg

CuCl2�2H2O, 15 mg Na2MoO4�2H2O, 15 mg

CoCl2�6H2O, and 10 mg KBr, and all per L. Bacteria were

grown on Miller’s lysogeny broth (LB) media at 25 °Cunder dark conditions. All reagents were obtained from

either Sigma-Aldrich (St. Louis, MO) or Fisher Scientific

(Pittsburgh, PA) unless otherwise specified. Specific fatty

acid methyl ester (FAME) standards were obtained from

Nu-Chek Prep, Inc. (Elysian, MN).

Isolation of algicidal bacteria

Algal strains were grown using the media described above

in 1.4-L tubes (c. 5 cm diameter 9 75 cm) with a 1-mm

glass capillary tube to provide constant aeration at a flow

rate of 0.3-L air per minute per L of culture media. Air was

provided via mass flow controllers providing compressed

air combined with 1% CO2. Cultures were grown for sev-

eral days following exhaustion of nitrate (limiting reagent)

as measured by nitrate indicator strips (Hach Company,

Loveland, CO). The culture was transferred to a standard

media bottle and inoculated with environmental samples

(10 mL) of pond sediments collected from select sites in

Minnesota. Cultures were placed in the dark and mixed

daily for several weeks until the algae showed visual signs

of stress or deterioration by microscopy. Aliquots were

then transferred to fresh culture of the same algal species

several additional times to enrich the culture for algal-

degrading bacteria. Under this regime, the algal cells pro-

vide the only source of carbon or nitrogen to any bacteria

growing in the culture. Fresh algal cells were then centri-

fuged and resuspended at 10X concentration in sterile agar,

and bacteria from the enriched cultures were plated on the

agar. Plates were visually inspected for zones of clearing

surrounding isolated colonies. These colonies were then

streaked several times to LB media plates to purify and pre-

pare frozen stocks. Genomic DNA was isolated using the

ZR Fungal/Bacterial DNA Kit (Zymo Research, Irvine,

CA). Strains were identified by amplifying the 16S rRNA

gene region of the genome using universal primers TPU1

(50 AGAGT TTGAT CMTGG CTCAG 30) and RTU8 (50

AAGGA GGTGA TCCAN CCRCA 30) and sequencing the

isolated PCR fragments with RTU8.

Determination of algicidal properties and

improvement in lipid extraction

Algal stock cultures were grown as above. Cultures were

divided into 10-mL aliquots and then inoculated with puri-

fied isolates of potential algicidal bacteria. These co-cul-

tures were mixed and incubated at room temperature in

the dark until the degradation of the algae cells was

observed as a change in culture color and cell morphology

compared with a control culture under the same conditions

but devoid of any added bacteria. Free lipids were extracted

from the aqueous phase by adding 2 mL hexane to the

10 mL culture and then vortexing the solution three times

at 5 s intervals. The hexane phase was then removed, and

50 lL of N-methyl-N-(trimethylsilyl)trifluoroacetamide

(MSTFA) was added to 1 mL of the hexane extract to

derivatize any free fatty acids. Samples were analyzed by gas

chromatography using conditions described previously and

compared with an external standard prepared from pal-

mitic acid and linoleic acid treated with MSTFA in the

same manner (Barney et al., 2012). Additionally, control

samples for total lipids of the same quantity of algae were

also extracted following the method of Bligh and Dyer

(Bligh & Dyer, 1959), and the obtained lipid fraction was

derivatized to FAMEs by treatment with methanol and sul-

furic acid for quantitation against an external FAME refer-

ence standard (Barney et al., 2012).

FEMS Microbiol Lett 354 (2014) 102–110 ª 2014 Federation of European Microbiological Societies.Published by John Wiley & Sons Ltd. All rights reserved

Algicidal bacteria 103

Scanning electron microscopy

One milliliter of solution was taken from freshly mixed

10 mL co-cultures. The solution was centrifuged to pellet

the cells which were then placed in 2% gluteraldehyde

and 0.1 M sodium cacodylate buffer for 2 h, rinsed in the

sodium cacodylate buffer, and then placed in 1% osmium

tetroxide and 0.1 M sodium cacodylate buffer for 2 h.

