Nitrogen Removal and Lipid Production from Secondary Wastewater Using Green Alga Chlorella ...

85 Pages·2012·0.8 MB·English

Nitrogen Removal and Lipid Production from Secondary
Wastewater Using Green Alga Chlorella vulgaris

By
Zhouyang Liu
B.S., Harbin Institute of Technology, 2007

Submitted in fulfillment of the requirements
for the degree of Master of Science
in Environmental Engineering
in the Engineering & Applied Science College of the
University of Cincinnati, 2012

Committee Members: Dr. Joo-Youp Lee (chair)
Dr. Tim Keener
Dr. Mingming Lu
Abstract
Increasing nitrogen discharges into natural water systems have caused more frequent
eutrophication and other water quality issues. Microalgae are fast growing photosynthetic
microorganisms that can assimilate nitrogen and phosphorus from water. Also, lipid content of
certain strains of microalgae could reach over 60%, making microalgae excellent feedstock for
biodiesel production. Green alga Chlorella vulgaris was tested for nitrogen removal and lipids
production using secondary wastewater from municipal wastewater treatment plant. Around 60%
of NH -Nitrogen was removed after 48 hour, and removal rate was further increased to 75%
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when carbon dioxide gas was added periodically to control pH. When more active algae seeds
were used, NH -Nigrogen removal rate of 97.1% was achieved. Chlorella vulgaris was also
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very effective for removing low concentration of phosphate from secondary wastewater. When
growing at normal conditions, Chlorella vulgaris contained more polar lipid than neutral lipid.
Total lipid content of Chlorella vulgaris ranged from 10.6% to 14%, and fatty acids were mainly
C16 and C18, making it good biodiesel stock. A freshwater microalgae survey in southwest
Ohio was also conducted to provide useful information for future outdoor algae cultivation. 24
genera of cyanobacteria and 49 genera of eukaryotic algae were identified, with Synechococcus
sp. and Cryptomonas sp. being the predominant prokaryotic and eukaryotic microalgae,
respectively.

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Acknowledgments

I would like to express my sincere gratitude to Dr. Joo-Youp Lee for his guidance and support
throughout this study. I would like to thank Dr. Tim Keener and Dr. Mingming Lu for their
helpful suggestions and feedbacks.

I would also like to thank Dr. Daniel Oerther and Dr. Mau-Yi Wu for assisting and teaching me
molecular biology techniques, and all the members in the lab especially Jinsoo Kim for their help
with my experiments.

Lastly, I want to thank my mother who never lost faith in me, and Zhen who was there with me
when I was at the lowest point of my life, for which I am forever grateful.

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Table of Contents
Chapter 1. Introduction ................................................................................................................... 1
1.1 Nitrogen and Phosphorus Pollution ...................................................................................... 1
1.2 Nutrients Removal Techniques ............................................................................................. 2
1.3 Nutrients Removal Using Microalgae .................................................................................. 3
1.4 Biofuels from Microalgae ..................................................................................................... 4
1.5 Objective of This Study ........................................................................................................ 6
Chapter 2: Molecular Survey of Freshwater Microalgae in Southwest Ohio Area ........................ 7
2.1 Introduction ........................................................................................................................... 7
2.2 Materials and Methods .......................................................................................................... 8
2.2.1 Sites Description and Sampling ..................................................................................... 8
2.2.2 Genomic DNA Extraction .............................................................................................11
2.2.3 PCR Amplification ........................................................................................................11
2.2.4 Cloning of rRNA Gene Fragments .............................................................................. 12
2.2.5 Sequence Analysis of Clones ....................................................................................... 13
2.3 Results and Discussions ...................................................................................................... 14
2.3.1 Cyanobacteria .............................................................................................................. 14
2.3.2 Eukaryotic Algae .......................................................................................................... 15
2.3.4 Comparison between Autumn and Winter Samples ..................................................... 18
2.4 Conclusions ......................................................................................................................... 22
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Chapter 3. Methods and Materials ................................................................................................ 24
3.1 Algae Cultivation ................................................................................................................ 24
3.2 Gravimetric Analysis of Lipids ........................................................................................... 25
3.2.1 Collection of Algae Biomass ....................................................................................... 25
3.2.2 Solvent Extraction of Total Lipids ............................................................................... 25
3.2.3 Silica Gel Purification .................................................................................................. 26
3.2.4 Esterification ................................................................................................................ 26
3.3 GC Analysis ........................................................................................................................ 26
3.4 Algae Cell Density Measurement ....................................................................................... 29
3.5 Ammonia Nitrogen and Phosphorus Analysis .................................................................... 29
3.5.1 Ammonia Nitrogen Measurement ................................................................................ 29
3.5.2 Orthophosphate Measurement ..................................................................................... 30
3.6 Total Inorganic Carbon Measurement ................................................................................. 30
Chapter 4. Ammonia Removal Using Chlorella vulgaris ............................................................. 31
4.1 Wastewater Properties ......................................................................................................... 31
4.2 Growth of Chlorella vulgaris in Closed Reactors .............................................................. 33
4.3 Nitrogen Removal Using Chlorella vulgaris ...................................................................... 36
4.3.1 Preparation of Algae Seeds .......................................................................................... 36
4.3.2 Experiment Set-up ....................................................................................................... 36
4.3.3 Nitrogen Removal Without Using CO ........................................................................ 37
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4.3.4 Nitrogen Removal with CO ........................................................................................ 41
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Chapter 5. Lipid Analysis ............................................................................................................. 45
5.1 Lipids in Microalgae ........................................................................................................... 45
5.3 Lipid Extraction from Algae ............................................................................................... 47
5.2 Neutral and Total Lipids in Chlorella vulgaris ................................................................... 48
5.3 Polar Lipids in Chlorella vulgaris ...................................................................................... 52
Chapter 6. Conclusions ................................................................................................................. 56
References ..................................................................................................................................... 58
Appendix ....................................................................................................................................... 65

