Toward the sustainable bioremediation of table olive processing wastewaters coupled

with the generation of value-added products

Eugenia Papadaki, Fani Th. MantzouridouLaboratory of Food Chemistry & Technology,

School of Chemistry, AUTH

International Olive Oil Council (IOC), Economic: World table olive figures (2017)

Spain23%

Greece8%

Italy3%

Algeria9%

Argentina3%

Egypt13%

Iran2%

Morocco5%

Peru2%

Syria6% Turkey

15%

USA3%

2.6 million tons

Lyeeffluent

Spanish-style green olives

Lye treatment

Washings

Californian-style black-ripe

olives

Storage in acidified brine

or water (optional)

Lye treatment and air

oxidation

Washings and color fixing

Brining

Naturally black olives

Washing watereffluent

Brine

Packaging

Brining -Fermentation

Brining -Fermentation

Wastewaters

Papadaki & Mantzouridou, Biochem. Eng. J. 2016, 112, 103–113

0

1

2

3

4

5

6

Lye Washing waters Brine Total

0 0

0.57

1.270.59

2.24

0.60

3.73

1.41

3.31

0.64

5.71

mᵌ

was

tew

ate

rs /

to

n o

live

Naturally black olives Spanish-style green olives Californian-style black-ripe olives

Papadaki & Mantzouridou, Biochem. Eng. J. 2016, 112, 103–113

Spanish-style green olives

Lye Washing waters

Brine

12.1 10.0 4.0

12.1 10.2 81.4

18.8 16.1 15.9

1.8 2.3 2.8

Brine

4.3

111.5

32.3

2.8

Naturally black olives

Californian-style black-ripe olives

Lye Washing waters

Brine

11.9 9.9 –

– – –

2.4 4.1 35

0.4 0.3 –

Papadaki & Mantzouridou, Biochem. Eng. J. 2016, 112, 103–113European Parliament. Council Directive 91/271/EEC, Off. J. Eur. Communities 1991, L135, 40−52

❖Phytotoxic activity❖Antimicrobial activity

❖Organic matter

COD < 125 mg O2/L

Requirements for the discharge of agro–food industrial wastewaters set by the European Union

Parameter

pH

Electrical conductivity (mS/cm)

Chemical oxygen demand - COD (g O2/L)

Total phenolic compounds (g/L)

↓Eutrophication of aquatic ecosystems

↓Shifts in soil microflora

↓Production of malodorous gases

✓Wastewater detoxification (Ozonation, photocatalysis,

wet air oxidation,electrochemical oxidation)✓Recovery of value-added products (Ultrafiltration,

evaporation)↓Polluted side streams

↓High energy requirements↓High cost technology

✓Wastewater detoxification✓Production of high value-

added products✓Green technology✓Low cost technology

Papadaki & Mantzouridou, Biochem. Eng. J. 2016, 112, 103–113

Pleurotus ostreatus in lye effluents

Aggelis et al., Appl. Microbiol. Biotechnol. 2002, 59, 353–360

LACCASE ACTIVITY

50% Color↓

MANGANESE PEROXIDASE ACTIVITY

NO REDUCTION OF PHYTOTOXICITY

52-76% Total phenolic compounds↓

Aspergillus niger in lye and washing water effluents1

Kyriacou et al., Process Biochem. 2005, 40, 1401–1408

Chemical Treatment

9000 L Air-lift Bioreactor

Biological treatment

Electrochemicaloxidation

1.6%H₂O₂

93% COD ↓

0.4%Ca(OH)₂

98% COD ↓

Sedimentation

71% COD↓65% Total phenolic compounds↓

RP−HPLC 280 nm

OH

OH

OHO

CH3

COOCH 3

OH

OHO

—Untreated —Treated

O

COOCH 3

CH3

OHO

O

RP−HPLC 240 nm

Papadaki et al., J. Agric. Food. Chem. 2018, 66, 4891–4901

Phytotoxicity

Lactuca sativa Lepidium sativum

84% Total phenolic compounds↓(initial concentration 1.5 g/L)

0.3-1.5 g/L Initial concentration of total phenolic compounds

↑ NaOH

↑ Phytotoxicity

↓ Phenolics

↓ Phytotoxicity

Aspergillus niger in lye and washing water effluents2

Activated sludge1

Ferrer-Polonio et al., Chem. Eng. J. 2015, 273, 595–602Aggelis et al., J. Agr. Eng. Res. 2001, 80, 283–292

Sequencing stirred batch reactorAir flow 550 L/h, dissolved oxygen 1.5-2.5 mg/L

No need for biomass acclimatization to salinity

Single-step biological process in brine

88% COD↓98% Total phenolic compounds↓

Multi-step biological process in lye effluents

10 L Draw-and-fill 1 L Stirred Draw-and-fill

Anaerobic process Aerobic process

84% COD↓28% Total phenolic compounds↓

50% COD↓13% Total phenolic compounds↓

In single step aerobic process,

no phenol degradation

was determined

Activated sludge2

Patsios et al., J. Chem. Technol. Biot. 2015, 91, 2253–2262

20 L submerged membrane bioreactorPore size 0.04 μm,

flux operation 10 L/m2 h,air flow rate 8 N dm3/min

Stable performance at moderatebiomass concentration (<10 g/L)