Specimens were rinsed in ultrapure water (NANOpure

Infinity) and dehydrated in an ethanol series. Once in

100% ethanol, they were put through two changes of hex-

amethyldisilazane for 5 min each. Drops of the suspen-

sion were placed on individual round glass coverslips

mounted on aluminum stubs and allowed to dry, sputter-

coated with gold-palladium, and observed in a scanning

electron microscope (S3500N; Hitachi High Technologies

America, Inc., Schaumberg, IL) at an accelerating voltage

of 10 kV at the University of Minnesota Imaging Center.

Real-time video imaging of bacteria and algae

cells

Algal and bacterial cells were grown together as described

above. After 4 days, several microliters of culture was

placed on a microscope slide and viewed using bright

field microscopy with an ix70 microscope (Olympus,

Center Valley, PA), and video was recorded with a SPOT

Insight Camera (Diagnostic Instruments, Sterling Heights,

MI) at the University of Minnesota Imaging Center.

Results

Isolation of algicidal bacteria

Bacteria from environmental samples were enriched using

an approach that provided algal cells as the sole carbon

and nitrogen source as described in the methods. Isolated

bacteria were then screened for algalytic activity in liquid

culture with specific algae vs. a control without any added

bacteria. Figure 1 shows an example of the differences in

culture appearance for one such experiment using algae

cells and specific isolated bacteria. Of the four algal

strains screened as part of this work, only N. oleoabun-

dans and D. tertiolecta were sufficiently prone to degrada-

tion using this approach with the isolated strains, while

S. dimorphus and C. sorokiniana showed no visible signs

of degradation within 14 days of inoculation of bacterial

communities that resulted in deterioration with the other

two strains. Two specific strains of algicidal bacteria, des-

ignated AD6 and AD9 (where AD is a strain designation

for an algal degrader), resulted in the most significant

degradation with N. oleoabundans and D. tertiolecta and

were selected for further study.

Identification of algicidal bacteria

Pure isolates of bacteria obtained from the enrichment

process were identified by similarity searches using 16S

rRNA gene sequencing. The two primary strains selected

for further study were identified as Pseudomonas pseud-

oalcaligenes AD6 and Aeromonas hydrophila AD9. Addi-

tional strains identified included several species of

Citrobacter, P. putida, Stenotrophomonas rhizophila,

Sphingomonas sp. and Azospirillum picis. The majority of

these strains were > 99.5% identical over a range of

c. 900 bases. Isolated strains were utilized to screen for

their algal cell degradation potential with fresh algal cell

cultures.

(a)

(b)

Fig. 1. Algicidal bacteria with select algae strains. The two images

above illustrate the visible deterioration of N. oleoabundans and

Dunaliella tertiolecta cells exposed to Pseudomonas pseudoalcaligenes

AD6 and Aeromonas hydrophila AD9. (a) shows the strains

immediately after inoculation with the specific bacteria strains. (b)

shows the same samples at the time of lipid extraction. Samples in (b)