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Chapter 1. Introduction
1.1 Nitrogen and Phosphorus Pollution
Increasing nitrogen and phosphorus discharge into natural water systems poses great threat to
water quality and human health. Excessive nutrients (nitrogen and phosphorus) in aquatic
systems lead to more frequent eutrophication, which is a worldwide problem affecting both fresh
and coastal marine water resources. In the US, point sources are responsible for more than 50%
of the nutrients in rivers and streams in urban areas (Carpenter et al., 1998). Research shows
that municipal wastewater treatment plants (WWTPs) account for about 75 percent of the
nutrients from point sources (USGS, 1994; EEA 2005).

Nitrogen and phosphorus are the main causes of surface water eutrophication. Freshwater
eutrophication leads to excessive growth of phytoplankton, depletion of dissolved oxygen, and
has negative impacts on drinking water supply (Smith, 2003). Increased turbidity caused by
algae will require extra chlorination, increasing not only the costs but also the levels of
disinfection by-products (USEPA, 2001). Unlike freshwater systems where phosphorus is the
limiting factor for eutrophication (Schindler, 1977), nitrogen is the most crucial element in
coastal marine water systems (Oviatt et al., 1995). Adverse effects of coastal marine
eutrophication include blooms of toxic algae, reduced yields of desirable fish species, threats to
endangered aquatic species, shifts in marine ecosystem structures (Smith, 2003).

Most wastewater treatment plants include primary treatment and secondary treatment. Primary
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wastewater treatment uses screening, sedimentation, and other physical treatment processes to
remove floating and suspended solids from raw sewage. Primary treatment can remove about
50% of the BOD but wastewater still contains a high amount of organic pollutants. After the
passage of Clean Water Act in 1972, most WWTPs in the U.S. adopted secondary wastewater
treatment. Secondary wastewater treatment processes involve biological treatments to remove
organic matters from wastewater. The most commonly used approach is using microorganisms
in activated sludge to break down organic matters, and a secondary settlement to separate
activated sludge from treated wastewater (USEPA, 1998). Advanced wastewater treatment
(tertiary treatment) is not required at all WWTPs, and there are many variations, depending on
the characteristics of wastewater and effluent water quality requirements.

1.2 Nutrients Removal Techniques
Biological Nitrogen Removal (BNR) is currently the most widely used technique in WWTPs.
The biological processes involve in nitrogen removal are nitrification and denitrification. First,
ammonia is converted into nitrite (NO -) by autotrophic bacteria group Nitrosomonas under
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aerobic conditions. Then nitrite is oxidized to nitrate (NO -) by another bacteria group, in most
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cases Nitrobacter. Nitrification process alone does remove nitrogen from wastewater.
Denitrification is required to convert nitrate into nitrogen gas by heterotrophic bacteria groups
such as Pseudomonas and Alcaligenes under anoxic conditions. Effluents from most
wastewater treatment plants (conventional activated sludge treatment including a nitrification
step) contain 1-10 mg/L of NH -N (USEPA, 2011). Organic nitrogen cannot be removed by
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biological process and only the non-soluble fraction can be removed using solids separations
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