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Industrial wastewater treatment to Polyhydroxyalkanoates as biodegradable polymers

Prof. Dr. Adel Mohamed Kamal El-Deana.eldean@aun.edu.eg

Assiut University

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كل} الماء من وجعلنااألنبياء { سورة حي شيء

We made from water every living thing

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Water and its important:In view of the increasing demand for water and in the light of water problems, which may be faced Egypt in the near future, the all types of waste water should be recycled and reuse. Sugar and paper mills consume huge amounts of water, which should recycled and reused.

•use of high quality drinking water for all applications• expensive sewer systems with a restricted live time• dilution and only elimination of nutrients•no recovery of resources (nutrients, energy, …)• enormous production of excess sludge

Major problems in the handling of water:

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This water is an ideal environment for the growth of micro-organisms. These microorganisms feed on organic material and water soluble sugars making it consume dissolved oxygen in the water. This leads to a higher level of Biological oxygen demand (BOD) in this waste water in addition to, these microbes may be pathogenic and /or toxinogenic sources. In mostly the water of these factories are disposal to the Nile River or into the sea.

Industrial wastewater in the sugar and paper mills contain many of dissolved organic matter, which is difficult to remove by water treatment, especially those which containing sugars.

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When BOD Levels are high, dissolved oxygen (DO) levels decrease because the oxygen that is available in the water is being consumed by the microorganisms. Since less dissolved oxygen is available in the water, fish and other aquatic organisms may not survive.The main purposes of wastewater treatment systems are to remove organic pollutants, but it would be very attractive if there were a way to recover the organic pollutants as valuable organic materials. One of the possible ways to recover organic pollutants in wastewater is to convert them into polyhydroxyalkanoates (PHAs), which are biodegradable plastics.

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Role of polymer in our life:

Today, polymers have become a necessary part of contemporary life pertaining to their durability and resistance to degradation [1].Worldwide production of petroleum based synthetic polymer was approximately 270.0 million tons in 2007]2] and these synthetic polymers are found to be recalcitrant to microbial degradation [3].

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1.5 million ton(total)

100 million ton(total)

250 million ton(only fossil resources)

60 years ago 20 years ago 2010

Nowadays, we live in the „Plastic Age“…

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Importance• 2003- North

America only– 107 billion pounds of

synthetic plastics produced from petroleum

– Take >50 years to degrade

– Improper disposal and failure to recycle overflowing landfills

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Quantities of Utilized Plastic Materials in Different Global Regions

250 Mtons / a World production & consumption of Plastic Materials

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TODAY: Polymers Predominately Deriving from Petro-Industry

•Disadvantages of fossil base polymer•Highly Resistant Polymeric Materials •No natural degradation •Insufficient performance of recycling systems •High risk connected to the thermal conversion of plastic by inceneration.

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What the material which can be used instead of plastic?

The answer is polyhydroxyalkanoates.

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PolyhydroxyalkanoastesPolyhydroxyalkanoastes (PHAs) are naturally-occurring polymers produced by bacteria. They are produced within the bacterial cell and can be extracted and processed adhesives, films, and polymer performance additives. As a family of polymers, PHAs have functional properties sufficient to replace a significant portion of the 300 billion pounds of petroleum-based plastics used worldwide today.

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•Polyesters accumulated by wide rage of bacteria• Intercellular carbon and energy storage compounds• Produced under condition of limiting nutritional elements such as N, P, S, Mg, etc•Properties depending on monomers and chain length: thermoplastic – elastomeric•PHA industrially produced by Metabolix (Cambrige, MA) using a pure culture of Ralstonia eutropha •Biologically degradable

Polyhydroxyalkanoate (PHAs)

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News of the commercial exploitation of PHB started in 1963 when Chemistry and Engineering News published an article concerning the development of a thermoplastic biopolymer material which was grown by fermentation. The article described how the polymer was extracted from bacterial cells where PHB grew in the form of sub-micron granules. A treatment of the dry bacteria with acetone was followed by a chloroform extraction which provided a polymer yield of 70–80% or more based on the dry weight of the bacteria [4].