are as follows, from left to right, N. oleoabundans and

P. pseudoalcaligenes AD6, N. oleoabundans and A. hydrophila AD9,

N. oleoabundans control, D. tertiolecta control, D. tertiolecta and

A. hydrophila AD9, D. tertiolecta and P. pseudoalcaligenes AD6.


104 E.M. Lenneman et al.

Screening of algicidal potential

Algal cells showed signs of distress in cultures contain-

ing the selected algicidal bacteria after a few days of

exposure. Incubation with A. hydrophila AD9 resulted in

rapid deterioration of algal cells for both N. oleoabun-

dans and D. tertiolecta, while P. pseudoalcaligenes AD6

took several additional days. The D. tertiolecta co-culture

resulted in a more dramatic color change in comparison

to the N. oleoabundans co-culture. When viewed under

bright field microscopy, cells of both algae were with-

ered and deflated and D. tertiolecta cells had lost motil-

ity (N. oleoabundans is not visibly motile using bright

field microscopy). The potential to improve lipid extrac-

tion in the treated co-cultures was evaluated by expos-

ing the cultures to a simple solvent extraction protocol,

and the lipid recovery was compared to algal cells with-

out bacteria. Figure 2 shows the quantity of lipids

(based on peak areas for all derivatized fatty acids

recovered compared to an external standard) obtained

from algal cells subjected to each of the primary bacte-

ria vs. a control incubated under the same conditions

but without any added bacteria. P. pseudoalcaligenes

AD6 showed only a minor increase in quantities of lip-

ids that were extracted from D. tertiolecta (twofold

increase), while A. hydrophila AD9 resulted in the great-

est increase in extractable lipids with D. tertiolecta

(nearly 12-fold). For N. oleoabundans, both P. pseudoal-

caligenes AD6 and A. hydrophila AD9 resulted in a simi-

lar increase (c. sixfold) in extractable lipids vs. the

control samples. Lipid profiles (retention times of spe-

cific derivatized fatty acids) were similar for the specific

algae strain regardless of whether the sample included a

specific bacterium or was simply the algal control sam-

ple. This indicates that the lipids are primarily derived

from the algae, and not related to the specific bacterium

in either case. Comparisons of the quantities of lipids

obtained via the mild hexane extraction vs. the quanti-

ties obtained when control samples containing the same

quantity of algae were treated with the more rigorous

solvent extraction protocol of Bligh and Dyer revealed

that c. 50% of total lipids were extracted using the mild

extraction conditions in the D. tertiolecta sample treated

with A. hydrophila AD9.

Evaluation of algal cells by electron

microscopy

Evaluation of cells using bright field microscopy revealed

some visual differences, especially for D. tertiolecta, where

motility can be easily observed at 4009 magnification. To

better understand the effects that certain bacteria were

having on various algae, the algal cells were further evalu-

ated using electron microscopy. Cells of D. tertiolecta and

N. oleoabundans co-cultured with P. pseduoalcaligenes

AD6 appeared highly deteriorated and fragmented with

many P. pseudoalcaligenes AD6 bacterial cells visible along

the cell wall of the algae (Fig. 3b and e). A different

change was found when cells were co-cultured with A.

hydrophila AD9, especially for N. oleoabundans which

appeared more withered and deflated than fragmented by

this technique (Fig. 3f).

Real-time video imaging of bacteria and algae

cells

Aeromonas hydrophila AD9 was also evaluated with

D. tertiolecta cells to visualize the behavior of bacteria

and algal cells in real time. Using bright field micros-

copy, video was obtained depicting bacterial cells

approaching algal cells at high speed, colliding with the

Fig. 2. Lipid extraction of algae subjected to specific bacteria. Shown

above are results from lipid extractions of 10 mLs of culture of

Dunaliella tertiolecta or N. oleoabundans cells exposed to the

bacterial strains Pseudomonas pseudoalcaligenes AD6 and Aeromonas

hydrophila AD9. Lipid quantities are based on peak areas of all

derivatized fatty acids found in each of the samples vs. an external

control of a known quantity of derivatized fatty acids. The results

presented are based on three separate experiments (n ≥ 3).



algal cells, and then swimming away. There were also a

number of events in which the bacteria would collide

into the algal cells, back up, then collide again, repeating

this a number of times before swimming away. The

durations of the interactions between algal cells and bac-

teria were longer than would be expected if these colli-

sions were a result of simple random motions and

collisions. Previous reports of a bacterium capable of lys-

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 3. Electron microscopy of algae subjected to specific bacteria. Shown above are several electron microscopy images of algae control cells (a,

d) and algae inoculated with specific bacterial strains (b, c, e and f) derived from the experiments shown in Fig. 1. Specific strains of algae and

bacteria are denoted on each image.



ing blue-green algae reported a similar feature where

close contact between the polar tip of the bacterium and

the algae was necessary for the lysis (Shilo, 1970). This

may indicate that the bacteria are using some sort of

chemotaxis to find cells and deliver specific algicidal

agents, such as a protease or lipase, although this obser-

vation is strictly speculative. The findings of zones of

clearing surrounding bacterial colonies when grown on

solid-media plates containing a lawn of algae are a

further indication of potential extracellular algicidal com-

pounds and have been noted by others as a screen for

algicidal compounds (Sakata et al., 2011). A video clip of

several interactions are provided as a Supporting Infor-

mation, Video S1.