Mixture of table olive processing wastewaters from various processings

92% TOC (Total organic carbon)↓83% Total phenolic compounds↓

Nannochloropsis gaditana in washing waters

Serrano et al., J. Environ. Sci. Health A 2017, 1–6

1 L glass bottleaeration rate 0.5 vvm

Sequential adaptation of microalga to wastewater at

increased substrate concentrations (10-80%) ensures

the process efficiency

69% TOC (Total organic carbon)↓72% Total phenolic compounds↓

Natural light of•14h of light•10h of dark

Methane (biogas) production using activated sludge1

Methane

Primary component of biogas (55-75%)

Mes et al., ISBN 9090171657, 2003, 58–102Papadaki & Mantzouridou, Biochem. Eng. J. 2016, 112, 103–113

In 2010 Europe’s biogas production reached 10.9 millions of tons

Biogas is a clean renewable fuel that can replace fossil fuels and reduce the negative environmental impact of the latter

During anaerobic digestion, organic compounds are converted into biogas through the enzymatic activities of naturally

occurring microorganisms under conditions of oxygen depletion

Beltran et al., Hazard. Mater. 2008, 154, 839–845; Zarkadas & Pilidis, Bioresour. Technol. 2011, 102, 4995–5003; Martín et al., Process Biochem. 1991, 26, 101–107; Intanoo et al., Bioresour. Technol. 2014, 173, 256–265; Prabhudessai et al., J. Energy 2013, 1–7

20 L Spherical magnetically stirred digester

81-94% COD ↓

Methane (295 mL/g CODdegraded)

50 L stainless steelcomplete mix anaerobic digester

81% TOC (total organic carbon) ↓17% Total phenolic compounds ↓

Methane (300 mL/g VSadded)

Methane (biogas) production using activated sludge2

Treatment of brine

Co-treatment of lye and washing water effluents with animal manure

Feed rate 1.66 L/d,agitation 60 rpm for 6 min/h, 55 °C

Batch operation, 35 °C

Coconut oil cake320 mL CH4/g VS

Cassava wastewaters183 mL CH4/g COD

Olive mill wastewaters260 mL CH4/g COD

Lactic acid and hydroxytyrosol production using lactic acid bacteria1

Hydroxytyrosol

Lactic acid

Acidulant, flavor enhancer, antimicrobial preservative mainly in food and pharmaceutical industries, as a raw material in plastics production

Antimicrobial, antioxidant, anticancer,anti-inflammatory, antidiabetic and neuroprotective properties

Wang et al., J. Biosci. Bioeng. 2015, 119, 10–18Fernández-Mar et al., Food Chem. 2012, 130, 797–813

Lactic acid and hydroxytyrosol production using lactic acid bacteria2

Concentrates (COD 399g/L)123.7 g/L lactic acid

36.4 g/L Hydroxytyrosol

Evaporation to 10%(After pH correction to 8.3)

12.5 g/L Lactic acid3.4 g/L Hydroxytyrosol20% COD ↓ to 38g/L

Brenes et al., Chem. Technol. Biotechnol. 2004, 79, 253–259; http://www.sigmaaldrich.com/catalog/substance/Linko & Yavanainen, Enzyme Microb. Technol. 1996, 19, 118–123; Wee et al., Enzyme Microb. Technol. 2004, 35, 568–573Kalogerakis et al., J. Environ. Manage. 2013, 128, 749–758

500 L Batch fermenterInitial pH value 5,

9 months, 20-25 °C

Treatment of washing waters with Lactobacillus pentosus

Distillates (COD 2.1g/L)Not detected lactic acid<0.01g/L Hydroxytyrosol

Lactic acid200-500 €/10 g (≥ 98%)

Hydroxytyrosol250 €/25 mg (≥ 98%)

Olive mill wastewaters (liquid-liquid extraction)1.2 g/L Hydroxytyrosol

Barley starch(Lactobacillus casei)162 g/L Lactic acid

Sugar molasses(Enterococcus faecalis)

96 g/L Lactic acid

Toward the development of lactic acid bacteria starter culturesfor table olive production

①High tolerance to acidic conditions②Extracellular polysaccharide production③Hydrolysis of oleuropein

① Tolerance to acidic conditions②Lipolytic activity③Extracellular polysaccharide production④Hydrolysis of oleuropein

①High tolerance to acidic conditions②Extracellular polysaccharide production③Lipolytic activity④Hydrolysis of oleuropein

Lactobacillus plantarum

Lactobacillusspp.

Leuconostocspp.

Enterococcusspp.

Lactic acid bacteria isolates from brine

Leuconostoc mesenteroides

Enterococcus faecium

Fendri et al., Environ. Technol. 2013, 34, 181–193

•Control of fermentation•Elimination of debittering step

Table olive processing wastewaters are high polluted, but also have the potential to be used as fermentation feedstocks.

The use of fungi, activated sludge and microalgae for the treatment of table olive processing wastewaters was really effective, causing important detoxification of these wastes. Findings are expected to provide useful information for the subsequent treatment of residual contaminants.

To ensure the economic feasibility of the process, the production/recovery of important to the market chemicalsthrough the wastewater treatment should be targeted.

Biotechnological production of methane, lactic acid and hydroxytyrosol is competitive to other wastes.

Oleuropeinolytic lactic acid bacteria isolates have the potential to be used as starter cultures for table olive fermentation by eliminating the debittering step.