Polyhydroxyalkanoate (PHAs) History

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Polyhydroxyalkanoates (PHAs) Structures:

n = 1 R = hydrogen poly(-3-hydroxypropionaten = 1 methyl poly(-3-hydroxybutyraten = 1 ethyl poly(-3-hydroxyvaleraten = 1 propyl poly(-3-hydroxyhexanoaten = 1 pentyl poly(-3-hydroxyoctanoaten = 1 nonyl poly(-3-hydroxydodecanoaten = 2 R = hydrogen poly(-4-hydroxybutyraten = 3 R = hydrogen poly(-5-hydroxybutyrate

O CH

R

(CH2)n C

O

100-30000

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Polyhydroxyalkanoates (PHAs): Produced under conditions of:Low limiting nutrients (P, S, N, O)Excess carbon2 different types:Short-chain-length 3-5 CarbonsMedium-chain-length 6-14 Carbons~250 different bacteria have been found to produce some form of PHAs

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Polyhydroxybutyrate (PHB)

• Example of short-chain-length PHA

• Produced in activated sludge

• Found in Alcaligenes eutrophus

• Accumulated intracellularly as granules (>80% cell dry weight) Lee et al., 1996

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Bioplastic Production Using Mixed Microbial Cultures

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PHA Biosynthesis

Ojumu et al., 2004

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Applications

Field ApplicationIndustry Products, films, paper laminates & sheets,

bags and containers– Automobiles

Medical Sutures, ligament replacements, controlled drug release mechanisms, arterial grafts…

Household Disposable razors, utensils, diapers, feminine hygiene products, containers…

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Main disadvantage of biodegradable materials, •Too high production costs•Synthetic plastics ~ 1€/kg•Polylactic acid ~ 3-4 €/kg•Starch compounds ~ 2-4 €/kg •Polyhydroxyalkanoates ~ 3.5-5€/kg

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Option to lower PHA production costs using mixing cultures:Pure Culture fermentation• Expensive raw materials• High investment and operational costs• High yields of PHA production (80%PHA/cell dry weight)Mixed Cultures (e.g. activated sludge)• Cheap substrates-waste materials• Low operational costs• Lower yield of PHA production (60%PHA/cell dry weight)

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Industrial wastewaters for PHA production

• Food waste• Olive and palm oil mills

effluents• Sugar-cane molasses• Diary effluent• Paper mill effluents• Fruit and tomato

cannery effluents• Brewery effluent• Municipal wastewaters

Acidogenic fermentation PHA

production

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Methane versus PHA Production

• Yield of methane: 0.350 m3/kg COD• Methane selling price: 0.2 € /m3

0.07€/kg COD

•Yield of PHA: 0.40 kg PHB/kg COD•PHB selling price: 5 €/kg

2 €/kg COD

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Production of polyhydroxyalkanoates (PHAs) in activated sludge treating wastewater using mixed cultures represents an economical and environmental promising alternative to pure culture fermentation. A process for production of PHA from a paper mill and sugar factories wastewater was examined at many research laboratories. Pulp and paper mills generate varieties of pollutants depending upon the type of the pulping process

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The wood pulping and production of the paper products generate a considerable amount of pollutants characterized by biochemical oxygen demand (BOD), chemical oxygen demand (COD), suspended solids (SS), toxicity, and color when untreated or poorly treated effluents are discharged to receiving water.The high water usage, between 20,000 and 60,000 gallons per ton of product, results in large amounts of wastewater generation [5]. The effluents from the industry cause slime growth, thermal impacts, scum formation, color problems, and loss of aesthetic beauty in the environment. They also increase the amount of toxic substances in the water, causing death to the zooplankton and fish, as well as profoundly affecting the terrestrial ecosystem [6].

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Carbon Cycle of Bioplastics

CO2

H2OBiodegradation

CarbohydratesPlastic Products

Plants

Fermentation PHA Polymer

Photosynthesis

Recycle

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The attempt to develop more cost effective processes for PHA production include the use of mixed cultures based processes and low cost substrates based on agro-industrial wastes and by-products.Selection of a stable culture with a high PHA storage capacity is of major importance for the effectiveness of the process.

Examples of using byproducts in production of PHAsFrom sugar cane molasses:In this example, the use of sugar cane molasses (a by-product of the sugar refinery industry with a very high sugar content and low cost) was investigated for the production of bioplastics by mixed microbial cultures. Two-stage process were developed for PHA production by mixed cultures from sugar cane molasses, comprising:

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1. continuous molasses acidogenic fermentation, 2. Selection of a PHA-accumulating culture and batch PHA accumulation using the enriched culture and the fermented molasses thus produced. Different strategies were investigated for culture selection on a fermented molasses feed in either a sequencing batch reactor or a continuous ADF system, in order to understand the impact of reactor configuration and operating mode on the “Feast and Famine” process. The cultures thus selected were compared in terms of microbial population composition and PHA storage capacity.