Discussion

Algae are viewed as a potential future feedstock for biofu-

els based on rapid growth and the production and accu-

mulation of lipids (Chisti, 2007; Hu et al., 2008). Many

algae are known to accumulate either neutral lipids such

as triacylglycerides as a form of energy storage under

nutrient-depleted conditions or polar lipids that compose

the extensive network of membranes in photosynthetic

cells (Chisti, 2007; Hu et al., 2008; Gouveia et al., 2009;

Tang et al., 2011; Hulatt et al., 2012). Either class of lipid

could serve as a feedstock of reduced carbon from which

to produce hydrocarbon fuels or other valuable bioprod-

ucts, although the polar lipids would be expected to be

more difficult to extract, as they are embedded in mem-

branes. One key hurdle to lipid isolation from algae is the

expense involved in a range of extraction approaches

(Mercer & Armenta, 2011; Halim et al., 2012). As part of

this work, we have investigated the potential of several

natural strains of bacteria to assist in the degradation of

the algal cell structure and improve the accessibility of

the cellular lipids for extraction. These studies with

environmental isolates of specific bacteria show some

promise for developing a natural process to improve the

extraction of certain lipids.

Several model strains of algae that have specific com-

mercial value or have shown promise for lipid produc-

tion were selected for these studies, including

D. tertiolecta, N. oleoabundans, S. dimorphus and C. soro-

kiniana (Borowitzka, 1999; Raja et al., 2007; Gouveia

et al., 2009; Araujo et al., 2011; Tang et al., 2011; Hulatt

et al., 2012). Each of these strains was obtained from cul-

ture collections, and they have been maintained in our

laboratory for several years as unialgal cultures with no

visible signs of contamination and are known to produce

high yields of neutral lipids under conditions of nutrient

stress. Following the isolation protocol detailed here, sev-

eral bacterial strains were identified and purified from a

series of enrichments followed by isolation on solid

media. The bacterial strains that showed the highest

degree or rate of cell deterioration for specific algae were

further studied and included the strains P. pseudoalcalig-

enes AD6 and A. hydrophila AD9. It is interesting that of

c. 20 bacteria that were isolated or identified, one of the

top strains identified in this study was a strain of

P. pseudoalcaligenes, which also had high similarity when

aligned with a strain of P. mendocina. Another strain,

P. mendocina DC10 isolated from Lake Dianchi in Yun-

nan Province, China, has also been implicated in other

reports as an algicidal bacterium acting on the cyanobac-

terium Aphanizomenon flos-aquae (Shi et al., 2009).

Broader studies with the two bacterial strains selected

here revealed a number of interesting features. Pseudomo-

nas pseudoalcaligenes AD6 and A. hydrophila AD9 showed

some degree of selectivity and specificity for the algae tar-

geted, as indicated by the concentrations of fatty acids

recovered from co-cultures following treatment vs. a con-

trol. It is possible that lower yields of lipid from D. tertio-

lecta treated with P. pseudoalcaligenes AD6 are the result

of P. pseudoalcaligenes AD6 utilizing the fatty acids as a

substrate for growth. Additionally, S. dimorphus and

C. sorokiniana seemed far less susceptible to deterioration

by these two strains, again indicating a certain degree of

specificity for these bacteria toward specific algae, while

other algae seemed resistant to attack. This feature of an

algicidal bacterium targeting specific strains of algae has

been observed in previous reports, and is not without

precedent (Kodani et al., 2002; Shi et al., 2009; Sakata

et al., 2011; Liu et al., 2013). D. tertiolecta was especially

susceptible to treatment by A. hydrophila AD9, resulting

in the highest levels of lipid extracted and a nearly 12-

fold improvement in extractable lipids vs. the control

devoid of any added bacteria. The liquid–liquid partition-

ing extraction selected here using hexane as a nonpolar

solvent is a mild technique and was chosen as it would

be representative of extraction processes that are less

energy intensive than using harsh solvents or high-energy

sonication.

Changes in cell morphology for each algal strain were

used as an additional measure of algalytic activity follow-

ing treatment with specific algicidal bacteria. Scanning

electron micrographs of the algae and bacteria co-cultures

show higher incidences of specific changes in cell

morphology of the two algal strains (Fig. 3). D. tertiolecta

treated with P. pseudoalcaligenes AD6 and A. hydrophila

AD9 showed similar changes, with the algae cells appear-

ing deteriorated and fragmented. The changes in cell

morphology were different for N. oleoabundans, as

N. oleoabundans cells exposed to A. hydrophila AD9

remained intact but deflated and withered. Dunaliella ter-

tiolecta and N. oleoabundans are generally cultured under



very different conditions (salt water vs. freshwater respec-

tively) and have many other features differentiating them

from one another. Certain Dunaliella species lack a rigid

cell wall, utilizing an elastic plasma membrane that allows

the cells to rapidly change in volume in response to

changes in osmolarity (Cowan et al., 1992). This potential

lack of a rigid cell wall may make D. tertiolecta more sus-

ceptible to biologic attack by algicidal bacteria in a differ-

ent manner than other algal strains. Recent reports found

that N. oleoabundans exhibited a much thinner cell wall

when grown under freshwater conditions vs. conditions

of high salinity (Baldisserotto et al., 2012). As the N. oleo-

abundans for this study was grown in a freshwater med-

ium, this might further explain the differences between

N. oleoabundans and the other green algae that were not

prone to degradation.

Algicidal activity has been reported previously for dif-

ferent strains of Aeromonas and Pseudomonas, and in cer-

tain cases, potential algicidal compounds have been

isolated, although the studies focused on harmful bloom-

forming cyanobacteria or diatoms (Kodani et al., 2002;

Sakata et al., 2011; Liu et al., 2013) and not on the

potential to improve lipid extraction. Potential algicidal

compounds from Aeromonas and Pseudomonas include

small extracellular compounds such as 2,3-indolinedione

(isatin), harmane, and lysine or clavulanate (Kodani

et al., 2002; Sakata et al., 2011; Liu et al., 2013). Reports

with alternative bacteria such as Kordia and Pseudoaltero-

monas have implicated extracellular proteases as being

responsible for algicidal activity (Lee et al., 2002; Paul &

Pohnert, 2011). Investigations of potential mechanisms

for activity seen here will be pursued in the future, once

genome sequencing is completed for these strains.

The two bacteria, P. pseudoalcaligenes AD6 and A. hy-

drophila AD9, based on this study with specific strains of

algae, should be classified as potential plant pathogens.

The further potential as pathogens in other organisms

was not a focus of this study and has not been thor-

oughly evaluated, although many strains of Aeromonas

are generally associated with human disease. Aeromonas

are ubiquitous in both brackish and fresh water, where all

these strains were isolated (Seshadri et al., 2006). Com-

mon diseases associated with Aeromonas include diarrhea

and wound infections (Pund & Theegarten, 2008), while

specific strains of A. hydrophila have been implicated in

more serious diseases such as necrotizing fasciitis (Min-

naganti et al., 2000), or flesh-eating bacteria syndrome,

generally linked to wound infections. Related strains of

P. mendocina are also common environmental strains that

can cause opportunistic nosocomial infections (Chi et al.,

2005). Thus, utilization of either of these strains for

large-scale application may not be suitable. However, as

both of these strains are ubiquitous in nature, it should

also be pointed out that this degradation of cells might

also be important in the biodegradation of additional

materials in the environments where these are found

(these strains were isolated from a shallow marsh sedi-

ment in Minnesota). Indeed, P. pseudoalcaligenes strains

are also highly relevant in the study of biologic methods

of polychlorinated biphenyl degradation and in many fur-

ther biotechnology applications (Triscari-Barberi et al.,

2012).

Genome sequencing projects have been initiated for

both of these bacteria and are currently in assembly with

a draft sequence to be released in the near term. Addi-

tional methods to characterize algicidal activity in strict

quantitative terms would enable more detailed studies of

the specific mechanism behind this algicidal feature and

will be the focus of future efforts. Initial screens have

revealed a number of potential extracellular proteins pro-

duced by each bacterium, and the availability of a

genome sequence should allow us to pursue more

detailed studies to look for causative agents of the algi-

cidal activity, so that the future potential application of

algicidal bacteria and their agents in algal harvest can be

fully evaluated.

Acknowledgements

This work was supported in part through a career award

from the Initiative for Renewable Energy and the Envi-

ronment, a program of the Institute on the Environment

at the University of Minnesota, supported by the State of

Minnesota and Minnesota electricity ratepayers, Minne-

sota Agricultural Experiment Station MIN-12-070 and

from the United States Department of Energy DE FG36-

08GO18161. We thank the Microbial Engineering pro-

gram at the University of Minnesota for additional sup-

port to Eric Lenneman. We thank Michael Tetzlaff for

assistance in growth of specific algae for the enrichment

process.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Video S1. A video file is included showing examples of

interactions between A. hydrophila AD9 and D. tertiolecta

under bright field microscopy.



potential application of algicidal bacteria for improved lipid recovery with specific algae

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