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The molasses acidogenic fermentation was carried out in a continuous reactor under anaerobic conditions (T=30ºC; HRT=10h and OLR = 1 g/l.h of sugars).In a first moment, the CSTR was operated at different pH values ranging from 5 to 7 (in order to assess the effect of different VFA profiles in the fermented molasses on PHA production).A pH of 6 was then selected to operate the reactor to produce the effluent used in all remaining experiments.The reactor effluent was clarified by ultrafiltration and the clarified fermented molasses were used as a feedstock for culture selection and PHA batch accumulation after pH adjustment.Culture selection was carried out in both anaerobic SBR subjected to feast and famine conditions and anaerobic continuous ADF system composed of two reactors and a settler.

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The SBR 12 hour cycles consisted of four discrete periods: fill (12.5 min); aerobiosis (feast and famine) (11 h); settling (38.5 min) and draw (9 min). Both systems – SBR and continuous – were operated under similar conditions of organic loading rate (60 – 120 Cmmol VFA/l.d), carbon to nitrogen ratio (C/N/P of 100/8/1), hydraulic (HRT of 1d) and sludge retention times (SRT of 10d).Moreover, similar feast to famine ratios were used in both systems, since the hydraulic retention times of the two continuous ADF reactors were designed to match the lengths of the feast and famine phases of the SBR cycle.Preliminary PHA accumulation tests were carried out feeding the clarified fermented molasses produced at different pH to a PHA-accumulating culture selected using acetate as the carbon source (the selection process was described [7]).

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In order to compare the accumulation efficiencies of the cultures selected using the two different reactor systems, PHA accumulation tests were carried out in a batch reactor inoculated with sludge from either one of the two culture enrichment systems (SBR or continuous ADF system) and fed with clarified fermented molasses produced in the anaerobic reactor (operated at pH 6).

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Cane molasses

AnerobicCSTR

Fermentedmolasses

Biomass

ClarifiedFermentedmolasses

SBR

Batchreactor

Acidogenic fermentation

Sequencing Batch

Reactor

PHA production process from sugar molasses by mixed cultures using a Sequencing Batch Reactor.

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Clarifiedfermentedmolasses

Feastreactor Famine

reactor

Batchreactor

PHA production

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Qiong Wu et al reported that a strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45◦C and in 250 g/l molasses although the optimal growth temperature was 35–37◦C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth associated process [8].

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From starchy wastewater:Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60–80%), propionic (10–30%) and butyric (5–40%) acids. The total volatile fatty acids reached 4000 mg/L at a chemical oxygen demand (COD) loading rate of 25–35 g/L day1.

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A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid compositionprofile was affected by COD loading rate: a medium rate around 9 g l1 day1 gave a high propionic acid content (29% wt) and a high rate around 26 g l1 day1 led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used.

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PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80–100 mg NH3 per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8–9 g l1) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate [9].

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Recovery of PHAs from CellsPHA producing microorganisms stained with Sudan black or Nile blue Cells separated out by centrifugation or filtrationPHA is recovered using solvents (chloroform) to break cell wall & extract polymerPurification of polymerNighat Naheed et al reported that three types of organic waste contaminated soils were selected for the isolation of polyhydroxyalkanoates producing bacteria that is, molasses, oil/ghee and sewerage. A total of 54 bacterial strains were isolated and screened for the PHA production [10].

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References:1. Abhishek Dutt Tripathi, Ajay Yadav, Alok Jha, S. K. Srivastava; , J Polym Environ (2012) 20:446–4532. Lazarevic D, Aoustin E, Buclet N, Brandt N (2010) Plastic waste management in the context of a European recycling society: comparing results and uncertainties in a life cycle perspective. Resour Conserv Recycling 55:246–2593.Flechter A (1993) Plastics from Bacteria and for Bacteria: PHA as matural, biodegradable polyesters. Springer, New York, pp 77–93. 4. R. H. Marchessault; Cellulose (2009) 16:357–359 5. Nemerow N. L., Dasgupta A. Industrial and hazardous waste management, New York: Van Nostrand Reinhold; 1991. 6. D. Pokhrel, T. Viraraghavan; Science of the Total Environment 333 (2004) 37– 58

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7. Serafim, L.S., Lemos, P.C., Oliveira, R. and Reis, M.A.M. (2004). Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions. Biotechnol. Bioeng. 87(2), 145-160. 8. Qiong Wu, Honghua Huang, Guohong Hu, Jinchun Chen, KP Ho & Guo-Qiang Chen; Antonie van Leeuwenhoek 80: 111–118, 2001.9. Jian Yu; Journal of Biotechnology 86 (2001) 105–11210. Nighat Naheed, Nazia Jamil, Shahida Hasnain and Ghulam Abbas; African Journal of Biotechnology Vol. 11(16), pp. 3321-3332,

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Polyhdroxyalkanoate as biopdegridable polymers

Biodegradable thermoplastics from renewable resources

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Thank you